Citation
A comparative study of evaporative water loss in three species of garter snakes of the genus thamnophis

Material Information

Title:
A comparative study of evaporative water loss in three species of garter snakes of the genus thamnophis implications for species drought tolerance along the Front Range of Colorado
Creator:
Henke, Clint Richard
Publication Date:
Language:
English
Physical Description:
vii, 51 leaves : ; 28 cm

Subjects

Subjects / Keywords:
Garter snakes -- Colorado ( lcsh )
Osmoregulation ( lcsh )
Garter snakes ( fast )
Osmoregulation ( fast )
Colorado ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 44-50).
General Note:
Department of Geography and Environmental Sciences
Statement of Responsibility:
by Clint Richard Henke.

Record Information

Source Institution:
|University of Colorado Denver
Holding Location:
Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
71778749 ( OCLC )
ocm71778749
Classification:
LD1193.L547 2005m H46 ( lcc )

Full Text
A Comparative Study of
Evaporative Water Loss in
Three Species of Garter Snakes
of the Genus Thamnophis:
Implications for species drought tolerance along the Front Range of Colorado
by
Clint Richard Henke
B.A., Fort Lewis College, 1996
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment of the
requirements for the degree of
Master of Science
Environmental Sciences
2005


This thesis for the Master of Science
degree by
Clint Richard Henke
has been approved
by
Ronald D. Beane
3f3>/0(c


Henke, Clint Richard (Master, Environmental Sciences)
A Comparative Study of Evaporative Water Loss in Three Species of Garter Snakes
of the Genus Thamnophis: Implications for species abundance along the Front Range
of Colorado
Thesis directed by Assistant Professor Michael J. Greene
ABSTRACT
Common garter snake (Thamnophis sirtalis) populations appear to be declining in
the State of Colorado. Coincidentally, the state has been undergoing a drought for the
past 4 years. In this study I analyzed this species ability to retain metabolic water in
relation to two highly abundant garter snake species (Thamnophis radix and
Thamnophis elegans) that overlap in range with T. sirtalis. Additionally, I measured
sprint speeds in all three species prior to and following dehydration to see if changes
in speed times occurred. Furthermore, evaporative water loss rates were measured
using unaltered and delipidized shed skin samples as a barrier. Thamnophis sirtalis
parietalis lost 21 percent of its body mass when deprived of water over a six day
period. This amount was 2.5 times the amount that similar species, T. radix and T. e.
vagrans, lost when exposed to the same environmental conditions. In the second
study, snakes sprinted at slower speeds when dehydrated. Significant differences
existed between sprint times prior to dehydration and following 6 days of dehydration
for all species when combined (p<0.01). Percentage of total extractable skin lipid
amounts ranged from 4 to 15 percent in this study. Evaporative water loss rates for T.
s. parietalis were higher than those for T. radix and T. e. vagrans, although
significant differences did not exist among the species in shed skins without lipid
extraction. Significant differences in EWL rates did exist in delipidized skins
between species.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
Signed

in


ACKNOWLEDGEMENT
My thanks to my advisor, Michael Greene for his patience with me for the past two
years and for taking me as his first graduate student at the University of Colorado,
Denver prior to arriving. I also would like to thank the Colorado Division of Wildlife
for its general interest in this project. Finally, I would ERO Resources for its support,
flexibility, and encouragement while I pursued this degree.
IV


CONTENTS
Figures
Tables
Chapter
1.0 Introduction 1
2.0 Methods and Materials 10
2.1 Animals 10
2.2 Captive Environmental Conditions................................10
2.3 Measurement of EWL in Snakes....................................10
2.4 Performance Analysis............................................13
2.5 EWL Measurement from Shed Skins/Skin Lipid Comparisons..........14
2.6 Statistical Analysis............................................15
3.0 Results 17
3.1 Live Snake Water Loss...........................................17
3.2 Sprint Speed Analysis...........................................27
3.3 Shed Skin EWL/Lipid Analysis....................................35
4.0 Discussion 37
4.1 Summary.........................................................37
4.2 Live Snake Water Loss...........................................37
4.3 Sprint Speed Analysis...........................................38
4.4 Shed Skin EWL/Lipid Analysis....................................40
4.5 Relevance to Conservation.......................................42
5.0 Bibliography 44
v


FIGURES
Figure 2.1: Screw-Top Vial Setup for EWL Measurements through Shed
Skin Samples.......................................................16
Figure 3.1 Average Percent Water Loss over a 6-day Period for Each
Species; Error Bars Represent Standard Error of the Mean..........19
Figure 3.6 Mass of snakes versus TEWL for All Three Species
Combined; Lines Fitted by Linear Regression; Equations for
Regression Lines are Given in the Text............................24
Figure 3.7 Percent Body Mass Loss due to TEWL over a 6-day Trial
versus Snake Mass(g); Lines Fitted by Linear Regression:
Equations for Regression Lines are Given in the Text..............25
Table 3.2 Sex, SVL and micro-habitat at time of capture for each
individual used in experiments.....................................27
Table 3.3 Average sprint times and sprint time change between day 1 and
day 6. Outlying data has been removed..............................28
Table 3.4 Average absolute velocities for each of the three species on day
1 and day 6. Outlying data has been removed.......................28
Figure 3.8 Percent Time Change for Each Species Following 6-Days of
Dehydration (outliers removed).....................................29
Figure 3.9 Linear Regression Fit for Sprint Speeds verses Percent Water
Loss (all data); Equation for Regression Lines is Given in the
Text...............................................................31
Figure 3.10 Sprint Speed Time Difference verses % TEWL for T. s.
parietalis; Lines Fitted by Linear Regression; Equation for
Regression Line is Given in the Text...............................32
Figure 3.11 Sprint Speed Time Difference verses % TEWL for T. radix;
Lines Fitted by Linear Regression; Equation for Regression
Lines is Given in the Text.........................................33
Table 3.5 Percent dry weight of extractable lipids following hexane and
chloroform extraction..............................................35
Figure 3.13 Mean EWL (mg/cm2/hr) from Vials Sealed with a Barrier of
Intact Skin from Three Species of Garter Snake.....................36
vi


TABLES
Table 3.1. Mean daily percent evaporative water loss and relative
standard deviations between T. s. parietalis, T. radix and T. e.
vagrans..............................................................18
Table 3.2. Sex, SVL and micro-habitat at time of capture for each
individual used in experiments.......................................27
Table 3.3. Average sprint times and sprint time change between days 1
and 6................................................................28
Table 3.4 Average absolute velocities for each of the three species on day
1 and day 6..........................................................28
Table 3.5 Percent dry weight of extractable lipids following hexane and
chloroform extraction................................................35
vii


1.0 Introduction
Prevention of water loss is one of the major evolutionary adaptations allowing
organisms to live in terrestrial habitats (Schmidt-Nielsen, 1975). Adaptations to dry
land include the development of the amniotic egg; lipid barriers on the epidermal
surfaces of animals; nasal turbinate structures; lipid-coated cuticle layers in insects;
thick impermeable skin (present in many desert lizards); and behavioral adaptations
such as coiling to reduce exposure of surface areas; or burrowing (Mayhew and
Wright, 1971; Burken et al., 1985; Andrade and Abe, 2000; Tu et al., 2002; DeNardo
et al., 2003). Physiological properties often correlate with habitat and may limit a
species ability to inhabit specific habitat types (Winne et al., 2001). Therefore,
understanding a species physiological and behavioral adaptations and constraints to
terrestrial life is crucial in understanding habitat maintenance and protection.
Early studies on water loss physiology assumed that skin was rather impermeable and
that most water was lost through respiration (Pettus, 1958). However studies
conducted by Bentley and Schmidt-Nielsen (1966) indicated that a significant amount
of water evaporates cutaneously. When studying temperature relations in the desert
iguana (Dipsosaurus dorsalis), Templeton (1960) found that water loss occurred
through respiration. It has become well-known in present times that terrestrial
animals (specifically ectotherms) primarily lose water cutaneously or via respiration
(Lillywhite and Sanmartino, 1993; DeNardo et al., 2003). Adaptations to prevent
water loss include physiological, morphological and behavioral development.
Physiological adaptations such as kidney structure, shedding cycle, blood circulation,
and body size aid in the prevention of excessive water loss (Gans et al., 1968; Cohen,
1975; Winne et al., 2001). For example, insects have developed lipid-coated cuticle
layers that, when removed, increase cuticular respiration, leading to increased water
loss (Ebeling, 1974; Beament, 1961). Many vertebrates have also developed lipid
barriers on the epidermal surface. This lipid matrix prevents desiccation and is
crucial to life outside of an aquatic environment (Elias, 1983; Elias and Menon, 1991;
Tu et al., 2002).
Additional physiological adaptations exist so that life can occur out of the water.
Many terrestrial animals have developed efficient kidneys (Schmidt-Nielsen, 1964;
Schmidt-Nielsen, 1975). Rodents of the family Heteromyidae are well known for
their ability to survive long periods of time with no drinking water due to the
development of long loops of Henle which allows for more reabsorbtion of water in
1


the kidney (Schmidt-Nielsen, 1964). Reptiles and birds are able to excrete
nitrogenous waste as uric acid, which is insoluble, as a solid, therefore conserving
water (Schmidt-Nielsen, 1975; Cogger and Zweifel, 1992). Additional adaptations
for life on land include the development of the amniotic egg and nasal turbinate
structures for burrowing (Hillenius, 1994; Rossi and Rossi, 1995; Wu et al., 2004).
Morphological acclimatization aids in the prevention of water loss. For example, the
Australian thorny devil (Moloch horridus) contains spiny scales on the cranium that
funnel water from rain and condensation to the mouth (Mayhew and Wright, 1971).
Another adaptation includes nasal structures that capture exhaled water (Perry et al.,
2000). Additional morphological adaptations that allow water conservation may
include thick impermeable skin or light body color (Porter and Tracy, 1983).
Mayhew and Wright (1971) found that the desert lizard Phymosoma mcalli has
water impermeable skin. Many desert iguanids possess the ability to change colors so
that at cooler temperatures, skin color is darker. Conversely, at higher temperatures,
skin colors may become lighter so that overheating does not occur (Atsatt, 1939;
Luke, 1994). Desert Heteromyid rodents have developed fur-lined pouches, which
minimize water loss when carrying seeds to burrows (Burt and Grossenheider, 1980).
This same family of rodents has developed elongated hind limbs with enlarged thigh
muscles, long tail, short neck and elongate hind feet which allow for saltatorial
locomotion which allows travel over long distances with fairly low energy
expenditure (Vaughan, 1986).
Behavioral mechanisms such as body position, and activity time may aid in the
prevention of water loss (Gans et al., 1968; Perry et al., 2000). Several species of
animal conserve water simply by living underground where temperature and humidity
are more consistent. Numerous species of reptiles, birds and mammals burrow to
escape heat and conserve moisture (Krakauer et al., 1968; Lahav and Dmiel, 1996).
Bulova (2002) found that desert tortoise (Gopherus agassizi) burrows contained a
much more consistent temperature and humidity compared to surface temperatures
and humidity levels. Certain species of desert kangaroo rats will seal off an
underground burrow to raise humidity levels and recycle moisture from breathing
(Randall, 1994). Several desert-dwelling snakes and lizards such as the sand boas of
Eurasia and Gila monsters and tortoises of the deserts of North America spend 80 to
90% of their lives underground (Krakauer et al., 1968; Brown and Carmony, 1991).
Some snake species have been known to coil to reduce surface area (Cohen, 1975), or
shift activity patterns to correspond with climatic conditions (Cohen, 1975; Mautz,
1982). Cohen (1975) found that the desert snake (Cerastes cerastes) lost significantly
less water when allowed to coil. Furthermore, dehydrated snakes of the same species
coiled readily when allowed (Cohen, 1975).
2


Evaporative water loss (EWL) is one physiological variable that potentially affects
habitat use. EWL is a combined measure of cutaneous and respiratory water loss.
Water lost through excretion is not included when measuring EWL (Krakauer et al.,
1968; Mautz, 1982; Winne et al., 2001). Previous studies suggest that cutaneous
water loss (CWL) contributes highly to total evaporative water loss (TEWL), whereas
respiratory water loss (RWL) does not contribute highly to TEWL. (Schmidt-
Nielson, 1964; Dmiel, 1972; Lahav and Dmiel, 1996; Moen et al., 2005). Dmiel
(1998) proved this by placing various snakes in glass tubes with head and body
separated by a latex membrane. Air was passed through each tube compartment
containing the body or head of the snake, at a specific velocity. Air passing over the
body of the snake was captured in a separate tube containing Drierite (anhydrous
CaC03). Differences in tubes with Drierite were analyzed. When various viperid
snakes from the Middle East were placed in glass tubes with head and body separated
by a latex membrane, CWL accounted for 61 to 66% of the TEWL (Dmiel, 1998).
Similarly, CWL contributed 71 to73% of TEWL in the desert snake Spalerosophis
diadema using similar methods (Dmiel, 1985). DeNardo (2003) et al. noted that
CWL resulted in 20 to71% TEWL and RWL consisted of approximately 7 to24% of
TEWL in Heloderma suspectum placed into environmentally controlled chambers.
Reptiles have adapted successfully to terrestrial environments in part by developing
physiological and behavioral mechanisms that prevent desiccation (Andrade and Abe,
2000). Early studies assumed that reptile scales inhibit EWL significantly (Pettus,
1958; Cloudsley-Thompson, 1971; Schmidt-Nielsen, 1975). However, it has been
noted that when comparing EWL in scaleless snakes verses snakes with scales, there
was virtually no difference in EWL amounts (Bennett and Licht, 1974).
The presence of epidermal lipid layers is thought to be the barrier that prevents
toxic intake, infection and dehydration in terrestrial beings (Tu et al., 2002). Data
suggest that this lipid barrier thickens following first and second ecdysis in snakes
(Tu et al., 2002). Epidermal lipid barriers vary from species to species. The amount
of lipids on each species differs and is most responsible for limiting EWL (Roberts
and Lillywhite, 1980; 1983; Tu et al., 2002). Roberts and Lillywhite (1983) noted
that within 11 snake species, lipid content comprised 2 to nearly 13 percent of dry
mass of the epidermal layer.
Lipid removal by immersion of shed snake skin samples into hexane and subsequent
chloroform baths has been found to increase water loss through the skin significantly
(Roberts and Lillywhite, 1980; 1983; Burken et al., 1985). For example, Burken et al
(1985) found that when 25 to 35% of lipids were removed from shed snake skins
from 8 species, transepidermal water loss occurred 3 to 10-times more quickly than
3


skins with 100% of lipids remaining. Shed skins that were 100% delipidized lost
water more quickly than evaporating water from an open free water cell. In this same
study, water loss rates prior to lipid removal ranged from 0.15 to 0.93 mg HaO/cm/hr.
Non-polar lipid-removal resulted in EWL rates of 1.5 to 2.4 mg H20/cm/hr. With
100% of lipids removed, EWL rates increased to a range of 20 to 32 mg FLO/cm/hr
(Burken et al., 1985). Additionally, Roberts and Lillywhite (1983) found that snakes
from arid environments contain significantly higher quantities of lipids than snakes
from more humid environments. Permeability of snake skin and habitat aridity was
also inversely related.
Abiotic environmental factors such as temperature and humidity also affect EWL
(Dmiel, 1998; Perry et al., 2000). Populations of reptiles apparently become adapted
to local environmental conditions to minimize EWL. For example, Dmiel (1998)
found that when the temperature is increased by 10F, nocturnal viperids from mesic
environments lost 2 to 3 times more water than viperids from xeric environments.
Similarly, EWL increased significantly in the desert lizard species Diposaurus
dorsalis when the temperature increased by 10F (Templeton, 1960). Furthermore,
desert reptiles that inhabit subterranean habitats are likely to lose more water when
forced to remain above ground than when placed in cooler, moister substrate
(Krakauer et al., 1968). Studies involving desert tortoise have revealed that EWL
decreased 16 to 54% when body temperatures dropped 6C (Bulova, 2002).
Furthermore, burrows utilized by tortoises were significantly cooler and more humid
than surface temperatures. This same study found that tortoises tend to be active for
very short periods of the day typically between the hours of 0900 and 1200 -
minimizing EWL (Bulova, 2002). Winne and Keck (2004) found similar shortened
activity times with whiptails within the genus Aspidoscelis (formerly
Cnemidophoris). Maximum daily activity lengths were typically 2 to 5 hours per day.
When placed into environmentally controlled chambers, lizards typically became
inactive by early afternoon, even if surface temperatures were not particularly high
(23C). Milstead (1957) found that five different species of Aspidoscelis retreated to
underground burrows when surface temperatures reached 26 to 30C.
The above information is essential when researching species habitat use and
conservation. Natural stressors such as drought, as well as anthropogenic stressors
such as rapid development and pesticide use may adversely affect one species, while
other closely-related species populations remain stable (Rossman et al., 1996).
For this study, it was my aim to compare the water loss physiology of three related
species of garter snakes, all native to Colorado, with the goal of offering insight into
the drought tolerance of each. The questions that I have asked include:
4


Is Thamnophis sirtalis parietalis, a species in decline in Colorado compared to
the other two species, less drought tolerant than the other two species of garter
snake studied that have stable populations?
What are the mechanisms the allow drought tolerance? Is performance
affected by prolonged dry periods?
An important aspect of such a study is the ability to compare congeners that live in
differing microhabitats. For example, Nielson (2002) compared water loss rates for
four closely related skinks on the north island of New Zealand and found the two rare
species lose more water than two more common species. Similarly Moen et al.
(2005) found that two closely related North American pit vipers (Agkistrodon
piscivorus and A. contortrix) lose significantly different amounts of water.
Garter snakes are excellent models for this type of comparative study. They are
among the widest ranging, and often most abundant reptile species found and are
relatively small snakes that tend to thrive well under captive conditions (Rossman et
al., 1996). They inhabit a variety of microhabitats ranging from aquatic systems to
deserts (Rossman et al., 1996; Hammerson, 1999). Different species may occur in a
variety of microhabitats within the same region, which suggests that EWL rates could
differ significantly between species, especially in more arid regions of the west.
The common garter snake (Thamnophis sirtalis) is one of the most widely distributed
snakes in the world and one of the few species that ventures into the Artie Circle
(Conant, 1975; Rossman et al., 1996;). Rossman et al. (1996) identifies eleven
subspecies within this species that may reach lengths up to 137.2 cm. Fitch (1965)
state that T. s. parietalis inhabits almost every mesic environment in Kansas.
Similarly, Rossman et al. (1996) state that western subspecies of T. sirtalis are more
aquatic than eastern counterparts. T. sirtalis is also classified as a generalist feeder.
Fitch (1982) mentions that in Kansas T. s. parietalis was recorded eating voles, deer
mice and harvest mice (Microtus, Peromyscus, and Reithrodontomys), in addition to
ranid frogs and earthworms.
In Colorado, T. s. parietalis historically inhabited the South Platte River watershed
from Denver north (Figure 1.1). This species has been known to inhabit riparian and
wetland areas throughout the northeastern portion of the state (Hammerson, 1999). In
recent years the populations of this species have begun to decline in Colorado
(Hammerson, 1999; CDOW, 2003). Reasons for the population declines are currently
unknown. However, recent severe drought, declines in local amphibian populations,
and rapid residential and commercial development are suspected. Between 2000 and
2004, Colorado endured one of the most severe droughts of modem times. In 2002
5


the City of Denver recorded its driest year on record with less than 7.5 inches (190.5
mm) of total precipitation for the year (NOAA, 2004). Above normal annual
precipitation has not been observed in the city since 1999. Furthermore, rural
communities north of Denver have undergone a rapid increase in human population.
Many riparian and wetland areas that once contained high numbers of this species
have been developed. In 2001, the Colorado Division of Wildlife (CDOW) listed the
common garter snakes as a State Species of Special Concern and has made it illegal
to collect specimens without proper permitting (CDOW, 2003).
Two additional species of garter snake overlap in range with the common garter
snake in Colorado. The plains garter snake (Thamnophis radix) is also a wide-
ranging species with a geographic distribution from southern Alberta and Manitoba to
northeastern New Mexico (Rossman et al., 1996). In Colorado, this species inhabits
areas east of the Front Range foothills (Hammerson, 1999) (Figure 1.2). T. radix is a
fairly long snake measuring at over 109 cm with distinct dorsal and lateral yellow to
orange stripes (Nero, 1957).
Thamnophis radix is classified as a generalized feeder (Rossman et al., 1996).
Gregory (1977) states that in Manitoba, this species feeds primarily on amphibia and
invertebrates. The diet of this species has been known to shift with the seasons,
feeding primarily on earthworms in the spring, but including ranid frogs and mice in
the summer months (Siegel, 1984).
Typically T. radix inhabits prairie and pastures near wetlands or streams (Gregory,
1977), but may often stray far from water (Hammerson, 1999; pers. obs.). In
Colorado, this species is fairly abundant throughout its historical range (Hammerson,
1999). This snake appears to have adapted readily to development, but is often killed
on roadways and by mowing practices (Dalrymple and Reichenbach, 1984;
Hammerson, 1999). During collection of this species for this research, many of the
specimens were collected in suburban neighborhoods within Metropolitan Denver.
Similarly, the western terrestrial garter snake (Thamnophis elegans ssp vagrans) is a
locally common snake that inhabits areas within the western 1/3 of North America
(Rossman et al., 1996). In Colorado T. e. vagrans overlaps in range with the
common and plains garter snakes (Figure 1.3). This snake appears to be very
adaptable to surrounding conditions. In the Sierra Nevada range in California this
species is reportedly very strongly associated with aquatic habitats (Jennings et al.,
1992). Similarly, in the higher elevations of the Rocky Mountains, Thamnophis
elegans appears to be semi-aquatic (Rossman et al., 1996). However, on Vancouver
6


Island, the ecological preferences of T. elegans compared to two similar species (T.
sirtalis and T. ordinoides) consists of areas lacking water or forest cover (Gregory,
1984). In Colorado, T. e. vagrans inhabits nearly every habitat type (Hammerson,
1999).
T. e. vagrans is the most generalized species in both food preferences and habitat
affinity in Colorado. Throughout most of its range, this species appears to have the
widest food range of any snake (Rossman et al., 1996). In areas where T. elegans is
the dominant species, food variety is fairly narrow (Kephart, 1982). However, in
areas such as Vancouver Island where this snake occurs sympatrically with
Thamnophis ordinoides and Thamnophis sirtalis, food habits are very generalized. In
Colorado, I have personally observed this snake feeding on montane voles {Microtis
montanus) and western chorus frogs (Pseuddacris triseriata) within meters of each
other. The ability of this snake to feed on a variety of different prey has likely
enhanced its ability to inhabit a wide range of habitats within the state. I was able to
find this snake along drainages and streams in central Denver.
Each of the three species used in this research have similar activity patterns in
Colorado. Hammerson (1999) reports that each of the three species typically emerge
from hibernation in mid-March and return to hibernation in late-September and early
October. I have personally have observed T. radix and T. elegans basking on warm
days in mid-February (Henke per. observation). Similarly, many of the T. sirtalis
collected for this study were collected in late October 2004.
In this study, experiments were conducted to compare EWL rates in T. s. parietalis,
T. radix and T. e. vagrans. The first consisted of a dehydration experiment in which
access to drinking water was removed and water loss was measured. Defecation and
urination was controlled by the lack of food and water provided and did not affect
EWL rates. EWL loss was measured by recording mass loss in each snake. The
purpose of this experiment was to analyze how each species responds physiologically
to dry conditions that could potentially occur in the natural environment.
Subsequently, performance was also measured in healthy snakes and dehydrated
snakes. Sprint speed and species performance have been measured by other
researchers as a general indicator of physiological performance (Hertz et al., 1982;
Pinch and Claussen, 2003). Many studies have analyzed the effects of certain abiotic
factors specifically temperature on an animals ability to flee (Templeton, 1960;
Bennett, 1980; Hertz et al., 1982; Claussen et al., 2001; Pinch and Claussen, 2003).
Specific studies have also observed the ability of an animal to move quickly when
7


faced with, predation, drought, and physically challenging topography (Scribner and
Weatherhead, 1995; Jayne and Irschick, 2000; Berry et al., 2002). Pinch and
Claussen (2003) found that the sprint speed of the lizard Sceloporus undulatus is
affected significantly by temperature and slope. The researchers set up testing tracks
(one with rounding comers, one linear) and measured the time for lizards to sprint
through the tracks. Similarly, Scribner and Weatherhead (1995) measured
antipredator escape speeds in three species of semi-aquatic snakes (Nerodia sipedon,
Thamnophis sauritus, and Thamnophis sirtalis). When chased by a researcher, snake
swim speed was measured through a 7-meter long trough full of water.
Performance in this study was measured by recording sprint speeds for each snake,
over a 2 meter track, prior to and following removal of water. It is hypothesized that
dehydrated snakes are faced with secondary adverse effects such as slowed responses
for fleeing predators.
In a final experiment, water loss was also measured through shed skins from the same
snakes used in the live experiment following methods outlined by Burken et al
(1985). Comparisons of water loss from skins containing lipids and skins, in which
the lipids have been removed, were made.
This study was derived following communication with the CDOW about T.s. sirtalis
populations in Colorado. Personal observation of declines also played a part in
developing this study.
8


Morgan County, CO Source: Colorado Natural Diversity Information System
Source: Colorado Division of Wildlife
Adams County, CO Source: Colorado Natural Diversity Information System
Figure 1.3: Thamnophis elegans vagrans and County Occurrence in Colorado
Boulder County, CO
Source: Colorado Natural Diversity Information System
9


2.0 Methods and Materials
2.1 Animals
Between May 2003 and October 2004, seven Thamnophis elegans, seven Thamnophis
radix and six Thamnophis sirtalis were collected from various field sites along the
northern Front Range of Colorado. All snakes were collected from sites in which
possible range overlap could occur (Denver, Jefferson, Arapahoe, Boulder, Weld and
Larimer Counties). Snakes were collected under CDOW permit numbers 03-HP918
and 04-HP981. Experiments were not performed on gravid snakes. The microhabitat
within which each snake was collected was noted. Microhabitat was classified into
three types: upland, riparian-upland, and wetland. Identification of microhabitat was
based on the U.S. Army Corps of Engineers (USCOE) criteria for wetland
classification (USCOE, 1987). Dominant vegetation, hydrology, and soil
characteristics were noted at each capture site (USCOE, 1987). Soil data was
obtained using the Soil Conservation Service (SCS) mapping (SCS, 1974). Each
snake was measured for length, sexed, and assigned an ID number.
2.2 Captive Environmental Conditions
To acclimate to captive conditions, snakes were housed for a minimum of three
months in the laboratory before experiments were conducted. All snakes were
housed individually on the Downtown Denver Campus of the University of Colorado
at Denver Health Sciences Center (UCDHSC) in the animal care facility in the North
Classroom. Animals were housed under Animal Care and Use Assurance number
A3659-01. Smaller snakes were housed in 5-gallon plastic sweater boxes and larger
snakes were housed in 10-gallon glass aquariums. Each snake was provided water ad
libidum. Newspaper was provided as bedding and each snake was provided a shelter.
Humidity and temperature were consistently measured in each cage to ensure no
differences in conditions in each cage type. No differences in temperature or
humidity existed in any of the cages. The temperature of the animal care facility
room was maintained at 24C and the relative humidity was maintained at
approximately 21% during experiments. Snakes were fed a mixture of earthworms,
small mice, and fish every 8 to 10 days except during experimental procedures. All
snakes regularly shed skin, were active and regularly ate food. All snakes were
healthy at the time of experiments.
2.3 Measurement of EWL in Snakes
Several methods for measurement of evaporative water loss in snakes have been
developed. Dmiel developed a method in which the animal is placed into a glass
tube with two compartments one for the head and one for the rest of the body. The
10


compartments are separated by a latex membrane. Dry air is then passed through the
compartments and captured by separate tubes containing anhydrous CaC03
(Drierite). The tubes containing Drierite are then weighed with the evaporative water
captured by the air passing through the tubes with the snakes (Dmiel, 1972, 1985,
1998).
Winne et al. (2001) and Moen et al. (2005) used a method where snakes are placed
into tubular steel mesh contraptions. Snakes in the tube are then placed into an
environmentally controlled chamber and weighed at periodic intervals.
A third common method was developed by Kattan and Lillywhite (1989) and
followed by Nielson (2002). This method involves placement of subjects onto a mesh
platform, which is then placed onto an electronic balance with draft shields. Drierite
is placed into the balance with the animal. Mass loss is measured at regular intervals
by the experimenter.
Methods used to measure EWL in live snakes in this study follow those outlined by
Chiszar (personal comm., 1996) and Perry et al. (2000). Total daily water loss was
measured in an environmental setting that simulated the dry conditions of the Front
Range for several days. Temperature and humidity were set to mimic Front Range
averages. Snake position and activity was not altered so that each snake could act as
it would under natural conditions. Snake cages contained hiding places and natural
props (rocks or small sticks) to try to imitate settings that would be sought out under
natural conditions throughout the experiments. This also resulted in minimal
disturbance to the snakes.
2.3.1 Preliminary Design
Between June 10, 2004 and June 16, 2004, preliminary studies with Thamnophis
elegans and Thamnophis radix were conducted to verify TEWL measurement
techniques following protocols outlined below. Methods used to measure TEWL
differences consisted of fasting the snakes 10 days prior to measurements. Drinking
water was removed from cages and snakes were dehydrated for a period of 7 days.
Measurements of body mass loss for each snake were recorded daily (Chiszar, 1996).
Water loss differences were noted although there was no statistical significance
between TEWL amounts in T. radix and T. elegans (F=0.069, n=14, df=13, p<0.8).
Over a period of 7 days, T. radix lost approximately 6 percent of its body mass,
whereas T. elegans lost approximately 5 percent of its total mass. Following 7 days
of dehydration, water was provided. Each snake was weighed 24 hours after re-
hydrating. Average weight-gain was approximately 6 percent for both species,
11


suggesting that all mass lost was that of metabolic water. Detailed preliminary data
including statistical results have been included in Appendix A.
Experiment 1: Comparison of evaporative water loss between three species of
garter snake
This experiment compared total EWL (TEWL) among Thamnophis sirtalis parietalis,
Thamnophis radix, and Thamnophis elegans vagrans individuals. The purpose of this
experiment was to compare evaporative water loss rates among three closely related
species that overlap in habitat use in the State of Colorado (Hammerson, 1999). I
hypothesized that T. s. parietalis would lose a greater amount of water, more quickly
than T. radix and T. e. vagrans.
Methods employed to measure TEWL differences consisted of dehydrating the snakes
for a period of 6 days by removing drinking water from cages, and then measuring the
body mass loss for each snake daily (Chiszar, 1996). In snakes that have been fasted
so that they do not defecate during the experiment, the only significant source of
water loss can come across the skin or from respiration. Snakes do not sweat and do
not urinate. For 10 days prior to the experiment, no food was offered to the snakes to
ensure that snakes were post absorptive during the experiment and did not have body
mass losses due to defecation (Winne et al., 2001). Following the 10-day period, and
after ensuring that all snakes had defecated, initial masses of each individual were
measured to O.Olg on an electronic balance with draft shields. Water was removed
from each cage for a total of 6 days and the snakes were weighed daily. Cutaneous
water loss (CWL) and respiratory water loss (RWL) were not differentiated in this
study. Both measures of water loss are included in measurements of EWL (Moen et
al., 2005). Metabolic loss of carbon (C02) was not accounted for since the loss of
C02 is approximately equal to the amount of 02 taken in (Roberts, 1968). To
normalize data for differences in total body mass of the snakes, loss in mass was
converted to percent daily mass loss. Daily percent mass loss for each snake was
averaged daily and grouped according to species (Table 3.1). Data for each species
were analyzed as a rate (percent loss in body mass/time) using a linear regression
model (Figures 3.3, 3.4, 3.5). Additional statistical measures used to analyze data
consisted of a one way analysis of variance (ANOVA) followed by a Tukey post-hoc
test of total percent water loss after 6 days.
Snakes were grouped according to sex, snout to vent length (SVL), and habitat at the
time of capture; snakes also were compared for TEWL using a one-way ANOVA.
Habitat was classified into three types: upland, riparian upland and wetland. Data
were grouped according to habitat type and analyzed for differences in total EWL
12


using a one way ANOVA. Table 3.2 lists snake ID, habitat found in, sex and initial
SVL.
2.4 Performance Analysis
The methods outlined in this section incorporate methods outlined by Scribner and
Weatherhead (1995), Finkler and Claussen (1999), and Pinch and Claussen (2003).
The goal of this experiment was to analyze whether lack of water affects the animals
ability to escape a simulated predation. The experiment consisted of quantifying
snake physical performance prior to, and following dehydration. Data were collected
by recording sprint speeds of individual snakes down a 2-meter track. I hypothesized
that sprint times following dehydration would be significantly slower than times
recorded prior to dehydration.
Fully hydrated individuals were removed from their cages and placed in a room at
23C. Animals were allowed to equilibrate to the temperature of the room for
approximately 20 minutes prior to sprint speed measurement. This was done to
ensure that temperature differences did not influence speed differences between
individuals. The track consisted of four 3.5-foot long 2x4s acquired at a local
hardware store. Boards were placed vertically approximately 12 inches apart to
replicate a visual barrier such as a wall that snakes were not likely to climb over.
Boards were placed lengthwise to form a 2.3 meter long track. Astro-turf was placed
along the bottom of the track to provide traction for the animals and to simulate
ground cover. Two marks were placed 2-meters apart to indicate the start and finish
of each snake.
Each individual was placed onto the track and lightly tapped on the tail to encourage
sprint escape prior to dehydration and following 6 days of dehydration. Each
individual snakes time from the start line to the finish line was measured using a stop
watch. Sprint times were measured three times for each individual prior to and
following dehydration and the mean time was calculated. At least 5 minutes passed
before each snakes second and third trial sprints. Data from the three trials were
averaged for statistical analysis.
Mean time differences between day 1 and day 6 were calculated. Average sprint
times and absolute velocities between day 1 and day 6 were analyzed within each
species and between each species using repeated measures ANOVA and paired t-
tests. Differences in velocities also were analyzed using a repeated measures
ANOVA. Percent EWL and sprint speed times were correlated within species using
Pearsons correlation. General trends between water loss and sprint speed times were
13


mapped using simple linear regression models. Data that consisted of at least a 1-
minute average increase between day 1 and day 6 trials were considered outlying data
and were not used during the statistical measurements. Such a magnitude of time
increase was caused by snakes that paused on the track or refused to move from the
start position.
2.5 EWL Measurement from Shed Skins/Skin Lipid Comparisons
This experiment compared EWL with shed skins (transepidermal water loss) using
methods outlined by Roberts and Lillywhite (1983) and Burken et al. (1985). Shed
skins were collected from T. s. parietalis, T. radix, and T. e. vagrans individuals used
in the previous two experiments. Skin samples that were shed in one piece were
used. Broken sheds were not used. EWL was measured through the skin samples
with lipids intact and with lipids removed by solvent extraction. I hypothesized that
EWL would be significantly higher in T .s. parietalis shed skin samples than in T.
radix and T. e. vagrans skin samples. I also predicted that EWL would be
significantly higher in delipidized skin samples verses those with lipids intact for all
three species used in the study.
i
Newly shed whole skins from each of the three species of snake were collected from
cages and stored in Ziploc baggies. Each bag was marked with the corresponding
snakes ID number and placed in a freezer (-20C) until use in the experiment.
EWL was measured with skins containing lipids and those without lipids using the
following methods. Skins were removed from the freezer and allowed to equilibrate
to room temperature. Two approximately 3 cm long pieces of the shed were clipped
from the dorsal mid-body portion of the shed skin. Half of the clipped skin samples
were left intact while lipids were extracted from the second set of clipped samples.
The amount of lipid extracted was measured by comparing the differences in mass
between intact samples and those samples after solvent extraction. Dried, unaltered
skin samples were weighed to the nearest 0.000lg on an electronic balance with draft
shields. Following lipid extraction, the same set of samples was weighed again after
drying. Percent lipid amount was calculated as dry lipid weight/dry weight of skin x
100.
Lipid extraction consisted of immersing each skin sample in a 100% hexane bath for
approximately 30 minutes, in individual 5 ml vials. Each skin sample was rinsed
twice with 100% hexane following the 30 minute immersion. The hexane bath was
used to remove non-polar lipids. Following extraction of non-polar lipids, skin
14


samples were immersed in a 1:1 chloroform-methanol solution for 30 minutes in
individual 5 ml vials, followed by two rinses of solvent, to remove polar lipids. Skins
were then dried and extracts were saved. Removal of lipids in solvents does not
damage the structure of the skin (Burken et al., 1985; Tu et al., 2002).
Permeability of each skin sample was measured using 8 ml screw top vial. Two
rubber septa were used to seal the skin samples in the vial screw-top lid. Holes 6 mm
in diameter were punched in each septum. Skin samples were trimmed using a 10
mm diameter cork cutter. The skin sample was then placed in between the two septa
so that the external or dorsal portion faced outwards Septa with skin samples exposed
through the holes were then carefully placed into the vial cap. Vials were filled with
4 ml of water and each cap containing septa and skin samples was carefully screwed
onto the vial (Figure 2.1). Each vial was checked for leaks, weighed initially, and
then placed inverted into an incubator at a temperature of 31C for 3 days so that
water would directly interface with the skin barrier. Weight measurements were
taken daily. Water loss was calculated and converted to a rate of mg H2O loss per
cm2 per hour.
Data were analyzed through use of a one-way ANOVA and differences in water loss
between delipidized skins and skins with lipids intact were measured using paired t-
tests.
2.6 Statistical Analysis
All ANOVA calculations were performed in Sigma Stat (Version 2.0). Linear
regression models, Pearsons correlation and paired t-tests were performed using
SPSS version 12.0 for Windows.
15


16


3.0 Results
3.1 Live Snake Water Loss
Thamnophis sirtalis parietalis lost evaporative water at a faster rate than did T. radix
or T. elegans vagrans (F = 5.393 n=18, df=17, p<0.02; Figure 3.1, Table 3.1). Post-
hoc analysis showed that, over a 6-day trial, T. s. parietalis lost a significantly higher
percentage of water than did T. radix (Tukey, p<0.03) and T. e. vagrans (Tukey,
p<0.04). There was no significant difference in percent water lost between T. radix
and T. e. vagrans (Tukey, p>0.05). On average over the 6-day dehydration trial, T. s.
parietalis lost water at 2.5 times the rate of T. radix and 2.4 times the rate of T. e.
vagrans. Figure 3.2 reports the average percent daily loss in body mass for each of
the three species.
Linear regression models indicate that T. s. parietalis loses 4.17 percent of body mass
per day when deprived of water (TEWL T. s. parietalis = -2.51 + 4.17*Day, R square
= 0.980, p<0.001; Figure 3.3). T. radix which loses approximately 1.54 percent of
body mass (TEWL T. radix = -0.51 + 1.54*Day, R square = 0.95, p=0.001; Figure
3.4) . T. e. vagrans loses approximately 1.65 percent body mass when deprived of
water (TEWL T. e. vagrans = -0.54 + 1.65*Day, R square = 0.95, p=0.001; Figure
3.5) . T. radix and T. e. vagrans lose significantly less body mass than T. s. parietalis
when denied water.
Regression analysis showed that larger snakes lose much more water than smaller
individuals when comparing mass of snake to grams of water loss for all species
combined (Water loss (g) = -0.20 + 0.12*Weight (g), R square = 0.65, p<0.001;
Figure 3.6). However, smaller individuals exhibited a slightly greater percent
(although not significant statistically) water loss than did larger individuals; percent
water loss decreased with increasing body mass (Water loss (%) = 14.36 + -
0.03*Weight (g), R square = 0.03 p>0.05; Figure 3.7). The modest slope is likely a
result of a greater surface area/volume ratio in the smaller snakes.
17


Table 3.1 Mean daily percent evaporative water loss and relative standard deviations
between T. s. parietalis, T. radix, and T. e. vaerans.___________________________
Date T. s. Darietalis T. radix T. e. vaerans
11/14/2004 0 0 0
11/15/2004 -3.6 2.5 -2.3 0.85 -2.9 1.7
11/16/2004 -7.1 2.1 -3.6 1.1 -3.7 1.5
11/17/2004 -10.7 3.3 -4.5 1.1 -5.1 1.9
11/18/2004 -14.5 5.0 -5.7 1.5 -6.1 1.7
11/19/2004 -18.6 7.7 -7 1.5 -7.5 1.5
11/20/2004 -21.5 8.1 -8.5 1.7 -9.1 1.8
18


Figure 3.1 Average Percent Water Loss over a 6-day Period for Each Species; Error
Bars Represent Standard Error of the Mean
in
tn
o
a>
CO
£
T.s.parietalis T. radix T.e.vagrans
Species
19


Figure 3.2 Graphical Representation of Mean Daily Percent Evaporative Water Loss
Between T. s. parietalis, T. radix and T. e. vagrans
T. sirtalis
T. radix
T. elegans
20


Figure 3.3 Average Percent TEWL per Day in T. s. parietalis; Equations for Regression
Lines are Given in the Text
Linear Regression with
99.00% Mean Prediction Interval
21


T.radix (% EWL)
Figure 3.4 Average Percent TEWL per Day in T. radix; Lines Fitted by Linear
Regression; Equations for Regression Lines are Given in the Text
Linear Regression with
99.00% Mean Prediction Interval
1.00 2.00 3.00 4.00
5.00 6.00
Day


T.e.vagrans (%EWL)
Figure 3.5 Average Percent TEWL per Day in T. e vagrans; Lines Fitted by Linear
Regression; Equations for Regression lines are Given in the Text
Linear Regression with
99.00% Mean Prediction Interval
1.00 2.00 3.00 4.00 5.00 6.00
Day
23


Water Loss (g)
Figure 3.6 Mass of snakes versus TEWL for All Three Species Combined; Lines Fitted
by Linear Regression; Equations for Regression Lines are Given in the Text
Linear Regression with
95.00% Mean Prediction Interval
24


yQ ssorj J0JB/\/v
Figure 3.7 Percent Body Mass Loss due to TEWL over a 6-day Trial versus Snake
Mass(g); Lines Fitted by Linear Regression: Equations for Regression Lines are Given
in the Text
Linear Regression with
95.00% Mean Prediction Interval
25


3.1.1 Analysis of Micro-Habitat and Sex Effects on TEWL
To ensure that covariates did not influence TEWL, sex, size and microhabitat
information associated with water loss was analyzed. Sex and micro-habitat selection
do not appear to be correlated to TEWL. A one-way ANOVA revealed that no
differences in TEWL exist between male and female snakes within any of the three
species (F=0.356 n=20, df=19, p >0.1) or between snout-vent-length (SVL) and mass
within or between any of the three species (Tukey, p>0.1). Similarly, no significant
TEWL differences exist between snakes from upland, riparian upland and wetland
areas (F=1.16, n=20, df=19, p>0.1). Table 3.2 shows each snakes individual ID
number, species, sex, SVL, initial mass and micro-habitat at the time of capture.
Individuals of each species of both sexes and from all three micro-habitats were
included in the TEWL experiment to control for these effects. Furthermore, there
was considerable overlap in both snout to vent length and mass among the individuals
of the three species used in the experiments.
26


Table 3.2 Sex, SVL and micro-habitat at time of capture for each individual used in
experiments.
Snake ID Suedes Sex SVL (cm) Initial Mass i&l Habitat at Canture
la T. s. parietalis F 66.1 106.1 Riparian Upland
2a T. s. parietalis F 73 156.0 Riparian Upland
3a T. s. parietalis F 24.9 6.0 Wetland
4a T. s. parietalis M 32.3 15.6 Riparian Upland
5a T. s. parietalis M 62.3 85.5 Wetland
6a T. s. parietalis M 55 71.9 Wetland
lb T. radix M 36.5 21.1 Upland
3b T. radix F 55 65.8 Upland
4b T. radix F 51.4 58.8 Wetland
5b T. radix M 54.3 49.0 Wetland
6b T. radix F 50.7 59.4 Riparian Upland
7b T. radix M 43.4 43.6 Upland
lc T. e. vagrans F 41 26.0 Riparian Upland
2c T. e. vagrans F 37 26.6 Riparian Upland
3c T. e. vagrans M 35.5 24.7 Riparian Upland
4c T. e. vagrans F 37.6 27.4 Upland
5c T. e. vagrans F 43 35.5 Wetland
6c T. e. vagrans M 43.4 34.5 Wetland
7c T. e. vagrans F 42.6 30.6 Riparian Upland
9c T. e. vagrans F 60.5 97.7 Wetland
3.2 Sprint Speed Analysis
One T. s. parietalis and one T. e. vagrans were excluded from the results of this
section due to vastly different sprint speeds that decreased by at least 60 seconds by
day 6 (see Methods). When all snake species data were combined, snakes sprinted at
significantly slower speeds when dehydrated over a 6-day period compared to speeds
when fully hydrated (repeated-measures ANOVA, Fi,i9=35.8, p<0.01). Significant
differences also existed in absolute velocities (measured in m/s) between day 1 and
day 6 (repeated-measures ANOVA, Fi,i9=l 1.7, p<0.05). Significant differences in
sprint speeds when fully hydrated compared to those when dehydrated did not exist in
27


T. s. parietalis (paired t-test, t=-1.483, df=4, p>0.1), T. radix (paired t-test, t=-0.908,
df=5, p>0.1), or T. e. vagrans (paired t-test, t=-4.132, df=6, p>0.1). Table 3.3 depicts
the average sprint times for day 1 and day 6 in the first two columns, and sprint time
change between day 1 and day 6 in the last two columns. Table 3.4 shows average
velocities for each species on day 1 and day 6. Figure 3.8 shows the average percent
time change for each species.
Table 33 Average sprint times and sprint time change between day 1 and day 6.
Outlying data has been removed._________________________________________
Species Avg. Sprint Time Day 1 (Seconds) Avg. Sprint Time Day 6 (Seconds) Sprint Time Change -Days 1-6 (Seconds) Sprint Time Change Days 1-6 (Percent)
T. s. parietalis 9.96 2.6 17.66 12.6 +7.7 11.6 69.1
T. radix 14.27 4.4 15.75 5.7 +1.5 4.0 21.3
T. e. vagrans 18.67 9.8 29.4 14.0 +10.7 6.9 57.5
Table 3.4 Average absolute velocities for each of the three species on day 1 and day 6.
Outlying data has been removed. ________________________________________________________________
Species N Average absolute velocity (m/s) Day 1 Average absolute velocity (m/s) Day 6
T. s. parietalis 5 0.19 0.041 0.14 0.103
T. radix 6 0.16 0.077 0.15 0.083
T. e. vagrans 7 0.12 0.050 0.07 0.040
28


Figure 3.8 Percent Time Change for Each Species Following 6-Days of Dehydration
(outliers removed)
0>
O)
co
O
CD
E
a)
o
>_
a>
Q_
T. s. parietalis T. radix
Species
T. e. vagrans
29


When all species data are combined, the general trend was a slower sprint time after a
period of dehydration. A linear regression model (95% mean prediction interval)
shows no significant correlation between EWL and sprint speed time change with
outliers removed, (Change in time = 2.19 + 0.39*%EWL Day 6, R square = 0.06;
Pearsons Correlation = 0.249, p>0.05) (Figure 3.9). If outlying data are included,
regression analysis shows a significant correlation between sprint speed time change
and EWL (Pearsons Correlation = 0.558, p<0.05).
Figures 3.10, 3.11, and 3.12 consist of simple linear regression models (95% mean
prediction interval) reflecting individual sprint speed time differences for each species
following 6 days of dehydration. Linear regression models show that this general
trend toward slower sprint times in dehydrated snakes exists in T. s. parietalis and T.
radix (Figures 3.10 and 3.11). However, regression models indicate a reverse trend
for T. e. vagrans (Figure 3.12). Simple linear regression models show no significant
correlation between EWL and sprint speed time change in T. s. parietalis (Change in
time = -12.97 + 1.10*%EWL Day 6, R square = 0.25; Pearsons Correlation = 0.503,
p>0.1) (Figure 3.10), no significant correlation between EWL and sprint speed time
change in T. radix individuals (Change in time = -4.00 + 0.65*%EWL Day 6, R-
square = 0.07; Pearsons Correlation = 0.270, p>0.05) (Figure 3.11) or T. e. vagrans
Change in time = 33.34 + -2.36*EWL Day 6, R-square =0.18; Pearsons Correlation
= -0.419, p>0.1) (Figure 3.12). Regression lines show an opposite trend for T. e.
vagrans however. If outlying data were included, correlations between EWL and
sprint speed would be considered significant for T. s. parietalis and T. e. vagrans.
30


Change in Time (Seconds)
Figure 3.9 Linear Regression Fit for Sprint Speeds verses Percent Water Loss (all
data); Equation for Regression Lines is Given in the Text
Linear Regression with
95.00% Mean Prediction Interval
31


Time Change (sec)
Figure 3.10 Sprint Speed Time Difference verses % TEWL for T. s. parietalis; Lines
Fitted by Linear Regression; Equation for Regression Line is given in the Text
Linear Regression with
95.00% Mean Prediction Interval
32


Time
Figure 3.11 Sprint Speed Time Difference verses % TEWL for T. radix; Lines Fitted by
Linear Regression; Equation for Regression Lines is given in the Text
Linear Regression with
95.00% Mean Prediction Interval
33


in Time
Figure 3.12 Sprint Speed Time Difference verses % TEWL for T. e. vagrans; Note the
Reverse Trend Compared to T. s. parietalis and T. sirtalis; Lines Fitted by Linear
Regression; Equation for Regression Lines is given in the Text
Linear Regression with
95.00% Mean Prediction Interval
34


3.3 Shed Skin EWL/Lipid Analysis
Percentage of total extractable lipids (TEL) removed with hexane and chloroform
from shed skins accounted for 4 tol5% of the dry weight of individual skin samples.
Average percentages of lipids extracted for each species are shown in Table 3.5.
Table 3.5 Percent dry weight of extractable lipids following hexane and chloroform
extraction.
Species N Percent dry weight of TEL
T. s. parietalis 5 13.28 + 9.73
T. radix 6 6.44 2.17
T. e. vagrans 7 8.34 3.62
No significant differences existed between species in EWL rates after 72 hours when
intact skins that had not had lipids extracted with solvent were used as a barrier to
water loss (ANOVA F2,3 = 0.97, p>0.1) (Figure 3.13). Significant differences did
exist in EWL rates in delipidized skin (ANOVA F2,3 = 5.9, p<0.05).
Table 3.6 shows the average transepidermal water loss rates for T. s. parietalis, T.
radix and T. e. vagrans through shed skin samples, with and without lipids extracted,
from inverted vials (water to air contact) at 31C. Water loss was measured over a
period of 72 hours.
Extraction of skin lipids led to significantly higher water loss rates for all three
species. Extraction of lipids caused T. s. parietalis samples to lose six times more
water than samples with intact lipids (paired t-test, t=-3.872, df=4, p<0.05).
Significant differences also exist between T. radix, which lost six times as much
water and T. e. vagrans which lost eight times as much water from skin sample with
lipids extracted versus those with lipids intact (T. radix paired t-test, t=-4.828, df=4,
p<0.01; T. e. vagrans paired t-test, t=-5.716, df=6, p<0.01). Rates of
transepidermal water loss are shown in Table 3.6.
35


Table 3.6 Rates of transepidermal water loss with intact skin samples.
Species N mg/cm2/hr no lipid extraction mg/cm2/hr lipid extraction
T. s. parietalis 5 0.923 0.80 5.67 2.76
T. radix 6 0.548 0.111 3.53 1.34
T. e. vagrans 7 0.534 0.433 4.33 1.78
Figure 3.13 Mean EWL (mg/cm2/hr) from Vials Sealed with a Barrier of Intact Skin
from Three Species of Garter Snake
<0
HS
o>
E
36


4.0 Discussion
4.1 Summary
1. T. s. parietalis lost 2.5 times the water as T. radix and T. e. vagrans. T. s.
parietalis also lost water at a significantly higher rate than T. radix and T. e.
vagrans.
2. When all species were combined, significant differences existed between
sprint speed times and absolute velocities. When each species was analyzed
independently, significant differences were not apparent in T. s. parietalis, T.
radix and T. e. vagrans. No significant correlation between EWL and sprint
speeds existed in any of the species.
3. Although not significant, T. s. parietalis did lose water at a higher rate
through unaltered shed skins than T. radix or T. e. vagrans. Significant
differences did exist in EWL rates between species in skins that had been
delipidized. Rates of water loss were consistent with previous studies.
4.2 Live Snake Water Loss
Based on known habitat associations, I predicted that T. s. parietalis would lose more
water than two congeners after a prolonged period of dehydration. State locality
records for this species occur near major rivers and streams (Hammerson, 1999).
Additionally, Rossman et al. (1996) state that western subspecies, including T. s.
parietalis populations in Colorado, tend to be more aquatic than eastern subspecies.
In this study, I found that T. s. parietalis lost 21% of its body mass when deprived of
water over a 6 day period. This amount was 2.5 times the amount that similar
species, T. radix and T. e. vagrans, lost when exposed to the same environmental
conditions. Significant differences also exist in the rate at which each species loses
water. Linear regression models show that T. s. parietalis loses approximately 4% of
body mass per day (Figure 3.3) when deprived of water and food, while T. radix and
T. e. vagrans lose approximately 1.5 to 1.6% of body mass per day when exposed to
the same conditions (Figures 3.4, 3.5). Sex, length and mass did not appear to affect
TEWL, thus consistent with other previous studies conducted by Dmiel (1998),
Winne et al. (2001) and Moen et al. (2005). No statistical significance occurred
between EWL amounts or rates and microhabitat collection. Microhabitat differences
may be more prominent with a larger sample size.
37


Other comparative studies have produced results analogous to those in this study.
Winne et al. (2001) noted similar results in the closely related Nerodia fasciata and
Seminatrix pygaea. Both snakes overlap in habitat use, but S. pygaea lost nearly
three times the amount of water. Moen et al. (2005) found that the species
Agkistrodon piscivorus lost twice as much water as A. contortrix. Nielson (2002) also
observed different water loss rates within skinks of the genera Oligosoma. Two
threatened species, the Chevron skink (O. homalonotum) and (O. striatum), lost 34%
more water than the relatively common speckled skink (O. infrapunctatum) and
Fallas skink (O.fallia).
One could argue that, because Thamnophis sirtalis inhabits wet areas across North
America (Sweeny, 1992) and in Colorado (Hammerson, 1999), it is rather intolerant
of longer dry periods and is susceptible to high TEWL. Researchers have
hypothesized numerous mechanisms that affect EWL rates in reptiles. Some argue
that differences in water loss rates between species and within species may be strictly
the result of habitat affinity (Nielson 2002; Perry et al., 2000; Dmiel, 1998). Several
previous studies have linked higher TEWL to animals that inhabit more humid
environments (Baeyens and Roundtree, 1983; Dmiel, 1985). Moen et al. (2005)
observed this scenario within the two species of Agkistrodon that inhabit different
habitats. Dmiel (1998) observed differing water loss rates within four species of
viperids that inhabit different habitats in the Middle East.
It is possible that evolutionary shifts have resulted in the differences in habitat
preference between T. sirtalis, T. radix and T. elegans. As stated above, Moen et al.
(2005) observed differing EWL rates within the genus Agkistrodon. The researchers
hypothesize that the different EWL rates may be the result of divergent evolutionary
transition. Differences also could be due to behavioral modifications such as varied
activity patterns. Both T. radix and T. elegans, normally diurnal, have been known to
become nocturnal during periods of hot weather (Heckrotte, 1962; Degenhardt et al.,
1996).
4.3 Sprint Speed Analysis
Snakes sprinted at slower speeds when dehydrated. Significant differences existed
between sprint times prior to dehydration and following 6 days of dehydration for all
species when all data were combined (p<0.01). However, intraspecific sprint speed
differences were not significant in all species. It is possible that small sample size
contributed to the non-significance with T. radix and T. e. vagrans, although both
species lose less water. Absolute velocities also differed significantly between days 1
and 6 (p<0.05). On average, T. s. parietalis had a velocity of 0.19 m/s prior to
dehydration that dropped to 0.14 m/s following dehydration. Similarly T. radix and
38


T. e. vagrans sprinted at speeds of 0.16 and 0.12 m/s initially and dropped to 0.15 and
0.07 m/s following dehydration. These data are consistent with data observed in
other semi-aquatic species. Finkler and Claussen (1999) note average sprint speeds
of 0.2 m/s in healthy Nerodia sipedon and Regina septemvittata at similar
temperatures. However, sprint speeds for all three species in this study were higher
than the 0.04 m/s recorded for T. sirtalis in a previous study (Heckrotte, 1967). In
addition to stressors such as drought, factors that potentially affect performance
include temperature, topography, and behavioral modifications.
Temperature is one factor that affects speed and stamina in reptiles. Pinch and
Claussen (2003) found that thermal variation affected Sceloporus undulatus sprint
speeds. Lizards sprinted at faster speeds on flat surfaces when measured at
temperatures of 25C and 30C respectively. Similarly, Scribner and Weatherhead
(1995) found that Nerodia sipedon and Thamnophis sirtalis relied more on defense
mechanisms (striking) than speed when tested at 10C, 20C and 30C. Individuals
fled at faster speeds at higher temperatures than when cooled. Finkler and Claussen
(1999) state that temperature was the single factor that affected swimming velocities
between Nerodia sipedon and Regina septemvittata. It is possible that temperature
may have been a factor with this study. At the time of both sprint speed trials,
temperatures were recorded at 23C in the lab, well within the active temperature
window for all three species. However, warmer temperatures may have resulted in
quicker speeds prior to dehydration.
Behavioral adaptations also may affect sprint speeds. Interestingly, in this study all
snakes did sprint and no individuals displayed any defense behavior, even when
normally aggressive snakes were provoked. However, T. e. vagrans was often
difficult to startle especially prior to dehydration. Regression models for T. e.
vagrans shows that trends toward slower sprint speeds were not extremely prominent
following dehydration (Figure 3.11). It is possible that T. e. vagrans relies more on
crypsis initially as Scribner and Weatherhead (1995) have observed with other semi-
aquatic snakes, whereas T. s. parietalis and T. radix are more likely to flee
(Degenhardt et al., 1996).
Topography and substrate apparently has influenced reptile sprint speeds in previous
studies. Pinch and Claussen (2003) note that the lizard Sceloporus undulatus sprints
at slower speeds when forced to move up steep slopes. Finkler and Claussen (1999)
note that the aquatic snakes Nerodia sipedon and Regina septemvittata generally
sprint at slower speeds than they swim. Their research is consistent with that of
Scribner and Weatherhead (1995) in that snake sprint and swim velocities are
positively correlated with temperature.
39


This portion of the experiment demonstrated notable differences in sprint speed times
prior to and following dehydration. An animals inability to flee due to a natural
stressor such as a prolonged dry period is a potential secondary effect that may
accelerate decline in that species population. Future studies combining a natural
stressor such as drought and temperature and snake size, in conjunction with sprint
speeds, may offer more insight to drought tolerance and secondary effects potentially
leading to higher declines. Future studies comparing sprint differences for individual
species should be compared with larger sample sizes.
4.4 Shed Skin EWL/Lipid Analysis
Percentage of total extractable lipid amounts ranged from 4 to 15% in this study
(Table 3.5). Surprisingly, the amount of extractable lipids for T. s. parietalis
averaged 13%, a higher number than expected. Percentages of extractable lipids for
T. radix averaged approximately 6% and were 8% for T. e. vagrans. Lipid quantities
were slightly higher than those reported in the study performed by Burken et al.
(1985). Extractable lipid quantities in Burkens study ranged from 4.9 to 5.7% dry
weight of shed skin samples for T. s. parietalis and averaged 4.9% to T. radix. Other
examples in the literature on extractable lipid concentrations could not be found for T.
e. vagrans. Differences in methods may have attributed to the conflicting numbers.
In this study, skins samples were dried in the laboratory hood. The mass difference
between the original unaltered skin sample and delipidized dry skin sample was
measured. The mass difference was then calculated into a percent. Burken et al.
(1985) extracted and dried the actual lipids in a vacuum and then calculated mass.
Other sources for the high variation found in this study may be due to frequency
between sheds and snake size and age. Tu et al. (2002) found that lipid barrier
formation rapidly increases in young Lampropeltis getula individuals and fluctuates
between sheds.
Although EWL rates for T. s. parietalis were higher than those for T. radix and T. e.
vagrans, significant differences did not exist in evaporative water loss amounts,
between species, in shed skins without lipid extraction. EWL rates from T. s.
parietalis skin samples averaged approximately 0.92 mg cm'2hr', whereas rates for T.
radix and T. e. vagrans averaged 0.55 and 0.53 mg cm" nr"1 respectively. EWL rates
in unaltered skins are consistent with those recorded in Burken et al. (1985). The
study performed by Burken et al. (1985) did not include water loss rates for T. e.
vagrans. However, EWL rates recorded in unaltered skin samples by Burken et al.
(1985) for T. s. parietalis and T. radix were 0.76 and 0.58 mg cm"2hr"'.
Significant differences did exist in EWL amounts between species in delipidized shed
skin samples. Extraction of lipids in shed skin samples led to significantly higher
40


water loss rates than those in unaltered skin samples, for each of the three species
analyzed. EWL rates in delipidized skins varied greatly from Burken et al. (1985),
but are more consistent with data from studies performed by Roberts and Lillywhite
(1980; 1983) in other semi-aquatic snakes. EWL rates in delipidized skins in this
study were 5.7 for T. s. parietalis, 3.5 for T. radix, and 4.3 mg cm'2hr 1 for T. e.
vagrans. Burken et al. (1985) recorded rates of 28 and 27 mg cm"2hr' for T. s.
parietalis and T. radix. Roberts and Lillywhite (1980; 1983) recorded EWL rates for
closely related semi-aquatic snakes Nerodia rhombifera and N. sipedon on the order
of 2.5 and 2.6 mg cm'^hr1, which are lower than numbers recorded in this study, but
closer in range than those in Burken et al. (1985). The differences between the study
performed by Burken et al. (1985) and this study may again be due to differences in
methods. In this study, the water diffused through epidermal samples into a closed,
temperature-regulated incubator, which may have humidified the closed area slightly,
leading to slower evaporative rates. In the previous study performed by Burken et al.
(1985), water diffused through epidermal samples into the open air. Evaporation of
water was likely greater in the open space. Similarly, studies performed by and
Roberts and Lillywhite (1980; 1983) involved water diffusing through skin samples
into a smaller apparatus than used in this study, thus requiring more time to
completely evaporate.
The high lipid content for T. s. parietalis was somewhat unexpected in this study
because water loss was higher in this species. However, it is known that some
reptiles have the ability to alter lipid content in the epidermal layers (Eynan and
Dmiel, 1993; Nielson, 2002; Tu et al., 2002; Smith pers. comm., 2005). It is
possible that factors such as temperature or shed cycles cause T. s. parietalis to lose
lipids frequently. Lipid chemical composition also may vary between T. s. parietalis,
T. radix and T. e. vagrans. Roberts and Lillywhite (1980) and Burken et al. (1985)
noted variations in chemical composition when comparing between different snake
species.
Lipid composition and amount have been proven to be directly correlated to EWL
(Roberts and Lillywhite, 1983; Burken et al., 1985; Landmann, 1988), rather than
scales as originally thought (Schmidt-Nielsen, 1964). Skin resistance and the
epidermal lipid barrier likely has evolved and is continuing to evolve for terrestrial
snakes. Such insight could allow for more accurate accounts of habitat affinity,
especially for species threatened with extinction (Nielson, 2002). Further studies
comparing live snake TEWL and EWL through shed skins should be conducted.
Other factors that could further understanding of the Thamnophis genera in the arid
West are lipid chemical composition, melting temperatures, and lipid fluctuations on
live snakes.
41


4.5 Relevance to Conservation
In Colorado, the common garter snake, Thamnophis sirtalis ssp. parietalis, is
declining throughout its historical range (CDOW, 2003). Two closely related species
that overlap in habitat and geographic range the plains garter snake (Thamnophis
radix) and the western terrestrial garter snake (Thamnophis elegans ssp. vagrans) are
not suffering from such declines. One possible factor that could be contributing to
the declines in T. s. parietalis populations is an inability to maintain proper water
balance during dry times. Declines may be secondarily enhanced due to an animals
inability to perform physically when presented with stressing conditions.
An animals ability to survive on land largely depends on how effectively it can
maintain proper metabolic water balance (Lahav and Dmiel, 1996; Perry et al.,
2000). In Colorado, the climate is dry and weeks go by without measurable
precipitation forcing many species to conserve metabolic water. Snakes, have
adapted to the climate, but may be forced to change behaviorally or physiologically to
minimize evaporative water loss during times of drought. One adaptation to reduce
evaporative water loss is to avoid normally dry micro-habitats (Wisley and Golightly,
2003). For example, Krakauer et al. (1968) found that burrowing animals are less
likely to lose high amounts of water when inhabiting burrows with high moisture
content. Additional studies conducted by Dmiel (1998), and Moen et al. (2005),
support this theory in finding that snakes able to tolerate drier conditions lose less
water, at a slower rate, than snakes from wetter areas. In general, garter snakes in
Colorado follow this trend. Although found considerable distances from water at
times (Hammerson, 1999), most species appear to favor wetter areas over dry areas.
This is especially true with Thamnophis sirtalis parietalis (Hammerson, 1999). This
species dependency on wetter microhabitats may offer one explanation as to why
individuals lost evaporative water at a significantly higher rate than T. radix and T. e.
vagrans in this experiment, thus offering insights to potential responses to recent
drought.
Water loss tolerance in species may aid in helping identify microhabitats and habitat
management for species that appear to be declining. To predict habitat use, Nielson
(2002) measured evaporative water loss in two species of endangered skinks
(Oligosoma homalonotum and O.striatum) endemic to New Zealand. EWL rates in
the two endangered species were much higher than those of common skinks found in
exposed, semiarid areas, suggesting that sheltered, more humid microhabitats may be
crucial to the endangered species survival. Webb and Shine (1998) researched the
thermal biology of the endangered broad-headed snake (Hoplocephalus bungaroides).
The researchers used radio telemetry to measure the snakes thermal preferences and
habitat use. Based on the data collected from the study, initiation of a habitat
42


restoration plan was formed. Dmiel (1985; 1998) and Lahav and Dmiel (1996) have
attributed skin resistance and EWL to habitat affinity with several species. A study
comparing a semi-aquatic snake species (,Matrix tessellata) to two terrestrial species
that inhabit mesic and dry habitats (Coluber rubriceps and Psammophis schokari)
attributed to higher EWL rates and lower skin resistance in Natrix versus the other
two species (Lahav and Dmiel, 1996).
The types of experiments conducted in this study are valuable for researching species
habitat use and conservation. Potentially detrimental natural stressors such as drought
or temperature extremes that may inhibit an animals physical ability may lead to
secondary effects, possibly enhancing declines in populations. The addition of
anthropogenic stressors, such as rapid development and pesticide use, may enhance
adverse effects to one species, while other closely related species populations remain
stable (Rossman et al., 1996).
It is likely that inter- and intraspecific rates of water loss differ due to a combination
of factors. Species populations and even individuals may be able to readily
physiologically adapt to changing conditions while other nearby populations or
individuals are unable to do this. Physiological mechanisms such as skin resistance
and epidermal lipid coatings may be the result of evolutionary trends in different
species. Behavioral adaptations such as shifting activity patterns and general habitat
affinities certainly affect water balance within individuals. Long-term studies
monitoring behavioral and physiological adaptations, as a result of EWL would offer
more insight to inter- and intraspecific trends, as well as possible shifts in habitat use.
These types of studies could lead to more accurate conservation and management of
habitat utilized by sensitive species such as Thamnophis sirtalis parietalis in
Colorado.
43


5.0 Bibliography
Andrade, D.V. and A.S. Abe. 2000. Water collection by the body in a viperid snake,
Bothrops moojeni. Amphibia-Reptilia 21:485-492.
Atsatt, S. R. 1939. Color changes as controlled by temperature and light in the
lizards of the desert regions of southern California. Biological Science, 1:237-276.
Baeyens, D.A. and R.L. Roundtree. 1983. A comparative study of evaporative water
loss and epidermal permeability in an arboreal snake Opheodrys aestivus, and a
semi-aquatic snake, Nerodia rhombifera. Comparative Biochemistry and
Physiology 76A:301-304.
Beament, J. W. L. 1961. The water relations of insect cuticle. Biological Review.
36:281-320.
Bennett, A.F. 1980. The thermal dependence of lizard behavior. Animal Behavior
28:752-762.
Bennett, A.F. and P. Licht. 1974. Evaporative water loss in scaleless snakes.
Comparative Biochemistry and Physiology 32A:213-215.
Bentley, P.J. and K. Schmidt-Nielsen. 1966. Cutaneous water loss in reptiles.
Science 151:1547-1549
Berry, K.H., K.E. Spangenberg, B.L. Homer, and E.R. Jacobson. 2002. Deaths of
desert tortoises following periods of drought and research manipulation. Chelonian
Conservation and Biology. 4:436-448.
Brown, D.E. and N.B. Carmony. 1991. Gila Monster: Facts and Folklore of
Americas Aztec Lizard, 2nd edition. High-Lonesome Books, Silver City, NM.
Bulova, S. J. 2002. How temperature, humidity, and burrow selection affect
evaporative water loss in desert tortoises. Journal of Thermal Biology, 27:175-189.
Burken, R.R., P.W. Wertz and D.T. Downing. 1985. The effect of lipids on
transepidermal water permeation in snakes. Comparative Biochemistry and
Physiology 31A:213-216.
Burken, R.R., P.W. Wertz and D.T. Downing. 1985. A survey of polar and nonpolar
44


lipids extracted from snake skins. Comparative Biochemistry and Physiology
31A:213-216.
Burt, W.H. and R.P. Grossenheider. 1980. Mammals (3rd ed.). Houghton Mifflin
Company, New York.
Chiszar, D. 1996. Personal Communication on methods for conducting dehydration
study on Colorado garter snakes.
Claussen, D.L., R. Lim, M. Kurz, and K. Wren. 2001. The effects of slope,
substrate, and temperature on the locomotion of the Ornate Box Turtle, Terrapene
ornate. Copeia 2002:411-418.
Cloudsley-Thompson, J.L. 1971. The Temperature and Water Relations of Reptiles.
Merrow Publishing Co., Watford Herts, England.
Cogger, H. and R.G. Zweifel (eds.). 1992. Reptiles and Amphibians. Smithmark
Publishing, New York, NY.
Cohen, A.C. 1975. Some factors affecting water economy in snakes. Comparative
Biochemistry and Physiology 51A:361-368.
Colorado Division of Wildlife (CDOW). 2003. Colorado Listing of Endangered,
Threatened, and Wildlife Species of Concern.
http://wildlife.state.co.us/species_cons/list.asp
Conant, R. 1975. A Field Guide to Reptiles and Amphibians of Eastern and Central
North America, 3rd ed. Houghton Mifflin, Boston.
Dalrymple, G.H. and N.G. Reichenbach. 1984. Management of an endangered
species of snake in Ohio, USA. Biological Conservation 30:195-200.
Degenhardt, W.G., C.W. Painter and A.H. Price. 1996. Amphibians and Reptiles of
New Mexico. University of New Mexico Press, Albuquerque, NM.
DeNardo, D.F., T.E. Zubal and T.C.M. Hoffman. 2003. Cloacal evaporative cooling:
a previously undescribed means of increasing evaporative water loss at higher
temperatures in a desert ectotherm, the Gila monster Heloderma suspectum. The
Journal of Experimental Biology 207:945-953.
45


Dmiel, R. 1972. Effect of activity and temperature on metabolism and water loss in
snakes. American Journal of Physiology 223:510-5116
Dmiel, R. 1985. Effect of body size and temperature on skin resistance to water loss
in a desert snake. Journal of Thermal Biology 10:145-149.
Dmiel, R. 1998. Skin resistance to evaporative water loss in viperid snakes: habitat
aridity versus taxonomic status. Comparative Biochemistry and Physiology Part A
121:1-5.
Ebeling, W. 1974. Permeability of insect cuticle. In The Physiology oflnsecta, Vol.
VI (edited by Rockstein M.) 2nd edn., pp 271-343. Academic Press, London.
Elias, P.M. 1983. Epidermal lipids, barrier function and desquamation. Journal of
Investigative Dermatology. 80 Suppl. 44s-50s.
Elias, P.M. and G.K. Menon. 1991. Structural and lipid biochemical correlates of the
epidermal permeability barrier. Adv. Lipid Res. 24:1-26.
Eynan, M. and T. Dmiel. 1993. Skin resistance to water loss in agamid lizards.
Oecologia 95:290-294
Finkler, M.S. and D.L. Claussen. 1999. Influence of Temperature, Body Size, and
Inter-individual Variation on Forced and Voluntary Swimming and Crawling
speeds in Nerodia sipedon and Regina septemvittata. Journal of Herpetology
33:62-71.
Fitch, H.S. 1965. An ecological study of the garter snake, Thamnophis sirtalis.
University of Kansas Publication, Museum of Natural History 15:493-564.
Fitch, H.S. 1982. Resources of a snake community in prairie-woodland habitat of
northeastern Kansas. In Herpetological Communities, (edited by J. Scott), pp 83-
97. U.S. Fish and Wildlife Service Region 13, Washington, D.C.
Gans, C.T., T. Krakauer, and C.V. Paganelli. 1968. Water loss in snakes:
interspecific and intraspecific variability. Comparative Biochemistry and
Physiology 27:747-761.
Gregory, P.T. 1977. Life-history parameters of the red-sided garter snake
(!Thamnophis sirtalis parietalis) in an extreme environment, the Interlake region of
Manitoba. Natural Museum Canada Publication Zoology 13:1-44.
46


-1984. Habitat, diet, and composition of assemblages of garter snakes
(Thamnophis) at eight sites on Vancouver Island. Canadian Journal of
Zoology 62:2013-2022.
Hammerson, G.A. 1999. Amphibians and Reptiles in Colorado. University Press of
Colorado and Colorado Division of Wildlife, Niwot, Colorado.
Heckrotte, C. 1962. The effect of the environmental factors in the locomotory
activity of the plains garter snake (Thamnophis radix). Animal Behavior 10:193-
207.
- 1967. Relations of body temperature, size and crawling speed of the common
garter snake, Thamnophis s. sirtalis. Copeia 1967:520-526.
Hertz, P.E., R.B. Huey, and E. Nevo. 1982. Fight versus flight: body temperature
influences defensive responses of lizards. Animal Behavior 30:676-679.
Hillenius, W. J. 1994. Turbinates in therapsids: Evidence for late Permian origins of
mammalian endothermy.
Jayne, B.C. and D.J. Irschick. 2000. A field study of incline use and preferred
speeds for the locomotion of lizards. Ecology 81:2969-2983.
Jennings, W.B., D.F. Bradford, and D.F. Johnson. 1992. Dependence of the garter
snake Thamnophis elegans on amphibians in the Sierra Nevada of California.
Journal of Herpetology 26:503-505.
Kattan, G.H and Lillywhite, H.B. 1989. Humidity acclimation and skin permeability
in the lizard Anolis carolinensis. Physiological Zoology 62:593-606.
Kephart, D.G. 1982. Microgeographic variation in the diets of garter snakes.
Oecologia 52:287-291.
Krakauer, T., C. Gans and C.V. Paganelli. 1968. Ecological correlation of water loss
in burrowing reptiles. Nature 218:659-660.
Lahav, S. and R. Dmiel. 1996. Skin resistance to water loss in colubrid snakes:
Ecological and taxonomical correlations. Ecoscience 3:135-139.
Landmann, L. 1988. The epidermal permeability barrier. Anatomy and Embryology
178:1-13.
47


Lillywhite, H.B. 1989. Circulatory adaptations of snakes to gravity. American
Zoology 27:81-95.
Lillywhite, H.B. and A.W. Smits. 1992. The cardiovascular adaptations of viperid
snakes. In J.A. Campbell and E.D. Brodie Jr. (editors), Biology of the Pitvipers,
pp. 143-153. Selva, Tyler, Texas.
Lillywhite, H.B. and V. Sanmartino. 1993. Permeability and water relations of
Hygroscopic skin of the file snakes Acrochordus granulatus. Copeia 1993:99-103.
Luke, C.A. 1994. Evolution of Color Change in Desert Lizards. In Herpetology of
the North American Deserts: proceedings of a symposium. Southwestern
Herpetologists Society, Van Nuys, California.
Mautz, W.J. 1982. Correlations of both respiratory and cutaneous water losses of
lizards with habitat aridity. Journal of Comparative Physiology 149:25-30.
Mayhew, W.W. and S.J. Wright. 1971. Water impermeable skin of the lizard
Phrynosoma m calli. Herpetologica 27:8-11.
Milstead, W.W. 1957. Observations of the natural history of four species of whiptail
lizard Cnemidophorus (Sauria, Teiidae) in Trans-Pecos Texas. Southwestern
Naturalist 2:105-121.
Moen, D.S., C.T. Winne and R.N. Reed. 2005. Habitat-mediated shifts and plasticity
in the evaporative water loss rates of two congeneric pit vipers (Squamata,
Viperidae, Agkistrodon). Evolutionary Ecology Research 7:759-766.
National Oceanic and Atmospheric Administration (NOAA). 2004. www.noaa.gov
Nielson, K.A. 2002. Evaporative water loss as a restriction on habitat use in
endangered New Zealand endemic skinks. Journal of Herpetology 36:342-348.
Nero, R.W. 1957. Observations at a garter snake hibemaculum. Blue Jay 15:116-
118.
Perry, G., R. Dmiel, and J. Lazell. 2000. Evaporative water loss in insular
populations of Anolis cristatellus (Reptilia: Sauria) in the British Virgin Islands.
III. Responses to the end of drought and a common garden experiment. Biotropica
32:722-728.
Pettus, D. 1958. Water relationships in Natrix sipedon. Copeia 3:207-211.
48


Pinch, F.C. and D.L. Claussen. 2003. Effects of Temperature and Slope on the
Sprint Speed and Stamina of the Eastern Fence Lizard, Sceloporus undulatus.
Journal of Herpetology 37:671-679.
Porter, W.P. and C.R. Tracy. 1983. Biophysical analyses of energetics, time-space
utilization, and distributional limits. In Lizard Ecology: Studies of a model
organism. Huey, Pianka and Schoener, eds. Harvard U. Pr., pp. 55-83.
Randall, J.A. 1994. Convergences and divergences in communication and social
organization of desert rodents. Australian Journal of Zoology 405-433.
Roberts, L.A. 1968. Water loss in the desert lizard Uta stansburiana. Comparative
Biochemistry and Physiology 27: 683-589.
Roberts, J.B. and H.B. Lillywhite. 1980. Lipid barrier to water exchange in reptile
Epidermis. Science 207:1077-1079.
-1983. Lipid permeability of Epidermis from Snakes. The Journal of
Experimental Zoology 238:1-9.
Rossi, J.A and R. Rossi. 1995. Snakes of the United States and Canada Vols. 1 and
2. Krieger Publishing Company, Malabar, FI.
Rossman, D.A., N.B. Ford and R.A. Seigel. 1996. The Garter Snakes Evolution and
Ecology. University of Oklahoma Press, Norman, Oklahoma and London Great
Britain.
Schmidt-Nielsen, K. 1964. Desert Animals. Physiological Problems of Heat and
Water. Oxford University Press, London.
Schmidt-Nielsen, K. 1975. Animal Physiology Adaptation and Environment.
Cambridge University Press, MA.
Scribner, S.J. and P.J. Weatherhead. 1995. Locomotion and antipredator behavior in
three species of semi-aquatic snakes. Canadian Journal of Zoology 73:321-329.
SCS (Soil Conservation Service). 1974. Soil survey of Adams County, Colorado.
USDA.
Siegel, R.A. 1984. The foraging ecology and resource partitioning patterns of two
species of garter snakes. Pd.D. diss., University of Kansas, Lawrence.
49


Smith, H. M. 2005. Personal communication on alteration of lipid content on reptile
skin.
Sweeny, R. 1992. Garter Snake Their Natural History and Care in Captivity.
Sterling Publishing, New York.
Templeton, J.R. (1960). Respiration and water loss at the higher temperatures in the
desert iguana, Dipsosaurus dorsalis. Physiological Zoology 33:136-145.
Tu, M.C., H.B. Lillywhite, J.G. Menon and G.K. Menon. 2002. Postnatal ecdysis
establishes the permeability barrier in snake skin: new insights into barrier lipid
structures. The Journal of Experimental Biology 205:3019-3030.
U.S. Army Corps of Engineers. 1987. Corps of Engineers Wetlands Delineation
Manual. Department of the Army. Vicksburg, MI. Y-87-1.
Vaughan, T.A. 1986. Mammalogy (3rd. ed.) Saunders College Publishing, New
York.
Webb, J.K. and R. Shine. 1998. Using thermal ecology to predict retreat-site
selection by an endangered snake species. Biological Conservation 86:233-242.
Winne, C.T., T. J. Ryan, Y. Leiden, and M.E. Dorcas. 2001. Evaporative water loss
in two natricine snakes, Nerodia fasciata and Seminatrix pygaea. Journal of
Herpetology 35:129-133.
Winne, C.T. and M.B. Keck. 2004. Daily activity patterns of Whiptail Lizards
(Squamata: Teiidae: Aspidoscelis): a proximate response to environmental
conditions or an endogenous rhythm? Functional Ecology 18:314-321
Wisely, S.M. and R. T. Golightly. 2003. Behavioral and Ecological Adaptations to
Water Economy in Two Plethodontid Salamanders, Ensatina eschscholtzii and
Batrachoseps attenuatus.
Wu, P., L. Hou, M. Plilus, M. Hughes, J. Scehnet, S. Suksaaweang, R. Widelitz, T.
Jiang and C. Chuong. 2004. Evo-Devo of amniote integuments and appendages.
The International Journal of Developmental Biology 48:249-270
50


Appendix A-
Preliminary Analysis Data
Mean Daily % Water Loss Comparison between
Thamnophis radix and Thamnophis elegans
Table 1. Mean daily percent evaporative water loss between T. radix, and T. e. vagrans.
Date T. radix T. e. vaerans
6-10-2004 0 0
6-11-2004 -1.2 -0.64
6-12-2004 -1.8 -2
6-13-2004 CM CO I -2.6
6-14-2004 -4.4 -4
6-15-2004 -5.8 -5
6-16-2004 1 1.1
6-17-2004 0* 0*
* Water returned to individuals
Figure 1. Graphical representation of mean daily percent evaporative water loss
between T. T. radix and T. e. vagrans and percent gain on Day 7
T. elegans
T. radix
Date
51