MIAMI UNIVERSITY The Graduate School

Certificate for Approving the Dissertation

We hereby approve the Dissertation of Gary W. Gerald, II

Candidate for the Degree: Doctor of Philosophy

______Director (Dennis L. Claussen)

______Reader (Alan B. Cady)

______Reader (Phyllis Callahan)

______(Nancy G. Solomon)

______Graduate School Representative (Robert L. Schaefer)

ABSTRACT

CONSEQUENCES OF ABIOTIC AND BIOTIC FACTORS ON LIMBLESS LOCOMOTION

Gary W. Gerald II

Snakes have the ability to move in a variety of ways depending on the habitat in which they are moving. All of these modes require some sort of lateral bending of the elongate body to generate the required force necessary for propulsion. However, the biomechanical mechanisms of each mode of limbless movement differ substantially among each other. Despite the potential importance of using multiple modes of movement in different ecological situations, we know very little about the influence of abiotic and biotic factors on multiple locomotor modes in these animals. The goal of this dissertation was to examine how temperature, habitat usage, and morphology affect four of the most common modes of limbless locomotion (lateral undulation, concertina, swimming, and arboreal) and shed light on the question of what ecological conditions most likely contributed to limb reduction in the early snake ancestor. The first chapter assessed the influence of temperature on different modes of locomotion. Decreasing temperature limits performance of each mode differently because of differences in the underlying physiological mechanisms governing each mode. The second chapter closely examined the combined effects of temperature and perch diameter on the speed and balance of limbless arboreal locomotion. Movement on perches was greatly limited by temperature, but not by decreasing perch diameter suggesting that snakes have a size-relative advantage compared to lizards when moving on narrow perches. The final chapter deals with assessing the relationships among microhabitat use, morphology, and locomotor performance of various modes. I found that species tend to perform better during modes they use most often in nature and perform more poorly during rarely-used modes suggesting that snakes do possess adaptations to enhance movement in preferred habitats. Moreover, morphological variables (mass, length, shape) significantly influenced each locomotor mode in somewhat similar ways. As a result, performance across various modes was either positively or not related to each other in most instances. My results suggest that morphological and physiological adaptations that promote movement via different modes do not conflict suggesting that a limbless body is beneficial in a number of different ecological situations.

CONSEQUENCES OF ABIOTIC AND BIOTIC FACTORS ON LIMBLESS LOCOMOTION

A DISSERTATION

Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Zoology

by

Gary W. Gerald, II Miami University Oxford, Ohio 2008

Dissertation Director: Dennis L. Claussen

TABLE OF CONTENTS

General Introduction ……………………………………………………………. 1 Temperature effects on locomotion ……………………………………...…… 2 Limbless locomotion ……………………………………………………….. 3 Arboreal locomotion ………………………….....………………………….. 4 Specializations and trade-offs ….………………………………………...….. 5 Direction of dissertation ……………………………………………………. 6 Literature Cited ……….…………………….…….……….………....……. 7 Chapter 1: Thermal dependencies of different modes of locomotion in neonate Brown snakes, Storeria dekayi …………………………………………….………. 12 Introduction ………………………...…………………………………….. 13 Materials and Methods …………………………………………….………. 14 Results …...... 16 Discussion ……………………………………………………………….. 17 Literature Cited …………………………….…………………………….. 21 Chapter 2: Effects of temperature and perch diameter on arboreal locomotion in the snake Elaphe guttata ……………………………………………………….…… 33 Introduction ………………………………………………………………. 34 Materials and Methods …………………………………………………….. 36 Results …………………………………………………………………... 39 Discussion ……………………………………………………………….. 40 Literature Cited …………………………………………………………... 46 Chapter 3: Relationships between morphology, microhabitat use, and limbless locomotion ……………………………………………………………… 55 Introduction ……………………………………………………………… 56 Materials and Methods ……………………………………………………. 58

ii Results …………………………………………………………………... 65 Discussion ……………………………………………………………….. 69 Literature Cited …………………………………………………………... 79 General Conclusions ………………………………………………………….…. 109 Conclusion ………………………………………………………………. 113 Literature Cited …………………………………………………………... 114

iii LIST OF TABLES

Chapter 1 Table 1. Pearson correlation coefficients for absolute velocities of 26 neonate brown snakes (Storeria dekayi) among locomotor modes and temperature. Pairwise α = 0.05. * p < 0.05. b Table 2. Allometric relationships (y = ax ) between log transformed SVL and log transformed mean and maximum absolute (m • s-1) velocities for 26 neonate brown snakes (Storeria dekayi) during 3 modes of locomotion at 10, 20, and 30 C determined by power functions. Numbers in parenthesis represent standard errors of the parameter estimates for the intercepts (a) and slopes (b). No significant (p < 0.0167) relationships were detected for any mode at any temperature.

Table 3. Q10 values for maximum absolute velocities attained by various species of snake during swimming (s) and undulatory crawling (c). 1 = Stevenson et al. (1985), 2 = Heckrotte (1967), 3 = Finkler (1995), 4 = Finkler and Claussen (1999), 5 = present study. Table 4. A summary of body length-relative velocities of concertina locomotion by

limbless squamates reported from this and previous studies. TL = total length, SVL = snout-vent length, WT = wide tunnel(s).

Chapter 2 Table 1. Parameters of least-squares regression models showing the relationship between number of body loops formed during movement/total body length (predictor) and mean head-tail distances/total body length (response) at three temperatures (10, 20, and 30 C) at three different perch diameters (3, 6, and 10 cm).

Chapter 3 Table 1. The reported habits and morphological data for the five snake species used in this study. Numbers represent means with the standard deviation in parentheses. SVL = snout-vent length, Body condition = total length (cm)/mass (g).

iv Table 2. Statistically significant differences in terrestrial, aquatic, and arboreal microhabitat use between five species of snake (n = 12/species). The order of use is listed from left to right with the species on the left using that particular microhabitat the most and the one on the right using it the least. Superscript letters denote statistical differences. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus. Table 3. Linear regression parameters for the relationships (y = α + βx) between uncorrected morphology and stamina of three different modes of limbless locomotion in 5 species of snake (n = 12/species). SVL = snout-vent length, Body condition = total length (cm)/mass (g). Table 4. Statistically significant differences in maximal snout-vent length (SVL)-relative speeds and stamina of multiple modes of limbless locomotion between five species of snake (n = 12/species). The order of use is listed from left to right with the species on the left displaying superior performance and the one on the right displaying poorer performance abilities. Superscript letters denote statistical differences. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus. Table 5. Linear regression parameters for the relationships (y = α + βx) between uncorrected data on performance during each of four locomotor modes and amount of time spent in each microhabitat type by 5 snake species (n = 12/species). Table 6. Pearson product-moment correlation coefficients between locomotor performance measures (maximal snout-vent length-relative velocity and stamina) of four modes of limbless locomotion in 5 snake species (n = 12/species). Arb = arboreal, LU = terrestrial lateral undulation, Conc = concertina, Swim = swimming. * = statistical significance at the 0.05 level.

v LIST OF FIGURES

Chapter 1 Figure 1. The influence of temperature on absolute mean (A) and maximum (B) velocities and snout-vent length (SVL) relative mean (C) and maximum (D) velocities attained during terrestrial lateral undulation, terrestrial concertina, and swimming by 26 neonate Brown Snakes (Storeria dekayi). Error bars represent ± SE. Figure 2. The influence of temperature on maximum snout-vent length (SVL) relative crawling speeds of 26 neonate Storeria dekayi (present study), 10 adult Nerodia sipedon (Finkler and Claussen 1999), 10 adult Regina septemvittata (Finkler and Claussen 1999), and 206 adult Natrix maura (Hailey and Davies 1986). Figure 3. The influence of temperature on maximum snout-vent length (SVL) relative swimming speeds in snakes, 26 neonate Storeria dekayi (present study), 10 adult Nerodia sipedon and 10 adult Regina septemvittata (Finkler and Claussen 1999). Figure 4. The relationship between body width relative to tunnel width and maximum snout-vent length relative speeds of concertina locomotion in 5 adult Coluber constrictor estimated from Jayne and Davis, 1991 (▲) and 26 neonate Storeria dekayi (●) estimated from the present study. Error bars represent ± one standard deviation.

Chapter 2 Figure 1. Diagram showing a lateral view of differences in head-tail distances (arrows) of snakes moving on a horizontal perch. Shorter head-tail distances (a) represent snakes looped around either side of the perch, a body position that provides more balance when moving. Larger head-tail distances (b) denote a more elongated body position that provides less balance when moving. Figure 2. Effect of perch diameter and temperature on (a) maximum speed and (b) head- tail distances relative to body length during arboreal locomotion by cornsnakes (Elaphe guttata). Bars represent ± 1 SE.

vi Figure 3. The relationship between mean speed and head-tail length relative to total body length, which is a measure of body posture during arboreal locomotion by Elaphe guttata on 10-cm (a), 6-cm (b), and 3-cm (b) diameter perches at 10 C, 20 C, and 30 C. Regression lines represent the relationship at each temperature with dashed lines representing 30 C, dotted lines representing 20 C, and solid lines representing 10 C. Figure 4. Proportion of arboreal locomotor trials in which individual cornsnakes (Elaphe guttata) fell off of a perch at different temperatures and perch diameters.

Chapter 3 Figure 1. A topology of 5 snake species based on both mitochondrial genes and morphology (Keogh 1996; Rodriquez-Robles and De Jesus-Escobar 1999; de Queiroz et al. 2002; Kelly et al. 2003; Hibbits and Fitzgerald 2005; Lawson et al. 2005) used to determine independent contrasts of morphological measurements, locomotor performance, and microhabitat use. Numbers represent the nodes for which each contrast was calculated. Figure 2. Proportion of time spent in terrestrial, aquatic, and arboreal microhabitats within artificial enclosures for individuals of five species of snake (n = 12/species) during 3 hrs of observations. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus. Figure 3. Average maximal absolute (A) and snout-vent length (SVL)-relative (B) velocities for 5 species of snakes (n = 12/species) combined for arboreal (Arb), terrestrial lateral undulation (LU), concertina (Conc), and aquatic (Swim) locomotion. Both performance measures were statistically different across all modes (p < 0.0001) except for the difference between arboreal and concertina (p = 0.20). Error bars represent ± SE. Figure 4. Average stamina times for 5 species of snake (n = 12/species) for terrestrial lateral undulation (LU), concertina (Conc), and aquatic (Swim) locomotion. Differences among modes were not statistically significant (p = 0.076). Error bars represent ± SE.

vii Figure 5. Relationship between body mass and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D). Figure 6. Relationship between snout-vent length (SVL) and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D). Figure 7. Relationship between body condition (cm/g) and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D). Figure 8. Relationship between cross-sectional area (mm2) and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D). Figure 9. Relationship between number of pre-caudal vertebrae and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D). Figure 10. A comparison of uncorrected maximal velocities (snout-vent length (SVL)- relative (A,B) and absolute (C,D)) between five species of snake (n = 12/species) for each of four modes of locomotion. Because of differences in scale, average arboreal and concertina velocities are shown together (A,C), while lateral undulation and swimming velocities are shown together (B,D). Note the differences in the y-axis between the panels. Error bars depict ± SE. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus. Figure 11. Mean duration of stamina by five species of snake (n = 12/species) moving during three modes of locomotion. Error bars depict ± SE. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus. Figure 12. Residuals from the relationships between independent contrasts (Felsenstein 1985; Am. Nat. 125:1-15) of locomotor velocities (A) and stamina (B) and the independent contrasts of body mass for four modes (A) and three modes (B) of limbless locomotion in 5 species of snakes.

viii

Figure 13. Relationships between the independent contrasts (Felsenstein 1985; Am. Nat. 125:1-15) of locomotor velocities and independent contrasts of microhabitat use for arboreal (A), terrestrial lateral undulation (B), terrestrial concertina (C), and aquatic (D) locomotion and the respective microhabitats in which these modes are used in 5 species of snakes. Figure 14. Relationships between the independent contrasts (Felsenstein 1985; Am. Nat. 125:1-15) of stamina and independent contrasts of microhabitat use for terrestrial lateral undulation (A), terrestrial concertina (B), and aquatic (C) locomotion by 5 snake species and the respective microhabitats in which these modes are used. Figure 15. Interspecific comparisons of the uncorrected average maximal velocities attained by five species of snake (n = 12/species) during four modes of locomotion. Velocities are represented as a percentage of the fastest average velocity by a particular species within each locomotor mode (i.e. the velocities of the fastest species were set to 1.0) to facilitate comparisons across modes. Arb = arboreal, LU = lateral undulation, Conc = concertina, Swim = swimming.

ix DEDICATION

I wish to dedicate this work to my grandmother Virginia Smith (1916-1997). She encouraged and supported me at an early age and I likely would not be where I am today without her love and guidance.

x ACKNOWLEDGMENTS

I would first like to thank my advisor Dr. Dennis Claussen for his support and encouragement during this process. I also would like to thank my dissertation committee Drs. Alan Cady, Phyllis Callahan, Robert Schaefer, and Nancy Solomon for their suggestions, guidance, and their helpful feedback on experimental design. I would also like to thank former Claussen Lab members Mike Elnitsky, Michael Finkler, Melanie Gregory, Jimmy Kirschberger, and Lawrence Spezzano for their assistance and support over the years. A number of other faculty and fellow graduate students provided valuable support, assistance, and friendship including Lisette Torres, Aron Costello, Chris Distel, Todd Levine, Gregg Marcello, Tim Muir, Jennifer Purrenhage, David Russell, Shawn Wilder, and Kerri Wrinn. This project would not have been possible without the help of undergraduate students that assisted with data collection, data entry, and snake husbandry. These students include Andrea Collins, Marc Gelpi, John Kotcher, Matt Kovach, Mark Mackey, Lora Mengle, Courtney Miskell, Levi Pasma, Andrew Trout, Rachel Whynott, and Caitlin Zematis. I am thankful to John Lamb and the staff of ACS Conservation at Arnold Air Force Base, Tullahoma, TN for allowing me to collect snakes. Moreover, Will Bird and Phil Peak of the Kentucky Herpetological Society, and Garry Johnson of the Louisville Zoo helped in the field and with the acquisition of additional specimens. Funding for the project came from the Department of Zoology, Miami University and the Kentucky Herpetological Society. I especially want to thank my girlfriend Lisette and my parents for their love and support.

xi

General Introduction

1 Most studies examining organismal adaptations to their environment have focused on whole-animal performance (Arnold 1983; Bennett 1989; Garland and Losos 1994). One important measure is locomotor performance, which translates directly into fitness. Presumably, adaptations optimizing locomotor performance (i.e. speed, endurance, maneuverability) allow animals to more successfully capture prey, avoid predators, defend territories, and locate potential mates. Locomotor performance has been shown to be both repeatable and heritable (Van Berkum and Tsuji 1987; Garland 1988). Faster speeds also have been found to influence survivorship in garter snakes (Thamnophis sirtalis) (Jayne and Bennett 1990a). Hence, selection should favor adaptations that promote increases in speed and endurance capacities in most animals (Arnold and Bennett 1988). Since locomotion is well suited for studies investigating an individual’s performance in nature (i.e. fitness), such responses have been evaluated in a wide range of taxa (e.g. annelids: Quillin 1999; arthropods: Full and Herreid 1984; mollusks: Donovan and Carefoot 1997; fishes: Domenici and Blake 1991; amphibians: Marvin 2003; reptiles: Losos 1990; birds: Brigham et al. 1998; mammals: Alexander and Maloiy 1984). Locomotor abilities of most animals are not fixed, however, but rather are strongly influenced by various biotic and abiotic factors. Among ectotherms, intra- and interspecific variation in performance results from differences in body size, reproductive condition, temperature, nutritional state, injury, and structure of the habitat being traversed (Punzo 1982; Stevenson et al. 1985; Losos and Sinervo 1989;Wisco et al. 1997; Finkler and Claussen 1999; Claussen et al. 2002; Shine 2003).

Temperature effects on locomotion It has been known for a long time that body temperature has an enormous effect on all physiological processes with rates increasing at higher temperatures and decreasing at lower ones (Bennett 1990). Temperature is probably the single most important factor governing physiological activity (e.g. metabolism, digestion, growth) and whole-animal performance in ectotherms (Cossins and Bowler 1987; Huey and Kingsolver 1989). Though temperature can alter thermoregulatory behavior in ectotherms (Lillywhite 1987a), it more directly controls performance through its underlying effects on physiological and biochemical rates (Bennett 1990). These rates, and hence, performance, increase with increasing temperature up to a thermal optimum, above which performance capabilities quickly decrease. It should be noted that temperature does not influence all physiological rates and performance abilities in the same

2 manner (Bennett 1990). Thermal influence on locomotion in ectotherms has mostly been examined in lizards (Christian and Tracy 1981; Huey 1982; Marsh and Bennett 1986). Fewer studies have examined the thermal dependencies of snake locomotion (Stevenson et al. 1985; Scribner and Weatherhead 1995; Finkler and Claussen 1999). Moreover, these studies have only assessed temperature effects on two modes of limbless locomotion (terrestrial lateral undulation and swimming). Assessing differences in thermal dependencies among different modes of movement will shed light on the underlying physiological costs of each mode and the patterns of variation in performance abilities across modes at a given temperature.

Limbless locomotion Limblessness has evolved independently many times (62 according to Greer [1991]) among squamate reptiles with snakes being the primary example (Gans 1975). As a result, modern snakes display a variety of locomotor modes. Snakes can utilize lateral undulation, concertina, rectilinear, or sidewinding modes depending on the characteristics of the substrate and media being traversed (Gans 1974). The four modes used most often (terrestrial lateral undulation, swimming, concertina, and sidewinding) involve propulsive forces being generated by pushing against the substrate via lateral flexion (Gray 1946; Jayne 1986). Jayne (1985) has provided detailed descriptions of the kinematics of the different terrestrial modes of snake movement. Lateral undulation, the most common mode of locomotion in limbless reptiles, is usually employed during swimming and terrestrial crawling. In the water, snakes utilize this locomotor mode by producing a wave which progresses down the body in a posterior direction pushing the surrounding water and propelling the snake forward in a similar manner to anguilliform fishes (Hertel 1966; Jayne 1985). In terrestrial situations, lateral undulation is accomplished using body curves that push against irregularities on the substrate, thus generating forward thrust (Cundall 1987). Although undulatory movements used to propel snakes in aquatic and terrestrial habitats may seem superficially similar, the generation of force and utilization of body musculature are very different, thereby making adaptations for effective swimming likely to contradict adaptations for efficient undulatory terrestrial locomotion (Jayne 1982; Cundall 1987). Snakes can utilize concertina locomotion when moving through narrow passageways, climbing vertical surfaces, or crawling over low-friction substrates (Pough et al. 2000). This

3 type of movement consists of parts of the body becoming stationary by making static contact with the ground or sides of a tunnel while other parts of the body are moved forward (Gans 1974). Contrary to lateral undulation, regions of contracting muscles, which vary in length with the length of the stationary bend in the body, do not proceed down the trunk (Cundall 1987). Therefore, it is possible that morphological and physiological adaptations for efficient lateral undulation and concertina also conflict with each other. Thus, individuals that frequently utilize concertina for climbing or burrowing purposes may exhibit a reduction in locomotor performance during swimming or undulatory crawling. With greater than 2300 species worldwide, snakes occupy a wide variety of habitat types and must use different locomotor modes when traversing disparate habitats. Although most snakes are capable of traversing a variety of habitats, the majority maintain a terrestrial lifestyle (Greene 1997). However, even among this group, some are adapted for a fossorial lifestyle (e.g. Tantilla, Virginia), whereas others possess adaptations tuned for an arboreal (e.g. Opheodrys, Corallus) or semi-aquatic (e.g. Nerodia, Regina) existence (Greene 1997). Most studies of snake locomotion have assessed the influence of various factors such as body size, substrate, and gravidity on terrestrial crawling in snakes (Jayne and Bennett 1989; Kelley et al. 1997; Plummer 1997). Fewer studies have examined swimming performance (Jayne 1985; Aubret et al. 2005) and no study has looked at arboreal performance. However, Lillywhite et al. (2000) measured the ability to cantilever in 31 species of snake and found that arboreal species are better able to extend their body.

Arboreal locomotion Most animals have the ability to utilize available structural configurations that make up arboreal environments. Moreover, many animals possess specific adaptations that permit them to exploit these configurations. Despite frequent usage of these structures by many animals, arboreal locomotion is poorly studied relative to terrestrial locomotion with the exception of primates (reviewed in Strasser et al. 1998). Of the few studies investigating arboreal locomotion in reptiles, the majority have been conducted on Anolis lizards (Losos and Sinervo 1989; Spezzano and Jayne 2004) with others being conducted on Lacertid lizards (Van Damme et al. 1997), chameleons (Lilje and Fischer 2004), and geckos (Vanhooydonck et al. 2005). Several attributes of the arboreal environment have been shown to influence locomotion in lizards.

4 Turning angle of perches were found to decrease speeds and increase the number of stops in Anolis lizards as the angle increases (Higham et al. 2001). Perch diameter also has been found to influence arboreal locomotion. Losos and Sinervo (1989) found that speeds decreased as diameter decreased in Anolis lizards and that short-legged species move more easily on thinner perches than species with longer legs. Although smaller perch diameters caused a decrease in sprint speed, they had little effect on jumping abilities (Losos and Irschick 1996). In Anolis sagrei, perch diameter had a stronger influence on kinematic variables associated with the hindlimb than did incline, and speeds were reduced on lower diameter branches primarily due to a decrease in stride length (Spezzano and Jayne 2004). Despite these studies, very little, if any, information is available on how the arboreal environment influences locomotion in limbless reptiles.

Specializations and trade-offs Snakes with highly specialized patterns of habitat preference may only rarely encounter substrates that require different locomotor modes (e.g. the use of lateral undulation to swim across streams by tree-dwelling snakes; the use of concertina to crawl up vertical surfaces by sea snakes). In contrast, many species are generalists in terms of locomotor modes and frequently encounter different substrates during their lifetime within their home ranges. For example, many North American “terrestrial” colubrids are sometimes found in trees (e.g. Elaphe guttata, Coluber constrictor, Pituophis melanoleucus) (G. Gerald, personal observation). Presumably, these species are able to efficiently utilize many different locomotor modes. Snakes specialized for certain habitat types may possess morphological traits that reflect their specialized habits. For example, fusion of cephalic scales, slender bodies and long tails, and laterally compressed tails are characteristic of snakes that display fossorial, arboreal, and aquatic lifestyles, respectively (Savitsky 1983; Lillywhite and Henderson 1993). Structural modifications are seen in a variety of snake taxa with regards to specialization and are an excellent example of convergent evolution. Individuals with these traits are assumed to have a higher fitness (i.e. increased survival and reproductive success) compared to individuals with similar habits lacking these traits. However, very few studies have examined the validity of these assumptions. Although most of these traits may function to improve performance within preferred habitats, performance may be hindered in habitats not typically used. Hence,

5 locomotor abilities within one habitat or substrate may be governed by the proportion of time a species spends using a given habitat or substrate in nature. Likewise, species should display the poorest locomotor performance capabilities when moving on substrates least likely to be encountered and utilized. Shine and Shetty (2001) showed that terrestrial elapid snakes were faster on land but slower in water compared to sea kraits (Lauticauda colubrina). This trade-off also was observed for sea snakes (Lauticaudidae) among different species, though not among individuals of the same species (Shine et al. 2003). In a recent study on terrestrial locomotion in two species of Laticaudid sea snakes, the more terrestrial Laticauda colubrina had better cliff- climbing abilities and was stronger than the more aquatic Laticauda laticaudata (Bonnet et al. 2005). This study suggests that locomotor capabilities reflect patterns in terrestrial habitat use in these species. Other than these reports, no study has examined the influence of habitat specialization on locomotor performance in snakes.

Direction of dissertation The goal of this work was to investigate some of the biotic and abiotic factors responsible for setting limits on various modes of limbless locomotion. Because different modes require different biomechanical properties with corresponding differences in physiological and metabolic capacities, abiotic factors may influence each mode in contrasting ways. Moreover, different species are expected to show locomotor adaptations fine-tuned for preferred microhabitats they occupy in nature. Chapter one examines the influence of ambient temperature on three modes of snake locomotion (lateral undulation, concertina, swimming) in newborn brown snakes (Storeria dekayi). Then locomotor abilities measured in these snakes are compared to speeds reported in the literature for other species across the same experimental temperatures. The second chapter investigates how perch diameter and temperature influence arboreal locomotion in a semi-arboreal species (Elaphe guttata). These results are compared to other modes used by other species to scrutinize the ability of snakes to balance on perches of various diameters. The third chapter assesses the relationships among microhabitat use, morphology, and locomotor performance across locomotor modes in five different species. Performance data among species are compared and modes to determine if trade-offs exist among different locomotor modes. The concluding chapter discusses how the results from this study contribute to the existing knowledge about different modes of snake locomotion. The implications of this

6 work on snake distributions worldwide, the radiation of snakes into various habitats, and the evolution of limblessness also are discussed.

Literature cited Alexander R.M. and G.M.O. Maloiy. 1984. Stride lengths and stride frequencies of primates. Journal of Zoology 202:577-582. Arnold S.J. 1983. Morphology, performance and fitness. American Zoologist 23:347- 361. Arnold S.J. and A.F. Bennett. 1988. Behavioral variation in natural populations. V. Morphological correlates of locomotion in the garter snake (Thamnophis radix). Biological Journal of the Linnean Society 34:175-190. Aubret F., X. Bonnet, R. Shine, and S. Maumelat. 2005. Swimming and pregnancy in Tiger snakes, Notechis scutatus. Amphibia-Reptilia 26:396-400. Bennett A.F. 1989. Integrated studies of locomotor performance. In: Complex organismal functions: integration of evolution in vertebrates (Ed. by Wake D.B. and G. Roth). pp. 191-202. Wiley. Chichester, UK. Bennett A.F. 1990. Thermal dependence of locomotor capacity. American Journal of Physiology 259:R253-R258. Bonnet X., I. Ineich, and R. Shine. 2005. Terrestrial locomotion in sea snakes; the effects of sex and species on cliff-climbing ability in sea kraits (Serpentes, Elapidae, Laticauda). Biological Journal of the Linnean Society 85:433-441. Brigham R., M. Fenton, and H. Aldridge. 1998. Flight speed of foraging common nighthawks (Chordeiles minor): Does measurement technique matter? American Midland Naturalist 139:325-330. Christian K.A. and C.R. Tracy. 1981. The effect of the thermal environment on the ability of hatchling Galapagos land iguanas to avoid predation during dispersal. Oecologia 49:218-223. Claussen D.L., R. Lim, M. Kurz, and K. Wren. 2002. Effects of slope, substrate, and temperature on the locomotion of the ornate box turtle, Terrapene ornata. Copeia 2002:411-418.

7 Cossins A.R. and K. Bowler. 1987. Temperature biology of animals. London. Chapman and Hall. Cundall D. 1987. Functional Morphology. In: Snakes: ecology and evolutionary biology (Ed. by Seigel R.A., J.T. Collins, and S.S. Novak). pp. 106-140. Blackburn Press. Domenici P. and R.W. Blake. 1991. The kinematics and performance of the escape response in the angelfish (Pterophyllum eimekei). Journal of Experimental Biology 177:253-272. Donovan D.A. and T.H. Carefoot. 1997. Locomotion in the abalone Haliotus kamtschatkana: pedal morphology and cost of transport. Journal of Experimental Biology 200:1145-1153. 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. Full R.J. and R.F. Herreid. 1984. Fiddler crab exercise: the energetic cost of running sideways. Journal of Experimental Biology 109:141-161. Gans C. 1974. Biomechanics: an approach to vertebrate biology. University of Michigan Press. Ann Arbor, MI Gans C. 1975. Tetrapod limblessness: evolution and functional corollaries. American Zoology 15:455-467. Garland T. Jr. 1988. Genetic basis of activity metabolism. I. Inheritance of speed, stamina, and antipredator displays in the garter snake, Thamnophis sirtalis. Evolution 42:335-350. Garland T. Jr. and JB Losos. 1994. Ecological morphology of locomotor performance in squamate reptiles. In: Ecological morphology: integrative organismal biology (Ed. by P.C. Wainwright and S.M. Reilly). pp. 240-302. University of Chicago Press. Gray J. 1946. The mechanism of locomotion in snakes. Journal of Experimental Biology 23:101-120. Greene H.W. 1997. Snakes: the evolution of mystery in nature. University of California Press. Berkeley, CA.

8 Greer A.E. 1991. Limb reduction in squamates: identification of the lineages and discussion of the trends. Journal of Herpetology 25:166-173. Hertel H. 1966. Structure form and movement. Rheinold, New York. Higham T.E., M.S. Davenport, and B.C. Jayne. 2001. Maneuvering in an arboreal habitat: the effects of turning angle on the locomotion of three sympatric ecomorphs of Anolis lizards. Journal of Experimental Biology 204:4141-4155. Huey R.B. 1982. Temperature physiology and the ecology of reptiles. In: Biology of the reptilia, vol. 12 Physiology C, Physiological ecology (Ed. by Gans C. and F.H. Pough). pp. 25-91. Academic Press. Huey R.B. and J.G. Kingsolver. 1989. Evolution of thermal sensitivity of ectotherm performance. Trends in Ecology and Evolution 4:131-135. Jayne B.C. 1982. Comparative morphology of the semispinalis-spinalis muscle of snakes and correlations with locomotion and constriction. Journal of Morphology 172:83-96. Jayne B.C. 1985. Swimming in constricting (Elaphe g. guttata) and nonconstricting (Nerodia fasciata pictiventris) colubrid snakes. Copeia 1985:195-208. Jayne B.C. 1986. Kinematics of terrestrial snake locomotion. Copeia 1986:915-927. Jayne B.C. and A.F. Bennett. 1989. The effect of tail morphology on locomotor performance of snakes: a comparison of experimental and correlative methods. Journal of Experimental Zoology 252:126-133. Jayne B.C. and A.F. Bennett. 1990. Selection on locomotor performance capacity in a natural population of garter snakes. Evolution 44:1204-1229. Kelley K.C, S.J. Arnold, and J. Gladstone. 1997. The effects of substrate and vertebral number on locomotion in the garter snake Thamnophis elegans. Functional Ecology 11:189-198. Lilje K.E. and M.S. Fischer. 2004. Arboreal locomotion of the chameleon Chamaeleo calyptratus, Sauria. Journal of Morphology 260:307. Lillywhite H.B. 1987. Temperature, energetics and physiological ecology. In: Snakes: ecology and evolutionary biology (Ed. by Seigel R.A., J.T. Collins, and S.S. Novak). pp. 422-477. McGraw Hill Publishing Co.

9 Lillywhite H.B. and R.W. Henderson. 1993. Behavioral and functional ecology of arboreal snakes. In: Snakes: ecology and behavior (Ed. by Seigel RA and JT Collins). pp. 1-48. Backburn Press. Lillywhite H.B., J.R. LaFrentz, Y.C. Lin, and M.C. Tu. 2000. The cantilever abilities of snakes. Journal of Herpetology 34:523-528. Losos J.B. 1990. The evolution of form and function: morphology and locomotor performance in West Indian Anolis lizards. Evolution 44:1189-1203. Losos J.B. and B. Sinervo. 1989. The effects of morphology and perch diameter on sprint performance of Anolis lizards. Journal of Experimental Biology 145:23-30. Losos J.B. and D.J. Irschick. 1996. The effect of perch diameter on escape behaviour of Anolis lizards: laboratory predictions and field tests. Animal Behaviour 51:593-602. Marsh R.L. and A.F. Bennett. 1986. Thermal dependence of sprint performance of the lizard Sceloporus occidentalis. Journal of Experimental Biology 126:79-87. Marvin G.A. 2003. Effects of acute temperature and thermal acclimation on aquatic and terrestrial locomotor performance of the three-lined salamander, Eurycea guttolineata. Journal of Thermal Biology 28:251-259. Plummer M.V. 1997. Speed and endurance of gravid and nongravid green snakes, Opheodrys aestivus. Copeia 1997:191-194. Pough F.H., R.M. Andrews, J.E. Cadle, M.L. Crump, A.H. Savitsky, and K.D. Wells. 2001. Herpetology. 2nd Ed. Prentice Hall. Upper Saddle River, New Jersey. Punzo C. 1982. Tail autotomy and running speed in the lizards Cophosaurus texanus and Uma notata. Journal of Herpetology 16:331-332. Quillin K.J. 1999. Kinematic scaling of locomotion by hydrostatic animals: ontogeny of peristaltic crawling by the earthworm Lumbricus terrestris. Journal of Experimental Biology 202:661-674. Savitsky A.H. 1983. Coadapted character complexes among snakes: fossoriality, piscivory, and durophagy. American Zoologist 23:397-409. Scribner S.J. and P.J. Weatherhead. 1995. Locomotion and antipredator behaviour in three species of semi-aquatic snakes. Canadian Journal of Zoology 73:321-329. Shine R. 2003. Locomotor speeds of gravid lizards: placing ‘costs of reproduction’ within an ecological context. Functional Ecology 17:526-533.

10 Shine R. and S. Shetty. 2001. Moving in two worlds: aquatic and terrestrial locomotion in sea snakes (Laticauda colubrina, Laticaudidae). Journal of Evolutionary Biology 14:338-346. Shine R., H.G. Cogger, R.R. Reed, S. Shetty, and X. Bonnet. 2003. Aquatic and terrestrial locomotor speeds of amphibious sea-snakes (Serpentes, Laticaudidae). Journal of Zoology 259:261-268. Spezzano L.C. Jr. and B.C. Jayne. 2004. The effects of surface diameter and incline on the hindlimb kinematics of an arboreal lizard (Anolis sagrei). Journal of Experimental Biology 207:2115-2131. Stevenson R.D., C.R. Peterson, and J.S. Tsuji. 1985. The thermal dependence of locomotion, tongue flicking, digestion, and oxygen consumption in the wandering garter snake. Physiological Zoology 58:46-57. Strasser E., J. Fleagle, A. Rosenberger, and H. McHenry. 1998. Primate locomotion: recent advances. New York. Plenum Press. van Berkum F.H. and J.S. Tsuji. 1987. Among-family differences in sprint speed of hatchling Sceloporus occidentalis. Journal of Zoology 212:511-519. van Damme R., P. Aerts, and B. Vanhooydonck. 1997. No trade-off between sprinting and climbing in two populations of the lizard Podarcis hispanica (Reptilia: Lacertidae). Biological Journal of the Linnean Society 60:493-503. Vanhooydonck B., A. Andronescu, A. Herrel, and D.J. Irschick. 2005. Effects of substrate structure on speed and acceleration capacity in climbing geckos. Biological Journal of the Linnean Society 85:385-393. Wisco J., H. Matles, and D. Berigan. 1997. Is the scaling of locomotor performance with body size constant? Ecological Entomology 22:483-486.

11

Chapter 1:

Thermal dependencies of different modes of locomotion in neonate Brown Snakes, Storeria dekayi

12 Introduction The evolution of limblessness required the development of new modes of locomotion for animals, such as snakes, to successfully capture prey, avoid predators, and locate potential mates (Gans 1986). Because of the high amount of friction associated with this specialized morphology, most snakes have evolved the ability to utilize one of several locomotor modes depending upon the substrate or media being traversed (Jayne 1986; Cundall 1987). The most common mode used by snakes is lateral undulation, which is used both on land and in water. In terrestrial situations, lateral undulation is accomplished using body curves that push against irregularities on the substrate, thus generating forward thrust (Jayne 1986). In water, snakes utilize this locomotor mode by producing a wave which progresses down the body in a posterior direction, pushing the surrounding water and propelling the snake forward in a similar manner to that employed by anguilliform fishes (Hertel 1966; Jayne 1985). Although undulatory movements used to propel snakes in terrestrial and aquatic habitats are superficially similar, the generation of force and utilization of body musculature are very different (Cundall 1987). Snakes can also move via concertina locomotion when moving through narrow passageways, climbing vertical surfaces, or crawling over low-friction substrates (Jayne 1988; Jayne and Davis 1991). This type of movement consists of parts of the body becoming stationary by making static contact with the ground or sides of a tunnel while other parts of the body are moved forward (Gans 1974). Contrary to lateral undulation, regions of contracting muscles, which vary in length with the length of the stationary bend in the body, do not proceed down the trunk (Cundall 1987). Like most other performance capabilities, limbless locomotion is well known to be strongly influenced by temperature (Huey 1982; Bennett 1989; Finkler and Claussen 1999). Finkler and Claussen (1999) found that although swimming velocities exceeded terrestrial velocities in semi- aquatic snakes (Nerodia sipedon and Regina septemvittata), this difference was highly dependent on temperature. Other studies have shown that swimming and crawling velocities achieved by garter snakes (Thamnophis) also have different temperature sensitivities (Stevenson et al. 1985; Scribner and Weatherhead 1995). The thermal dependency of locomotion appears to reflect thermal effects on the underlying physiological mechanisms governing performance, such as maximal oxygen consumption and muscle activity (Bennett 1990). Consequently, the disparity

13 in thermal dependencies between aquatic and terrestrial undulation likely signifies differences in the properties of muscle contraction and force generation. Shine and Shetty (2001) documented the inability of snakes to simultaneously optimize locomotor performance of different modes, thereby suggesting that trade-offs exist due to the inability to evolve adaptations that simultaneously enhance both undulatory swimming and crawling. They found that Sea Kraits (Laticauda colubrina) swam 60% faster but crawled 80% slower than terrestrial snakes within the same family (Elapidae). In contrast, Shine et al. (2003) found no evidence of trade-offs when six taxa of sea-snakes were compared to each other. With the exception of these studies, no study has fully explored the potential trade-offs in performance during different modes in terrestrial snakes. This investigation aimed to examine the thermal dependencies of undulatory crawling and swimming and concertina crawling in neonate Brown Snakes (Storeria dekayi), a habitat generalist (Ernst and Ernst 2003), and to determine if trade-offs in maximum velocities exist among these three locomotor modes in this species. Relatively little information is available on locomotor performance in neonate snakes (Bennett and Jayne 1990). This is unfortunate since neonates are smaller, must maneuver in the same environment as adults, and experience higher mortality rates from predation than do adults (Arnold and Wassersug 1978; Carrier 1996). As a result, we hypothesize that neonate snakes will exhibit higher body length-relative velocities compared to adult snakes and we predict that velocities attained by neonates will increase as temperature increases in a similar fashion as previously observed in adults. Moreover, the thermal dependencies of terrestrial and aquatic undulation and concertina should differ because of kinematic differences among these three locomotor modes. We also hypothesized that aquatic undulatory velocities will exceed velocities of other modes because water offers little resistance to the forward thrust generated by waves at the posterior region of the body (Jayne 1985). Likewise, we expected movement by lateral undulation on land to be faster than movement by concertina because of the alternations of movements and pauses and because of the higher energetic costs associated with the latter mode of locomotion (Walton et al. 1990; Jayne and Davis 1991). Finally, we predicted that individuals exhibiting superior speeds during one locomotor mode will display slower speeds during other modes (i.e. trade-offs).

Materials and Methods

14 Two gravid female Storeria dekayi were hand collected during summer 2004 under woody cover objects located in an open loblolly pine stand on Arnold Air Force Base, Franklin County, Tennessee, USA and taken to the laboratory for an unrelated study. The first female (29.8 cm snout-vent length [SVL]; 8.14 g) gave birth to 19 offspring, whereas the second female (18.3 cm SVL; 2.62 g) gave birth to only 7 offspring. The availability of neonate S. dekayi provided an opportunity to assess the thermal dependencies of different locomotor modes in a habitat generalist species. Only neonates were used for this study and adults were later released at their point of capture. All neonates (N = 26; 6.2-7.6 cm SVL; 0.22-0.28 g) were housed individually at 24 ± 0.5 C, maintained on a 12:12 L:D cycle, and fed red wiggler worms (Eisenia foetida). Locomotor velocities of each individual snake moving via lateral undulation (crawling and swimming) and concertina were measured in an environmental chamber at three different temperatures (10, 20, and 30 C). Individuals and locomotor modes were randomized within each temperature and conducted in the following sequence: 20, 10, 30 C. All individuals were acclimated to test temperatures a minimum of 2 h before velocities were measured. For each locomotor mode, snakes were stimulated to move continuously by lightly tapping the tail with a small painter’s brush. All snakes were stimulated until three runs were completed in succession for each condition. Both absolute velocities (m/s) and body length-relative velocities (SVL/s) were determined for each run. Average velocities were calculated as the average of the three runs while maximum velocities were considered the fastest of the three runs. Terrestrial lateral undulation was measured using a linear racetrack containing infrared photocells every 25 cm that was connected to an automatic timer that recorded times to the nearest 0.1 s. The track (1.25 m long x 2.9 cm wide) was lined with an artificial substrate containing rough carpet that provided purchase points to facilitate the desired locomotor mode. This track width allowed snakes to crawl without contacting the sides of the track, forcing them to move down the track by pushing off of the small projections in the substrate; therefore, we are confident that snakes crawled by undulation during each terrestrial trial. Only runs in which no wall contact was made were used for analyses. Concertina locomotion was elicited by stimulating snakes to move on a linear track (50 cm long x 0.6 cm wide) containing infrared photocells every 25 cm to record times to the nearest 0.1 s. This track contained thin pieces of cardboard affixed to the sides of the track to allow snakes to make static contact with the sides. The floor of the track was a smooth piece of plastic used to

15 discourage undulation and force snakes to push and pull themselves forward following contact with the track sides. Swimming by S. dekayi occurred at the water’s surface and sub-surface swimming was not observed. Swimming velocities were attained by stimulating snakes to swim in a modified gutter (1.5-m long x 10.6 cm wide) marked off in 25-cm intervals and filled with water of the appropriate temperature. Large rocks were placed on either end of the gutter to give snakes something to swim towards. Because of the inconsistencies of infrared photocells when used in water, the time it took snakes to swim 1 m was measured with a stopwatch to the nearest 0.1 s.

To evaluate the influence of temperature on locomotor velocities during different modes, Q10 values were determined for maximum absolute velocities. Repeated measures ANOVAs (PROC GLM) were used to ascertain differences in velocities relative to temperature and locomotor mode and the Bonferroni method was used for multiple comparisons. Pearson product moment correlations tests were used to detect trade-offs in velocities among locomotor modes. Generalized linear models (GLM) were used to examine the relationships between SVL and velocities for each temperature and mode. Data were log-transformed and compared to a Bonferroni-adjusted significance level of α = 0.0167 to account for the three temperature treatments. Since all snakes used in this study originated from two litters, litter was included as a random effect in the GLM (PROC MIXED) to determine if heredity influenced velocities during each mode and temperature. These models were then compared to models without litter included using the differences in log likelihood values as a χ2 statistic to determine whether or not litter contributed significantly to the variance of each model. All statistical tests were run using Minitab (Minitab Inc., State College, Pennsylvania, U.S.A.) and SAS (SAS Institute, Version 9.1).

Results Both absolute and body length-relative velocities of all three modes increased significantly with temperature (df = 2, 170; p < 0.0001; Fig. 1). Snakes moved fastest during swimming and moved slowest during concertina at all temperatures (df = 2, 170; p < 0.0001; Fig. 1). The interaction between temperature and locomotor mode was also significant (df = 4, 170; p < 0.0001). Within each locomotor mode, velocities increased significantly from 10 to 20 C (p <

0.0001), but not from 20 to 30 C (p > 0.0167; Fig. 1). Q10 values were calculated for maximum

16 absolute velocities attained by neonates. Between 10 and 20 C, Q10 values were 2.36, 3.00, and 1.69 for concertina, terrestrial lateral undulation, and swimming velocities, respectively.

Between 20 and 30 C, Q10 values were 1.21, 1.11, and 1.20 for concertina, terrestrial lateral

undulation, and swimming velocities, respectively. Differences in Q10 values among locomotor modes indicate that the influence of temperature on speed was mode dependent. Absolute velocities of individual neonates were compared to determine if individuals displaying faster velocities during one mode were slower while moving during other modes (Table 1). Despite this prediction, negative correlations among modes and throughout temperatures were not detected. In contrast, concertina velocities were not correlated with any other mode and terrestrial lateral undulation and swimming were positively correlated at 30 C, meaning that individuals that crawled faster via undulation on land were also faster swimmers. Some positive correlations were also detected between terrestrial undulation and swimming across temperatures and within modes across temperatures (Table 1). Litter had no effect on mean or maximum absolute velocities attained for any mode of locomotion at any temperature (p > 0.25 for all modes and temperatures). The models examining the relationships between body length and speed were reduced to power functions when litter and interaction effects were not significant. No significant relationships were observed between size and absolute velocities for any mode of locomotion at any temperature (Table 2).

Discussion It is well documented that locomotor performance, like most physiological processes, is temperature dependent (Bennett 1990). The present study supports both this notion and previous observations that temperature influences disparate modes of limbless locomotion in different ways (Stevenson et al. 1985; Scribner and Weatherhead 1995; Finkler and Claussen 1999). This study is the first to examine thermal effects on velocities attained by neonate snakes and the first to compare the thermal dependencies of undulation (terrestrial and aquatic) and concertina locomotion. As expected, absolute velocities by neonates were much lower than those found for larger snakes for all modes examined. It is possible that the velocities measured during this study represent a small portion of the variation found in this species since only two litters were used. However, the observed variation in velocities was very similar to variation in velocities of

17 terrestrial and aquatic undulation in related species (Scribner and Weatherhead 1995; Finkler and Claussen 1999). Swimming, undulatory crawling, and concertina velocities by S. dekayi displayed differential

responses with increasing temperature (Fig. 1). Overall, Q10 values were generally around 2-3 between 10 and 20 C and <2 between 20 and 30 C as found in other ectothermic vertebrates

(Bennett 1990). The observed Q10 values for swimming were similar to those reported for Thamnophis elegans but slightly smaller than those found for Nerodia sipedon and Regina septemvittata between 10 and 20 C (Table 3). The Q10 values for terrestrial undulation between 10 and 20 C were similar to those found in previous studies; however, the values between 20 and 30 C were slightly smaller than those found in other snake species (Table 3). Differences in thermal responses among the three modes investigated here likely represent differences in physiological requirements associated with mechanical properties of each mode (Jayne 1986). For example, the relative ease of wave propagation in aqueous environments, increased surface area for force generation, and decreased friction associated with moving through water are likely the major reasons snakes achieve relatively higher speeds at lower temperatures during

swimming (Jayne 1985). This hypothesis is supported by the lower Q10 values observed between 10 and 20 C for swimming compared to those of the other two modes. Large increases in speed with temperature would not be expected if faster speeds can be attained at low temperatures. The disparities in thermal responses may also be due to differences in transport costs among modes. Terrestrial undulation is likely a more energetically expensive task than aquatic undulation for the reasons already stated. Although the net costs of transport for terrestrial -1 -1 lateral undulation have been measured in one species of snake (1.15 ml O2 g km in Coluber constrictor; Walton et al. 1990), the costs of transport of swimming by snakes have yet to be quantified. From an ecological standpoint, neonates will be at a disadvantage relative to adults when a predator is encountered because they are slower in an absolute sense. Consequently, only body length-relative velocities should be used when comparing velocities from this study with those from other studies on larger snakes. Admittedly, one must be cautious when comparing velocities of juveniles with those of conspecific and heterospecific adults given the potential differences in the physiological capacities and morphologies associated with locomotion. Nonetheless, terrestrial undulatory velocities of neonate S. dekayi were similar to those of other

18 snakes previously examined at all test temperatures (Fig. 2). At 10 C, neonate S. dekayi travel at approximately the same relative speed as larger snakes (Fig. 2). Differences become apparent as temperature increases, with the neonates being relatively slower than N. sipedon and R. septemvittata but faster than Natrix maura. Swimming velocities of neonates were also similar to others at 10 C, but slower than those of N. sipedon and R. septemvittata at 20 and 30 C (Fig. 3). Differences in body length-relative velocities among species likely reflect differences in the use of that particular locomotor mode in nature. Storeria dekayi is typically found in a variety of terrestrial habitats and is common to areas close to freshwater wetlands, ponds, and streams (Wright and Wright 1957; Ernst and Ernst 2003). Moreover, this species feeds primarily on slugs and earthworms located under cover objects or leaf litter (Ernst and Ernst 2003). Nerodia sipedon and R. septemvittata are semi-aquatic species that feed on a variety of aquatic prey and crayfish, respectively. Interspecific variation in the physiological capacities for terrestrial and aquatic undulation at various temperatures among these species may be driven by differences in activity levels, preferred habitats, and foraging mode. Concertina is used by many snakes to travel through tunnels or climb vertical surfaces. It is -1 -1 very slow and much more energetically costly (8.49 ml O2 g km net cost of transport in C. constrictor; Walton et al. 1990) than terrestrial lateral undulation. As expected, concertina velocities achieved by neonate S. dekayi were much lower than terrestrial and aquatic lateral undulation. Temperature coefficients between 10 and 20 C were higher than those for swimming but lower than those for terrestrial undulation. Relatively smaller increases in concertina velocities compared to terrestrial undulation result from the differences in kinematic properties between these two locomotor modes. Specifically, frequencies of movement for concertina locomotion are 2.4 times higher than those for terrestrial lateral undulation (Walton et al. 1990). Increased frequencies of movement result in more momentum changes due to the stop-and-go nature of concertina. Greater numbers of momentum changes associated with higher concertina velocities at warmer temperatures could limit speed increases at higher temperatures (Davis and Jayne 1991). Since the high transport costs of concertina locomotion appear to be caused primarily by large movement frequencies (Walton et al. 1990), larger increases in transport costs with speed for concertina relative to terrestrial undulation likely contributed to the differences in temperature coefficients observed between these modes.

19 The underlying mechanism of concertina locomotion in limbless squamates appears to be similar across the few observed species (Jayne 1988; Gans and Gasc 1990). Within snakes, the activity of the muscles in relation to vertebral flexion and points of static contact are very similar for all species previously examined (Jayne 1988). Therefore, we believe it is appropriate to compare body length-relative velocities of neonates from this study with those of adults of other limbless squamates from previous studies. Neonate S. dekayi generally attained faster concertina velocities relative to body length compared to most other snakes and Ophisaurus (Table 4). It has been suggested that selection is likely strong for higher speeds in neonates compared to adults because of an increased probability of mortality (Carrier 1996). One might also expect neonates to display relatively faster concertina speeds if they utilize concertina more in nature. However, neonates in this study were much slower than the amphisbaenid Rhineura floridana but had similar relative velocities to those of racers (Coluber constrictor) moving through 3, 5, and 7 cm wide tunnels (Table 4). Jayne and Davis (1991) found that differences in tunnel width resulted in significant effects on concertina endurance capacities of C. constrictor. Though differences were non-significant, concertina velocities increased with tunnel width for C. constrictor (Jayne and Davis 1991). Velocities observed in neonate S. dekayi moving in a 0.6 cm tunnel fall in line with relative velocities of C. constrictor moving at different tunnel widths (Fig. 4). This trend suggests that body width at mid-body relative to tunnel width could be a good predictor of maximal concertina speeds in limbless reptiles. Additional studies of concertina locomotion by various species of limbless reptiles are needed to test this hypothesis. Contrary to our predictions, we found no significant relationships between size and velocities of concertina, terrestrial lateral undulation, or swimming. This is not surprising considering the very small range of body sizes (6.2 – 7.6 cm SVL) of the neonates used in the study. Future studies should utilize a much larger range of body sizes to determine how size influences multiple modes of locomotion in snakes. An additional aim of this study was to examine the hypothesis that adaptations favoring enhanced locomotor abilities during one mode of limbless locomotion would be incompatible with adaptations promoting movement via other modes because of differences in kinematic properties, muscle activity, and force generation (Jayne 1986; Cundall 1987; Shine et al. 2003). We found no negative correlations among different locomotor modes in S. dekayi, thus indicating that an enhancement of one locomotor mode does not inhibit another (Table 1). In

20 contrast, positive correlations were detected within several modes at different temperatures and between terrestrial and aquatic lateral undulation at 30 C. This contradicts predictions that individual snakes that are superior swimmers cannot simultaneously be superior undulatory crawlers relative to other individuals (Jayne 1982; Cundall 1987). Variation in metabolic capacity among individual S. dekayi could explain correlations observed between modes and temperatures (Chappell et al. 1995). The non-existence of a trade-off could simply be the result of inter-individual variation in body condition or response to handling which could falsely produce positive rather than negative correlations (Shine and Shetty 2001). Finkler and Claussen (1999) documented some positive correlations between maximum undulatory crawling and swimming velocities in Regina septemvittata. When comparing individuals within species, Shine and Shetty (2001) and Shine et al. (2003) found no evidence of a trade-off between terrestrial and aquatic locomotion in sea-snakes (Laticaudidae), but did observe trade-offs in velocities when comparing sea-snakes to terrestrial elapids. Despite differences in the mechanisms of force generation between crawling and swimming in snakes, these modes may be similar enough so that adaptations that enhance one will enhance the other. Because of high intraspecific variation found in this and other studies, future studies designed to detect potential trade-offs in locomotor abilities should focus on interspecific comparisons (Finkler and Claussen 1999; Shine and Shetty 2001). Moreover, additional studies should relate performance via different modes of limbless locomotion to the actual use of these modes in nature.

Acknowledgments This research was supported by the Department of Zoology, Miami University. Protocols for this study were approved by the Miami University IACUC (Protocol 634). Snakes were collected under Tennessee Wildlife Resources Agency Scientific Permit 1948. We would like to thank Robert Schaefer for providing assistance with statistical analyses.

Literature Cited Arnold S.J. and R.J. Wassersug. 1978. Differential predation on metamorphic anurans by garter snakes (Thamnophis): social behavior as a possible defense. Ecology 59:1014-1022.

21 Bennett A.F. 1989. Integrated studies of locomotor performance. In: Complex organismal functions: integration of evolution in vertebrates (Ed. by Wake D.B. and G. Roth). pp. 191-202. Wiley, Chichester, UK. Bennett A.F. 1990. Thermal dependence of locomotor capacity. American Journal of Physiology 259:R253-R258. Carrier D.R. 1996. Ontogenetic limits on locomotor performance. Physiological Zoology 69:467-488. Chappell M.A., G.C. Bachman, and J.P. Odell. 1995. Repeatibility of maximal aerobic performance in Belding’s ground squirrels, Spermophilus beldingi. Functional Ecology 9:498-504. Cundall D. 1987. Functional morphology. In: Snakes: ecology and evolutionary biology (Ed. by Seigel R.A., J.T. Collins, and S.S. Novak). pp. 106-142. McGraw Hill, New York. Ernst C.H. and E.M. Ernst. 2003. Snakes of the United States and Canada. Smithsonian Books, Washington and London. Finkler M.S. 1995. Effects of temperature, body size, substrate and season on the locomotor performance of three species of Colubrid snake (Nerodia sipedon, Regina septemvittata, and Thamnophis sirtalis). Unpubl. M.S. thesis, Miami University, Oxford, Ohio. 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. Gans C. 1974. Biomechanics: an approach to vertebrate biology. University of Michigan Press, Ann Arbor, Michigan.

Gans C. 1986. Locomotion of limbless vertebrates: pattern and evolution. Herpetologica 42:33-46. Gans C., and J.P. Gasc. 1990. Tests on the locomotion of the elongate and limbless reptile Ophisaurus apodus (Sauria: Anguidae). Journal of Zoology (London) 220:517- 536.

22 Hailey A. and P.M.C. Davies. 1986. Effects of size, sex, temperature and condition on activity metabolism and defence behaviour of the viperine snake, Natrix maura. Journal of Zoology (London) 208:541-558. Heckrotte C. 1967. Relations of body temperature, size and crawling speed of the common garter snake, Thamnophis s. sirtalis. Copeia 1967:520-526. Hertel H. 1966. Structure form and movement. Rheinold, New York. Huey R.B. 1982 Temperature, physiology, and the ecology of reptiles. In: Biology of the reptilia, vol. 12 (Ed. by Gans C. and F.H. Pough). pp. 25-93. Academic Press, London. Jayne B.C. 1982. Comparative morphology of the semispinalis-spinalis muscle of snakes and correlations with locomotion and constriction. Journal of Morphology 172:83-96. Jayne B.C. 1985. Swimming in constricting (Elaphe g. guttata) and non-constricting (Nerodia fasciata pictiventris) colubrid snakes. Copeia 1985:195-208.

Jayne B.C. 1986. Kinematics of terrestrial snake locomotion. Copeia 1986:915-927.

Jayne B.C. 1988. Muscular mechanisms of snake locomotion: an electromyographic study of the sidewinding and concertina modes of Crotalus cerastes, Nerodia fasciata and Elaphe obsoleta. Journal of Experimental Biology 140:1-33. Jayne B.C. and A.F. Bennett. 1990. Scaling of speed and endurance in garter snakes: a comparison of cross-sectional and longitudinal allometries. Journal of Zoology (London) 220:257-277. Jayne B.C. and J.D. Davis. 1991. Kinematics and performance capacity for the concertina locomotion of a snake (Coluber constrictor). Journal of Experimental Biology 156:539-556. Scribner S.J. and P.J. Weatherhead. 1995. Locomotion and antipredator behaviour in three species of semi-aquatic snakes. Canadian Journal of Zoology 73:321-329.

Shine R. and S. Shetty. 2001. Moving in two worlds: aquatic and terrestrial locomotion in sea snakes (Laticauda colubrina, Laticaudidae). Journal of Evolutionary Biology 14:338-346.

23 Shine R., H.G. Cogger, R.R. Reed, S. Shetty, and X. Bonnet. 2003. Aquatic and terrestrial locomotor speeds of amphibious sea-snakes (Serpentes, Laticaudidae). Journal of Zoology (London) 259:261-268. Stevenson R.D., C.R. Peterson, and J.S. Tsuji. 1985. The thermal dependence of locomotion, tongue flicking, digestion, and oxygen consumption in the wandering garter snake. Physiological Zoology 58:46-57. Walton M., B.C. Jayne, and A.F. Bennett. 1990. The energetic cost of limbless locomotion. Science 249:524-527. Wright A.H. and A.A. Wright. 1957. Handbook of snakes of the United States and Canada. Comstock Publishing Associates, Ithaca, New York.

24 Table 1. Pearson correlation coefficients for absolute velocities of 26 neonate brown snakes (Storeria dekayi) among locomotor modes and temperature. Pairwise α = 0.05. * p < 0.05.

Lateral Undulation Concertina Swimming Mode Temp. 10 C 20 C 30 C 10 C 20 C 30 C 10 C 20 C 30 C 10 C 1.000 Lateral 0.569* 1.000 Undulation 20 C 30 C 0.297 0.292 1.000

Absolute 10 C 0.226 -0.278 -0.130 1.000

Mean -0.094 -0.010 -0.453 0.333 1.000 Concertina 20 C Velocities 30 C 0.233 -0.109 -0.017 0.550* 0.688* 1.000

10 C 0.077 0.042 0.298 0.027 -0.114 0.205 1.000

0.138 0.160 0.355 -0.053 -0.290 -0.107 0.582* 1.000 Swimming 20 C 30 C 0.422 0.528* 0.785* -0.037 -0.566 -0.208 0.153 0.129 1.000

10 C 1.000 Lateral 20 C 0.223 1.000 Undulation 30 C -0.131 0.494 1.000

Absolute 10 C 0.213 -0.322 -0.398 1.000

Maximum 20 C 0.113 0.033 -0.210 0.464 1.000 Concertina Velocities 30 C 0.363 -0.013 -0.161 0.443 0.729* 1.000

10 C 0.020 0.296 0.475* -0.017 -0.132 0.083 1.000

20 C -0.095 0.452 0.338 0.005 -0.137 -0.170 0.509* 1.000 Swimming 30 C 0.095 0.223 0.535* -0.023 -0.359 -0.100 0.303 0.218 1.000

25 b Table 2. Allometric relationships (y = ax ) between log transformed SVL and log transformed mean and maximum absolute (m • s-1) velocities for 26 neonate brown snakes (Storeria dekayi) during 3 modes of locomotion at 10, 20, and 30 C determined by power functions. Numbers in parenthesis represent standard errors of the parameter estimates for the intercepts (a) and slopes (b). No significant (p < 0.0167) relationships were detected for any mode at any temperature.

Absolute Average Velocities Absolute Maximum Velocities

Temperature Mode A b R2 a b R2 (C) 10 0.75 (0.18) -0.14 (0.08) 0.051 0.71 (0.14) -0.26 (0.06) 0.078 Concertina 20 0.79 (0.10) -0.13 (0.05) 0.046 0.76 (0.08) -0.14 (0.04) 0.056 30 0.80 (0.15) -0.21 (0.08) 0.086 0.80 (0.14) -0.42 (0.08) 0.102 10 0.81 (0.10) -0.02 (0.06) 0.007 0.88 (0.07) 0.02 (0.05) 0.011 Lateral 20 0.83 (0.12) -0.01 (0.10) 0.001 0.85 (0.10) 0.01 (0.08) 0.001 Undulation 30 0.88 (0.07) 0.03 (0.06) 0.014 0.86 (0.06) 0.02 (0.06) 0.004 10 0.84 (0.08) -0.01 (0.06) 0.001 0.87 (0.06) 0.02 (0.05) 0.006 Swimming 20 0.82 (0.06) 0.02 (0.06) 0.006 0.84 (0.05) 0.01 (0.06) 0.002 30 0.86 (0.08) 0.02 (0.09) 0.003 0.87 (0.05) 0.03 (0.06) 0.008

26 Table 3. Q10 values for maximum absolute velocities attained by various species of snake during swimming (s) and undulatory crawling (c). 1 = Stevenson et al. (1985), 2 = Heckrotte (1967), 3 = Finkler (1995), 4 = Finkler and Claussen (1999), 5 = present study.

Temperature Thamnophis Thamnophis Thamnophis Nerodia Regina Storeria

range (C) elegans1 sirtalis2 sirtalis3 sipedon4 septemvittata4 dekayi5 Swimming 10-20 1.86 - 2.2 2.3 2.5 1.69 20-30 1.16 - 1.4 1.3 1.1 1.20

Crawling 10-20 2.75 - 2.5 3.0 3.2 3.00 20-30 2.00 1.6 1.7 1.4 1.4 1.11

Sample 9s, 16c 33 16 10 10 26 sizes

27 Table 4. A summary of body length-relative velocities of concertina locomotion by limbless

squamates reported from this and previous studies. TL = total length, SVL = snout-vent length, WT = wide tunnel(s).

Size No. Species Range SVL • s-1 TL • s-1 Condition Source individuals (cm TL)

Storeria 0.103- 0.079- 0.6-cm 8.1-10.3 26 Present study dekayi 0.240 0.184 WT

Coluber 3,5,7-cm Jayne and Davis, 88-101 5 0.18-0.28 - constrictor WT 1991

Nerodia 8,10-cm 84-110 2 - 0.02-0.05 Jayne, 1986 fasciata WT

Nerodia 5,7.5,10- 100 1 - 0.04-0.11 Jayne, 1988 fasciata cm WT

Elaphe 5,7.5,10- 159 1 - 0.02-0.05 Jayne, 1988 obsoleta cm WT

Acrochordus 74 1 - 0.04-0.11Linoleum Jayne, 1986 javanicus Pegboard Rhineura 29 1 - 0.18-0.31 with 2.5,5 Jayne, 1986 floridana spacing Ophisaurus Gans and Gasc, 56.5-59.2 2 - 0.03-0.05 4-cm WT apodus 1990

28

Figure 1. The influence of temperature on absolute mean (A) and maximum (B) velocities and snout-vent length (SVL) relative mean (C) and maximum (D) velocities attained during terrestrial lateral undulation, terrestrial concertina, and swimming by 26 neonate Brown Snakes (Storeria dekayi). Error bars represent ± SE.

29

Figure 2. The influence of temperature on maximum snout-vent length (SVL) relative crawling speeds of 26 neonate Storeria dekayi (present study), 10 adult Nerodia sipedon (Finkler and Claussen 1999), 10 adult Regina septemvittata (Finkler and Claussen 1999), and 206 adult Natrix maura (Hailey and Davies 1986).

30

Figure 3. The influence of temperature on maximum snout-vent length (SVL) relative swimming speeds in snakes, 26 neonate Storeria dekayi (present study), 10 adult Nerodia sipedon and 10 adult Regina septemvittata (Finkler and Claussen 1999).

31

Figure 4. The relationship between body width relative to tunnel width and maximum snout- vent length relative speeds of concertina locomotion in 5 adult Coluber constrictor estimated from Jayne and Davis, 1991 (▲) and 26 neonate Storeria dekayi (●) estimated from the present study. Error bars represent ± one standard deviation.

32

Chapter 2:

Effects of temperature and perch diameter on arboreal locomotion in the snake Elaphe guttata

33 Introduction Locomotion is vitally important to many animals for accomplishing daily activities such as finding suitable habitats, searching for food, locating mates, and avoiding potential predators. Hence, traits that enhance locomotor performance, especially speed, are expected to be strongly influenced by natural selection (Irschick and Garland 2001; Kingsolver et al. 2001; Irschick 2003). Locomotor performance (e.g. speed and endurance) is considered to be a link between an animal’s morphology and fitness (Arnold 2003). Therefore, factors that influence performance will indirectly dictate reproductive success (Jayne and Bennett 1990a). One important factor that affects locomotion is the habitat being traversed. Many species, such as snakes, possess adaptations that enhance locomotion in one or more different habitats. The elongated, limbless body plan of snakes allows for the utilization of several different locomotor modes depending on habitat type (Gans 1986; Jayne 1986; Cundall 1987). Most frequently, snakes use lateral undulation when moving on terrestrial surfaces or during swimming (see Jayne 1986). Snakes can also move via concertina locomotion, which is used when snakes climb vertical surfaces or move through narrow passageways (Jayne 1988; Jayne and Davis 1991). Concertina is slower and more energetically expensive relative to terrestrial lateral undulation (Walton et al. 1990). Although several studies have examined the kinematics and performance of lateral undulation and concertina locomotion in snakes (Jayne 1985; Gans 1986; Jayne 1986, 1988; Jayne and Bennett 1990b; Finkler and Claussen 1999; Gerald and Claussen 2007), very few studies have assessed movements by snakes in arboreal environments (Mullin and Cooper 2002; Astley and Jayne 2007), despite the fact that many species alternate activity between terrestrial and arboreal environments or exhibit arboreal habits almost exclusively (Lillywhite and Henderson 1993; Jayne and Riley 2007). Arboreality in snakes has evolved independently in numerous lineages and is accompanied by adaptive morphological modifications (Lillywhite and Henderson 1993; Pizzato et al. 2007). Arboreal snakes tend to have attenuated bodies relative to body length (Guyer and Donnelly 1990; Pizzatto et al. 2007), which, presumably, improves locomotor performance on branches by reducing the amount of weight applied to a particular point on a branch. Moreover, this specialized body form enhances cantilevering for movements across gaps in arboreal environments (Lillywhite et al. 2000; Lin et al. 2003; Jayne and Riley 2007). An elongated body length relative to body thickness may also be important when snakes are supporting the body or

34 moving on only one branch. Longer snakes would be able to wrap more body loops around alternating sides of a perch, providing more support against gravity which is an extremely important force that must be accounted for when traveling in arboreal habitats (see Fig. 1). The formation of alternating body loops down the length of the body would provide better support, allowing snakes to grip the perch in various places to prevent listing if the snake’s center of mass moved lateral or below the center of the perch (Astley and Jayne 2007). However, maximizing the number of alternating loops on branches may constrain forward movement on that branch. There thus appears to be a trade-off between balance (in terms of number of alternating loops) and locomotor performance on a perch, and attainable speeds on branches are likely limited by the need for simultaneous balance. This trade-off between balance and maximum speed may be highly affected by a number of abiotic factors including the physical attributes (e.g. branch diameter, branch strength, protruding parts) and microclimate (e.g. ambient temperature, exposure to wind and precipitation) of branches being negotiated. Losos and Sinervo (1989) suggested that variation in leg length among Anolis lizards likely represents a trade-off between maximal speed and balance on thin perches, with longer-legged species moving faster on thicker perches but whose speed was reduced more on thinner perches compared to shorter-legged species. Overall, maximal speed appears to increase non-linearly with increased perch diameter in lizards (Losos and Sinervo 1989; Losos and Irschick 1996; Spezzano and Jayne 2004). No study has assessed the effects of perch diameter on locomotor performance in snakes. The thermal dependence of locomotor performance of ectotherms has been extensively studied. The typical patterns of temperature dependence of performance capacities are very similar in most ectotherms studied (Huey 1982; Hertz et al. 1983; Bennett 1990; Garland et al. 1991), with species tending to exhibit higher performance capabilities at higher temperatures up to some maximal level of performance at an optimal temperature, and then declining at very high temperatures. Fewer studies have examined the thermal dependence of limbless relative to limbed locomotor performance. Thermal sensitivities of limbless locomotion have been found to vary greatly depending on the mode of locomotion being used (Stevenson et al. 1985; Scribner and Weatherhead 1995; Finkler and Claussen 1999; Gerald and Claussen 2007). Gerald and Claussen (2007) found differences in thermal responses among terrestrial lateral undulation, swimming, and concertina locomotion in brown snakes (Storeria dekayi), which, potentially signifies the underlying physiological differences associated with muscle contraction and force

35 generation among modes. Unfortunately, no information is available for temperature effects on other modes of limbless locomotion, such as arboreal locomotion. The present study was designed to assess the influence of temperature on arboreal locomotion on different perch diameters by cornsnakes (Elaphe guttata). We measured maximum velocities and body posture of snakes crawling on horizontal perches that varied in diameter at different temperatures to answer the following questions: 1. How fast can snakes move on a perch and how do these velocities compare to velocities achieved via other locomotor modes (terrestrial lateral undulation, swimming, concertina)? 2. What influences do temperature and perch diameter have on arboreal locomotion and how does the thermal dependency of arboreal locomotion compare to those of other limbless locomotor modes? 3. Is there a trade-off between balance and velocity when snakes are moving on perches and how is this relationship influenced by perch diameter and temperature?

Materials and Methods Study Species The study species used was the cornsnake (Elaphe guttata), a North American “semi- arboreal” colubrid that inhabits a wide range of terrestrial habitats and is frequently observed climbing and basking in vegetation (Lillywhite et al. 2000; Ernst and Ernst 2003). Unfortunately, precise data on the use of arboreal microhabitats by E. guttata is not available. However, this species will use arboreal habitats to forage for fledgling birds, treefrogs, and lizards (Gibbons and Dorcas 2005), where maximizing speed is likely important for catching prey. This species has also been observed moving quickly up a tree and onto branches in response to a potential predator (G.W. Gerald, pers. obs.). Presumably, maximizing velocity of movements on perches should be selected for as that would increase this species’ chances of catching prey and escaping would-be predators. The use of a “semi-arboreal” species to assess arboreal limbless locomotion is more appropriate than using strictly arboreal or non-arboreal species since these species might display abnormally high or low arboreal performance, respectively, compared to most other snake species. Hence, a semi-arboreal species should show

36 an average ability to move in a three-dimensional environment, which should serve as a better comparison between limbless and limbed arboreal locomotion. Data were obtained from 15 sub-adult E. guttata. Sub-adults were used because of the difficulties in measuring locomotor velocities and with cooperation of adult E. guttata. It is very likely that differences in locomotor velocities between sub-adults and adults are due only to differences in body size. Gerald and Claussen (2007) found similar performance values relative to body size among various locomotor modes between neonate and adult snakes. To mitigate the effects of body size, all specimens included in the analyses were similar in body length (mean ± SE: 50.4 ± 0.8 cm; range: 45.6-57.5 cm snout-vent length [SVL]) and tail length (mean ± SE: 8.1 ± 0.3 cm; range: 6.9-10.3 cm). Though similar in body length, body mass was slightly variable among individuals (mean ± SE: 35.5 ± 3.1 g; range: 26.0-64.5 g). Snakes were acquired from a commercial supplier (Glades Herp Inc.; Bushnell, Florida) and kept individually in plastic containers 48 x 18 x 9 cm (L x W x H) within a custom-made rack. Snakes were provided with newspaper or paper towels for substrate and water ad libitum and housed in an environmental chamber at 25 ± 0.5°C with a 12:12 L:D photoperiod. Snakes were fed pre-killed mice (Mus musculus) of the appropriate size (i.e. the maximum width of the mouse was approximately equal to the maximum width of the snake) once per week and no trials were performed for 2 days following feeding. Animals were acclimated for 3-weeks before trials were conducted.

Arboreal Trials Arboreal locomotion was assessed using an artificial perch designed to manipulate perch diameter. Perches consisted of 1-m long PVC pipes wrapped in a rough-textured, burlap carpet substrate affixed to the periphery to provide purchase points for snakes to use for force generation and to prevent slipping. The burlap contained numerous projections (1.4 mm high) scattered randomly throughout the material (6-22 mm apart). Though the texture of perches can vary dramatically among different tree species, the burlap carpet created a texture similar to that of perches E. guttata has been observed using in nature (G.W. Gerald, pers. obs.). Perches were inserted horizontally into one of two metal bases positioned ca. 1.6-m off the ground. Bases were positioned directly across from each other so that two perches would come together to form a linear 2-m long perch. A large cushion was placed on the ground directly underneath the perch

37 to prevent injury when snakes fell. However, snakes were most often caught by hand when they slipped off the perch and rarely fell to the cushion. No snake was injured during this study. The Institutional Animal Care and Use Committee at Miami University approved all experiments (IACUC protocol 634). We assessed arboreal performance by video-taping (Sony DCR-TRV460 Digital8; New York, NY, USA) the dorsal view of all snakes moving at 10°C, 20°C, and 30°C and at three different perch diameters (3, 6, and 10 cm) in a repeated-measures experimental design. Individuals and perch diameters were randomized within each temperature and conducted in the following sequence: 20°C, 10°C, and 30°C. All individuals were acclimated to test temperatures a minimum of 2 h before velocities were measured. Snakes were stimulated to move continuously by lightly tapping the tail with a finger. All snakes were stimulated until three runs were completed in succession for each condition. Average and maximum absolute velocities (cm • s-1) and body length-relative velocities (number of body lengths per second; SVL • s-1) were determined for each run. Average velocities were calculated as the average of the three runs while maximum velocities were the fastest of the three runs. Additionally, we measured head-tail distances (defined as the linear distance between the tip of the snout and the tip of the tail), at the temporal midpoint of each run for each condition. The ratio of head-tail distance to total body length was used as an indirect quantitative measure of balance on a perch when snakes are moving, with higher ratios, which represent more elongated postures, indicative of less stability or balance (Fig. 1). The number of loops formed by snakes around either side of the perch was also determined for each run to verify the validity of the head-tail distance/total body length ratio for measuring balance and posture. Because the maximum number of loops snakes are capable of generating is constrained by the snake’s total body length, a ratio of number of loops/total length was determined. A more direct measure of balance was also obtained by recording the number of falls for each run for each individual.

Statistical Analyses We used repeated-measures two-way analyses of variance (RM-ANOVA) to assess the effects of temperature and perch diameter on (average and maximum) velocities and head-tail distance ratios. The Bonferroni multiple comparisons method was used to assess differences.

38 Q10 values, which represent the change in the rate of a process for every 10°C change in temperature, were determined to compare the thermal dependencies of various performance measures across conditions. Simple linear regressions were utilized to ascertain the relationships between maximum velocities and body length and between maximum velocities and head-tail distance ratios across conditions. Binary logistic regression models were used to examine the influence of temperature, perch diameter, and speed on the likelihood of falling off a perch to ascertain the potential trade-off between balance (falling frequency) and arboreal velocities. Data analyses were performed using Minitab (Minitab Inc., State College, Pennsylvania, U.S.A.) and SAS 9.1 (SAS Institute, Cary, North Caroline, U.S.A). All means are reported as ± SE.

Results Our analyses revealed that the four measures of velocity (average and maximum absolute velocities and average and maximum SVL-relative velocities) exhibited very similar relationships with the independent variables (perch diameter and temperature) assessed. Consequently, we provide the results for maximum absolute velocities only to avoid redundancies unless otherwise stated. To compare our results with those of other snake species, we used only SVL-relative maximum velocities since body size has a large influence on locomotor performance. As predicted, maximum arboreal velocities of E. guttata increased significantly with increasing experimental temperatures; repeated-measures two-factor ANOVA with temperature

and perch diameter as factors (F2, 134 = 148.3, P < 0.0001). Least squares mean tests indicated differences across all temperatures (p < 0.001; Fig. 2A). Perch diameter had no significant

influence on arboreal velocities (Same ANOVA; F2, 134 = 0.21, p = 0.81; Fig. 2A), nor did the

interaction between temperature and perch diameter (F4, 134 = 1.67; p = 0.16). Q10 values for

maximum absolute velocities were calculated for each perch diameter. Between 10-20°C, Q10 values were 2.77, 1.88, and 2.35, whereas those for 20-30°C were 1.44, 1.60, and 1.21 for perch diameters 3, 6, and 10-cm, respectively. Head-tail distance/total body length ratios were highly negatively correlated with the number of body loops formed on perches at all temperatures and perch diameters indicating that more body loops resulted in lower head-tail distance ratios (Table 1). Therefore, we used head-tail distance ratios to assess the relationships between performance and posture/balance in all

39 subsequent analyses. Head-tail distance ratios were significantly affected by temperature

(repeated-measures two-factor ANOVA; F2, 134 = 6.74, p = 0.002; Fig. 2B). Longer head-tail distance ratios and, hence, more elongated postures tended to be displayed by snakes while moving on perches at higher temperatures. This pattern was consistent at all diameters except for the largest diameter in which snakes maintained larger head-tail distances at the lowest experimental temperature (Fig. 2B). Although perch diameter alone had no significant effect on head-tail distances (Same ANOVA; F2, 134 = 1.08, p = 0.35), the interaction between temperature

and diameter was significant (F4, 134 = 4.99, p = 0.001; Fig. 2B) due to the increased head-tail distance ratios observed on the largest diameter branch at 10ºC. Neither body mass or total body length had a significant effect on arboreal velocities attained by cornsnakes at any temperature or on any perch diameter (Least-squares regressions: p > 0.15). Larger head-tail distance ratios resulted in significantly faster velocities during all conditions except when moving on the largest diameter at the lowest experimental temperature (Fig. 3). Average velocities tended to show a larger increase with head-tail distances (i.e. higher slopes) at higher experimental temperatures regardless of perch diameter (Fig. 3). Balance on a perch while moving tended to improve at higher temperatures and on smaller diameter perches (Fig. 4). A binary logistic regression with falling as the dependent variable and temperature and perch diameter as independent factors indicated that snakes were more likely to fall at lower temperatures (z = -4.58, p < 0.001) and off of larger diameter perches (z = 4.71, p < 0.001). Though not significant, both mean arboreal velocities and head-tail distance ratios tended to be positively related with the number of falls at all temperatures and perch diameters (p > 0.15).

Discussion The assessment of arboreal locomotion on horizontal cylinders represents a simplified arboreal situation due to the fact that the configuration of perches in nature most often contains projections of various kinds (e.g. smaller branches, leaves, protruding leaf buds), is rarely straight, and is often arranged at various inclines. However, this experimental set-up allows for the quantification of movement on perches without these confounding factors. This study is the second to quantify arboreal locomotor performance by snakes. Astley and Jayne (2007) examined the kinematic and performance variables of four E. guttata moving on

40 different perch diameters and inclines. They found that, on their artificial perches, snakes used a mode of locomotion similar to concertina due to the alternating left and right bends, the stop- and-go momentum changes, and the points of static contact made by some part of the body that is similar to concertina movement through tunnels. Astley and Jayne (2007) described arboreal locomotion as a combination of lateral undulation and concertina with movement being more similar to concertina for the reasons just mentioned. The present study found very little periodic stopping or regions of static contact unless snakes needed to increase their grip when they began to slip off the perch. Although we agree that arboreal locomotion is most likely a blending together of lateral undulation and concertina, the movement patterns we observed were more similar to lateral undulation. Snakes were generating thrust by pushing against the small projections in the burlap covering. Snakes moved continuously past the projections and formed body loops by alternating sides of the perch on which the body moved (Fig. 1). However, snakes did use lateral movements to form loops for the purpose of gripping the perch to maintain balance. The reason for the differences in findings between the present study and those from Astley and Jayne (2007) are likely due to the material used to cover the artificial perches. Astley and Jayne (2007) covered their perch with strips of duct tape, creating a grid of ridges approximately 0.1 mm high. This grid was used to assess arboreal movement in adult snakes averaging 125 cm in total length, 2.1 cm in body width, and 421 g in mass. The present study used perches covered with burlap that contained protrusions 1.4 mm high for sub-adult snakes averaging 58.5 cm in total length, 1.4 cm in body width, and 35 g in mass. Consequently, Astley and Jayne (2007) used smoother perches and larger snakes than the present study, which used rough-textured burlap to simulate bark that E. guttata has been observed using in nature (G.W. Gerald, pers. obs.). The presence of purchase points on our perches likely shifted the balance between lateral undulation and concertina more towards lateral undulation. And, since lateral undulation is less energetically expensive than concertina (Walton et al. 1990), snakes should utilize the projections on a rough-textured perch to generate forward thrust rather than generating points of static contact. Future studies should closely examine this potential trade-off between undulatory and concertina movements on different perch diameters. The velocities attained by E. guttata on a horizontal perch in this study are much slower than those of terrestrial lateral undulation and swimming and somewhat slower than concertina

41 velocities recorded in E. guttata (G.W. Gerald, unpublished data) and other species across the same experimental temperatures (Hailey and Davies 1986; Finkler and Claussen 1999; Gerald and Claussen 2007). Our data show that locomotor velocities increased with temperature across all perch diameters. This is not surprising since temperature is an instrumental factor that can both severely limit and enhance the performance capacities of most ectothermic animals (Reynolds and Casterlin 1979; Huey and Kingsolver 1989; Bennett 1990). However, the thermal sensitivities of arboreal velocities differed among the three perch diameters examined. Although

Q10 values were similar between 20-30°C for all diameters (1.2-1.6), values between 10-20°C

differed among the diameters. The lowest Q10 value (1.88) was observed for the intermediate diameter and the highest value (2.77) was observed for the smallest diameter. It is difficult to determine the reason for the disparity among diameters, though it may reflect actual differences in the physiological requirements needed to produce force while simultaneously supporting the body against gravity on different diameters. In general, arboreal locomotion appears to display similar thermal dependencies to other modes of limbless locomotion measured in other species

based on Q10 values (Hailey and Davies 1986; Finkler and Claussen 1999; Gerald and Claussen 2007). As previously mentioned, it has been suggested that arboreal locomotion involves a combination of undulatory and concertina movements for force generation (Gans 1974;

Lillywhite and Henderson 1993; Astley and Jayne 2007). It is worth noting that Q10 values of arboreal locomotion on the smallest (3-cm) diameter perch is most similar to the average values of terrestrial undulation for three other snake species (Nerodia sipedon, Regina septemvittata, and Storeria dekayi); whereas, Q10 values on the largest (10-cm) diameter perch closely resemble those of concertina locomotion in Storeria dekayi (Hailey and Davies 1986; Finkler and Claussen 1999; Gerald and Claussen 2007). This further suggests that movement on a 3-cm and 10-cm perch requires patterns of movement that are more similar to lateral undulation and concertina, respectively, in terms of the muscular properties necessary for balancing and movement. Body posture during locomotion on perches was affected by temperature with larger head-tail distance ratios (i.e. more elongated postures) being displayed at higher temperatures. Snakes were better able to produce faster velocities at higher temperatures, presumably due to the increase in muscle efficiency associated with higher, more optimum temperatures (Bennett 1990). This suggests a trade-off between a more looped posture (i.e. smaller head-tail distance

42 ratios) and maximum velocity. Because a higher percentage of the epaxial musculature can be used to grip a perch, a more looped posture is likely to increase balance while hindering forward velocity on a perch to prevent falling relative to a more elongated posture. This relationship varies with temperature similarly across diameters with more looped postures inhibiting attainable speeds more so at higher temperatures (Fig. 3). Overall, snakes completely fell off of perches in only 15% of the trials during this study. Although we found no significant relationships between head-tail distance ratios or velocity and falling, snakes frequently slowed or paused completely when postures were more elongated in order to re-situate themselves to prevent slipping off the perch. We did, however, detect a significant effect of temperature and perch diameter on falling, with snakes falling more often at lower temperatures and while moving on larger diameter perches (Fig. 4). It is interesting to note that the trend of snakes falling off of larger diameter perches can only continue to a point where the curvature of the perch would become more flat, decreasing the importance of balance. At lower temperatures, the reduction in the efficiency (i.e. rate and force) of muscle contractions likely causes snakes to fall more often. Therefore, a reduction in temperature not only causes a decrease in attainable locomotor velocities in arboreal situations, but also increases the likelihood of falling off a perch (i.e. decreased ability to grip a perch). This makes temperature a significant abiotic factor that could potentially limit the use of arboreal environments by many snake species. The large influence of temperature on arboreal locomotion, along with the problems associated with thermoregulation above ground, such as increased heat loss and exposure to wind (see Henderson and Lillywhite 1993), suggests that temperature could be a critical factor in determining the movement ecology and microhabitat selection of arboreal snakes. Moreover, temperature may be one of the main factors responsible for the limited occurrence of arboreal species in temperate compared to tropical environments where arboreal species can constitute over 50% of the snake fauna compared to less than 10% in temperate areas (Fitch 1982; Shine 1983). Snakes that frequently exhibit arboreal habits typically possess slender bodies relative to length and are characterized by greater relative length of epaxial muscles (Jayne 1982; Pizzato et al. 2007). Attenuated bodies also facilitate the crossing of discontinuous gaps between perches (Jayne and Riley 2007). We mitigated the potential influence of body size and shape on movement by using similarly-sized individuals of one species. An interspecific comparison of

43 arboreal performance would be helpful in quantifying the effects of morphology that have been widely cited as influencing arboreal locomotor abilities (Guyer and Donnelly 1990; Henderson and Lillywhite 1993, Pizzato et al. 2007). The limbless body plan appears to have several advantages over limbed animals (e.g. lizards) when moving through a network of branches. First, the elongated body of a snake can spread out mass more easily, permitting movement on lighter branches without weighing them down, thereby allowing for a relatively large mass to move on smaller diameter branches. We found no significant effect of perch diameter on arboreal speeds in E. guttata. Astley and Jayne (2007) found that E. guttata displayed reduced arboreal speeds on larger perch diameters when moving on relatively smooth perches. The present findings, along with those of Astley and Jayne (2007), differ from previous studies on lizards that indicate that sprint speeds decrease sharply with decreasing diameter (Losos and Sinervo 1989; Losos and Irschick 1996). In lizards, decreasing diameter has substantial effects on multiple postural and kinematic variables associated with the hindlimb used to improve balance, which essentially hinders maximal sprint speeds (Higham and Jayne 2004; Spezzano and Jayne 2004). Second, snakes are likely to be less affected by obstructions in an arboreal habitat that could interrupt locomotion due to the fact that there are no limbs to bump into obstacles and that the entire ventral surface (if moving on only one perch) is in complete contact with the arboreal substrate. In lizards, the clearance between the ventral surface of the body and the surface of the perch increases with decreasing diameter (Spezzano and Jayne 2004), which, increases the likelihood of obstacles from adjacent branches interfering with movement on smaller perches. Third, there is a higher tendency for longer-limbed lizards moving on narrow perches to tip to one side and potentially fall to the ground relative to shorter- limbed species. It has been shown that shorter limbs have evolved multiple times within some lineages of lizards (e.g. Anolis) that frequently utilize narrower perches (Moermond 1979; Losos 1990). For these reasons, it is likely snakes have an advantage over lizards relative to size when moving on narrow perches. However, more data on locomotor performance, kinematics, and energetics are needed to confirm this hypothesis. The biomechanics of arboreal locomotion differs from that of other locomotor modes because the need for simultaneously balance during movement is more important. This leads to a potential trade-off between speed and balance where it appears that selection cannot simultaneously maximize both performance measures. Chameleons (Chamaeleonidae) are an

44 excellent example of a specialized arboreal lizard that has maximized balance by evolving a very slow walking gait (Peterson 1984). Frustratingly, there are virtually no published data on arboreal speeds of snakes. It is widely noted that some lineages of arboreal snakes (e.g. boids, some pit-vipers) are heavy-bodied and move slowly over a network of branches to ambush potential prey. These snakes possess specializations, such as partially prehensile tails that allow for maximal stability on a branch. Other lineages of arboreal snakes (some colubrids, some elapids) are more slender-bodied, and considered to move very quickly on branches while actively foraging for prey (Pough et al. 2004). These differences are likely related to whether or not snakes constrict their prey since certain morphological features (e.g. short vertebrae, robust axial musculature and short tendons) that facilitate prey constriction appear to limit speed (Jayne 1982). Future comparative data on maximal speeds and balance would be very helpful in evaluating the relationship between arboreal locomotor performance and foraging mode in snakes. Finally, the thermal dependency of arboreal locomotion, along with morphological characteristics associated with different feeding strategies (e.g. constriction, venom), appear to form a selective regime that has favored or limited the evolution of arboreal habits in different lineages of snakes. Presumably, the use of arboreal habitats resulted in several internal and external morphological modifications that simultaneously optimized locomotor abilities and reproductive output (Lillywhite and Henderson 1993). Pizzatto et al. (2007) found that morphological adaptations to arboreal locomotion include modified reproductive morphology (e.g. asymmetry in ovarian position) and reduced relative clutch sizes in snakes. Phylogenetic constraints on body shape and selection for speed versus balance in three-dimensional habitats are likely to strongly determine morphological adaptations needed to enhance movement while optimizing reproductive success.

Acknowledgments This research was supported by the Department of Zoology, Miami University. We wish to thank Barry Landrum and the staff of the Instrumentation Laboratory at Miami University for design and construction of the artificial perches. We would also like to thank Lisette Torres, Andrea Collins, the Ecology and Evolution Group at Miami University, and three anonymous reviewers for providing useful comments that improved this manuscript.

45

Literature Cited Arnold S.J. 2003. Performance surfaces and adaptive landscapes. Integrative and Comparative Biology 43:367-375. Astley H.C. and B.C. Jayne. 2007. Effects of perch diameter and incline on the kinematics, performance and modes of arboreal locomotion of corn snakes (Elaphe guttata). Journal of Experimental Biology 210:3862-3872. Bennett A.F. 1990. Thermal dependence of locomotor capacity. American Journal of Physiology 259:R253-R258. Cundall D. 1987. Functional morphology. In: Snakes: ecology and evolutionary biology (Ed. by Seigel R.A., J.T. Collins, S.S. Novak). pp. 106-142. New York, NY: McGraw Hill. Ernst C.H. and E.M. Ernst. 2003. Snakes of the United States and Canada. Washington D.C.: Smithsonian Books. 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. 1982. Resources of a snake community in prairie-woodland habitat of northeastern Kansas. In: Herpetological communities (Ed. by Scott N.J.). pp. 83-97. U.S. Fish & Wildlife Service, Wildlife Research Rep. 13. Gans C. 1974. Biomechanics: an approach to vertebrate biology. Ann Arbor, Michigan: University of Michigan Press. Gans C. 1986. Locomotion of limbless vertebrates: pattern and evolution. Herpetologica 42:33-46. Garland T. Jr., R.B. Huey, and A.F. Bennett. 1991. Phylogeny and coadaptation of thermal physiology in lizards—a reanalysis. Evolution 45:1969-1975. Gerald G.W. and D.L. Claussen. 2007. Thermal dependencies of different modes of locomotion in neonate Brown Snakes, Storeria dekayi. Copeia 2007:577-585. Gibbons W. and M. Dorcas. 2005. Snakes of the Southeast. Athens, Georgia: University of Georgia Press.

46 Guyer C. and M.S. Donnelly. 1990. Length-mass relationships among an assemblage of tropical snakes in Costa Rica. Journal of Tropical Ecology 6:65-76. Hertz P.E., R.B. Huey, and E. Nevo. 1983. Homage to Santa Anita: thermal sensitivity of sprint performance in agamid lizards. Evolution 37:1075-1084. Higham T.E. and B.C. Jayne. 2004. Locomotion of lizards on inclines and perches: hindlimb kinematics of an arboreal specialist and a terrestrial generalist. Journal of Experimental Biology 207:233-248. Huey R.B. 1982. Temperature, physiology, and the ecology of reptiles. In: Biology of the reptilia, vol. 12 (Ed. by Gans C. and F.H. Pough). pp. 25-93. London: Academic Press. Huey R.B. and J.G. Kingsolver. 1989. Evolution of thermal sensitivity of ectotherm performance. Trends in Ecology and Evolution 4:131-135. Irschick D.J. 2003. Studying performance in nature: implications for fitness variation within populations. Integrative and Comparative Biology 43:396-407. Irschick D.J. and T. Garland Jr. 2001. Integrating function and ecology in studies of adaptation: Investigations of locomotor capacity as a model system. Annual Review of Ecology and Systematics 32:367-396. Jayne B.C. 1982. Comparative morphology of the semispinalis-spinalis muscle of snakes and correlations with locomotion and constriction. Journal of Morphology 172:83-96. Jayne B.C. 1985. Swimming in constricting (Elaphe g. guttata) and non-constricting (Nerodia fasciata pictiventris) colubrid snakes. Copeia 1985:195-208. Jayne B.C. 1986. Kinematics of terrestrial snake locomotion. Copeia 1986:915-927. Jayne B.C. 1988. Muscular mechanisms of snake locomotion: an electromyographic study of the sidewinding and concertina modes of Crotalus cerastes, Nerodia fasciata and Elaphe obsoleta. Journal of Experimental Biology 140:1-33. Jayne B.C and A.F. Bennett. 1990a. Selection on locomotor performance capacity in a natural population of garter snakes. Evolution 44:1204-1229. Jayne B.C and A.F. Bennett. 1990b. Scaling of speed and endurance in garter snakes: a comparison of cross-sectional and longitudinal allometries. Journal of Zoology (London) 220:257-277.

47 Jayne B.C. and J.D. Davis. 1991. Kinematics and performance capacity for the concertina locomotion of a snake (Coluber constrictor). Journal of Experimental Biology 156:539-556. Jayne B.C. and M.A. Riley. 2007. Scaling of the axial morphology and gap-bridging ability of the brown tree snake, Boiga irregularis. Journal of Experimental Biology 210:1148-1160. Kingsolver J.G., H.E. Hoekstra, J.M. Hoekstra, D. Berrigan, S.N. Vignieri, C.E. Hill, A. Hoang, P. Gilbert, and P. Beerli. 2001. The strength of phenotypic selection in natural populations. American Naturalist 157:245-261. Lillywhite H.B. and R.W. Henderson. 1993. Behavioral and functional ecology of arboreal snakes. In: Snakes: ecology and behavior (Ed. by Seigel R.A. and J.T. Collins). pp. 1-48. New York, McGraw Hill. Lillywhite H.B., J.R. LaFrentz, Y.C. Lin, and M.C. Tu. 2000. The cantilever abilities of snakes. Journal of Herpetology 34:523-528. Lin Y.C., J.C. Hwang, and M.C. Tu. 2003. Does the saccular lung affect the cantilever ability of snakes? Herpetologica 59:52-57. Losos J.B. 1990. The evolution of form and function: morphology and locomotor performance in West Indian Anolis lizards. Evolution 44:1189-1203. Losos J.B. and D.J. Irschick. 1996. The effect of perch diameter on escape behavior of Anolis lizards: lab predictions and field tests. Animal Behaviour 51:593-602. Losos J.B. and B. Sinervo. 1989. The effects of morphology and perch diameter on sprint performance of Anolis lizards. Journal of Experimental Biology 145:23-30. Moermond T. 1979. Habitat constraints on the behavior, morphology, and community structure of Anolis lizards. Ecology 60:152-164. Mullin S.J. and R.J. Cooper. 2002. Barking up the wrong tree: climbing performance of rat snakes and its implications for depredation of avian nests. Canadian Journal of Zoology 80:591-595. Peterson J.A. 1984. The locomotion of Chamaeleo (Reptilia: Sauria) with particular reference to the forelimb. Journal of Zoology (London) 202:1-42. Pizzatto L., S.M. Almeida-Santos, and R. Shine. 2007. Life-history adaptations to arboreality in snakes. Ecology 88:359-366.

48 Pough F.H., R.M. Andrews, J.E. Cadle, M.L. Crump, A.H. Savitsky, and K.D. Wells. 2004. Herpetology, 3rd edition. Upper Saddle River, NJ: Prentice Hall. Reynolds W.W. and M.E. Casterlin. 1979. Behavioral thermoregulation and the "final preferendum" paradigm. American Zoologist 19:211-224. Scribner S.J. and P.J. Weatherhead. 1995. Locomotion and antipredator behaviour in three species of semi-aquatic snakes. Canadian Journal of Zoology 73:321-329. Shine R. 1983. Arboreality in snakes: ecology of the Australian elapid genus Hoplocephalus. Copeia 1983:198-205. Spezzano L.C. Jr. and B.C. Jayne. 2004. The effects of surface diameter and incline on the hindlimb kinematics of an arboreal lizard (Anolis sagrei). Journal of Experimental Biology 207:2115-2131. Stevenson R.D., C.R. Peterson, and J.S. Tsuji. 1985. The thermal dependence of locomotion, tongue flicking, digestion, and oxygen consumption in the wandering garter snake. Physiological Zoology 58:46-57. Walton M., B.C. Jayne, and A.F. Bennett. 1990. The energetic cost of limbless locomotion. Science 249:524-527.

49 Table 1. Parameters of least-squares regression models showing the relationship between number of body loops formed during movement/total body length (predictor) and mean head-tail distances/total body length (response) at three temperatures (10º, 20º, and 30º C) at three different perch diameters (3, 6, and 10 cm).

Condition 95% Confidence Slope R2 P (Temp/Diameter) Limits (±)

10ºC / 3-cm -3.75 0.736 0.89 < 0.0001

10ºC / 6-cm -3.21 0.704 0.86 < 0.0001

10ºC / 10-cm -2.82 0.838 0.78 < 0.0001

20ºC / 3-cm -3.51 0.944 0.81 < 0.0001

20ºC / 6-cm -2.56 0.588 0.85 < 0.0001

20ºC / 10-cm -2.45 0.696 0.79 < 0.0001

30ºC / 3-cm -3.47 0.874 0.83 < 0.0001

30ºC / 6-cm -2.55 0.516 0.88 < 0.0001

30ºC / 10-cm -3.18 0.648 0.88 < 0.0001

50

Figure 1. Diagram showing a lateral view of differences in head-tail distances (arrows) of snakes moving on a horizontal perch. Shorter head-tail distances (a) represent snakes looped around either side of the perch, a body position that provides more balance when moving. Larger head- tail distances (b) denote a more elongated body position that provides less balance when moving.

51

Figure 2. Effect of perch diameter and temperature on (a) maximum speed and (b) head-tail distances relative to body length during arboreal locomotion by cornsnakes (Elaphe guttata). Bars represent ± 1 SE.

52

Figure 3. The relationship between mean speed and head-tail length relative to total body length, which is a measure of body posture during arboreal locomotion by Elaphe guttata on 10-cm (a), 6-cm (b), and 3-cm (b) diameter perches at 10ºC, 20ºC, and 30ºC. Regression lines represent the relationship at each temperature with dashed lines representing 30ºC, dotted lines representing 20ºC, and solid lines representing 10ºC.

53

Figure 4. Proportion of arboreal locomotor trials in which individual cornsnakes (Elaphe guttata) fell off of a perch at different temperatures and perch diameters.

54

Chapter 3:

Relationships between morphology, microhabitat use, and limbless locomotion

55 Introduction Natural selection should favor traits in organisms enhancing their ability to survive and successfully reproduce. Effective locomotor performance is one trait that, under most circumstances, should be strongly selected because individuals that are faster, have extended endurance, or can maneuver better are more likely to escape predators, find food, locate potential mates, and defend territories. Selection pressures should act directly on the morphological and physiological traits governing whole-animal performance (Arnold 1983). The main selection pressure shaping these underlying traits determining locomotor abilities is an individual’s patterns of habitat usage (Garland and Losos 1994). Since different combinations of morphological and physiological parameters could result in differential performance abilities throughout a range of ecological situations, one would expect to see strong relationships among morphology, locomotor performance, and structure of habitats most frequently used (Pough 1989; Losos 1990; Garland and Losos 1994; Irschick 2002; 2003; Irschick et al. 2005). Morphological modifications are found in animals that utilize well-defined microhabitats to optimize locomotion in that particular microhabitat. For example, in arboreal Anolis lizards, species with longer limbs are usually faster runners and can jump greater distances, and are found more often on thicker branches compared to species with shorter limbs (Losos and Sinervo 1989; Losos and Irschick 1996; Mattingly and Jayne 2004). Kohlsdorf et al. (2001) found that although species of Tropidurus lizards that inhabited sand dunes, rock outcroppings, and semiarid grasslands were very similar in body size and shape, they possessed very different performance capabilities strongly associated with habitat preference. These sibling species exhibited rapid divergence in performance (likely due to physiological differences) in response to selective pressures optimizing movement on frequently-used substrates (Kohlsdorf et al. 2004). If interspecific performance variation is guided by habitat use variation, then variation in performance should be predictable across a gradient of habitat types because species specializing in one particular habitat should possess adaptations enhancing their locomotor capabilities there. For example, desert-dwelling lizards in the genus Uma possess toe fringes that prevent the feet from sinking into the sand (Irschick and Jayne 1998). Similarly, arboreal species should be superior climbers compared to a terrestrial species adapted to move more efficiently on terrestrial

56 substrates. Although these associations have been noted in lizards, the relationship between locomotor capacity, performance, and habitat use by snakes is poorly understood. It might be expected that variation in performance by snakes will be substantially less than those of lizards because of morphological constraints resulting from their similar condition of limblessness. As a consequence, snakes have evolved a variety of locomotor techniques associated with the substrate or medium (Gans 1986). Snakes typically move via lateral undulation when traveling on land or swimming in water (Jayne 1985; Cundall 1987). When climbing vertical surfaces or traversing narrow passageways, snakes can utilize a slow, energetically expensive mode called concertina that involves an alternation of stop-and-go changes in momentum (Jayne 1988; Walton et al. 1990). Snakes also are capable of arboreal locomotion, which is a combination of lateral undulation and concertina movements combined with the need for simultaneous balance (Astley and Jayne 2007; Gerald et al. 2008). Much less common modes, such as sidewinding, rectilinear, slide-pushing, and thrust creeping, are used by relatively few species and only in certain specialized ecological situations. Differences in the biomechanics of these various modes of limbless locomotion likely means that trade-offs in performance abilities exist within and among species across these modes. For example, adaptations that enhance movement by snakes in terrestrial situations (i.e. lateral undulation) likely conflict with adaptations that enhance movement in water (Jayne 1985; Cundall 1987). If selection cannot simultaneously optimize performance in more than one habitat, one would expect animals to perform best in their most-frequently used habitat, and perform poorly in rarely-used habitats compared to other species (Losos 1990; Losos et al. 1993; Shine et al. 2003). Studies on snakes have mostly compared the performance characteristics of terrestrial lateral undulation with that of swimming. Shine and Shetty (2001) found that aquatic and terrestrial speeds of marine and terrestrial snakes (Elapidae) mirrored the use of those habitats and that snakes that performed well during one mode performed more poorly in the other (i.e. exhibited a trade-off). The aquatic snake Natrix maura is a faster swimmer but moved more slowly on land compared to the semi-aquatic Natrix natrix (Isaac and Gregory 2007). Bonnet et al. (2005) found that variation in climbing ability (a typical reef cliff) of sea-snakes reflected how frequently each species and sex climbed in nature.

57 Only one previous study has examined the influence of habitat use on the performance of multiple modes of locomotion. Aubret and Shine (2008) compared terrestrial lateral undulation, swimming, and arboreal performance in tiger snakes (Notechis scutatus) and found that individuals did better during that mode if they had been previously exposed to that habitat. However, this study did not examine interspecific differences in performance across various modes in relation to habitat preferences. No study has examined the relationships among interspecific variation in habitat use, morphology, and variation in performance across multiple modes of limbless locomotion. Moreover, no study has assessed the potential presence of trade- offs across multiple modes of locomotion. In the present study, I examined the relationships among habitat use, morphology, and locomotor performance in five species of colubrid snakes that vary in patterns of basic habitat usage. I assessed performance during four distinct modes of limbless locomotion (lateral undulation, concertina, arboreal, and swimming) to test the potential links between habitat use and performance. I hypothesized that snake species would exhibit faster velocities and better stamina during modes of locomotion used most often in nature. Second, interspecific variation in performance should be related to interspecific variation in morphology, with larger snakes performing relatively better during all modes. Lastly, adaptations enabling optimal performance during one mode will be incompatible with adaptations increasing performance during other modes. Hence, trade-offs should be detected across the various locomotor modes with the direction of the trade-off reflecting patterns of traditional habitat use both within and among species.

Materials and Methods Study Species The five North American snake species used in this study vary in their patterns of habitat use (Wright and Wright 1957; Conant and Collins 1998; Ernst and Ernst 2003). Three of the species (Elaphe alleghaniensis, E. guttata, Pituophis catenifer) belong to the family Colubridae, and hence, are more closely related to each other than the other two species (Nerodia sipedon and Thamnophis sauritus), which belong to the family Natricidae (Lawson et al. 2005). Four of the five species (except P. catenifer) can generally be found throughout the eastern United States in either continuous and/or disjunct populations.

58 The yellow ratsnake (Elaphe alleghaniensis quadrivittata, Holbrook, 1836) is found in a variety of wooded habitats, but is primarily a climber that forages for squirrels, birds, and bird eggs and basks high up (up to 15 m) in a number of tree species (Wright and Wright 1957; Burbrink 2001; Ernst and Ernst 2003). This species has been described on numerous occasions as either arboreal or semi-arboreal (Wright and Wright 1957; Tenant 1997; Conant and Collins 1998), though, to my knowledge, no study has actually quantified how much time they spend displaying arboreal vs. terrestrial “habits.” The congeneric cornsnake (Elaphe guttata, Linnaeus, 1766) is a denizen of many types of terrestrial habitats, though they are mostly associated with open-canopy areas (e.g. pine barrens, fire-maintained, around buildings). Although this species is, for the most part, considered terrestrial, it has been reported to be an excellent climber that will bask in tree cavities and forage for birds and bird eggs (Conant and Collins 1998; Ernst and Ernst 2003) and frequently will climb trees to escape a potential predator (Wright and Wright 1957; G.W. Gerald, pers. obs.). Seemingly, it is not uncommon for this species (especially newborns) to be found in or near bodies of water as they also have been described as being “good swimmers” (Ernst and Ernst 2003). Although sometimes referred to as semi-arboreal (Lillywhite et al. 2000, Ernst and Ernst 2003), its use of multiple habitats would suggest that E. guttata should be considered a habitat generalist throughout most of its range. No study has quantified the amount of time E. guttata spends displaying terrestrial versus arboreal versus aquatic behaviors. The bullsnake (Pituophis catenifer sayi, Schlegel, 1837) occupies a large variety of terrestrial habitats throughout its extensive range (from southwestern Canada to northern Mexico; Conant and Collins 1998). Ernst and Ernst (2003) described this species as an “accomplished burrower” that will go underground to escape extreme temperatures or to forage for burrowing mammals. Bullsnakes likely spend most of their time underground foraging. The conspecific gopher snake (Pituophis catenifer catenifer) has been reported to spend as much as 90% of its time underground (Rodriguez-Robles 2003) and the congeneric pinesnake (Pituophis melanoleucus) has been shown to spend approximately 78% of its time underground (Gerald et al. 2006). Although mostly a terrestrial /fossorial species, Pituophis c. sayi has been reported to occasionally climb (Marr 1985) and swim (Howitz 1986). The northern watersnake (Nerodia sipedon, Linnaeus, 1758) is considered a semi-aquatic species that can be found in or near almost any type of freshwater habitat throughout its

59 geographical range and has been described many times as an excellent swimmer (Conant and Collins 1998; Finkler and Claussen 1999; Ernst and Ernst 2003). It is interesting to note that N. sipedon spends much of its time basking on vegetation overhanging the water, which allows for quick escape into the water if a predator approaches (Wright and Wright 1957; Brown and Weatherhead 2000). Ribbon snakes (Thamnophis sauritus) differ morphologically from the previously mentioned species. Ribbon snakes have long, thin bodies and an extremely long tail (up to 38.8% of the total body length), presumably an adaptation to enhance arboreal locomotor abilities (Rossman et al. 1996). Throughout its range, this species is strongly associated with freshwater habitats (Wright and Wright 1957) and seldom wanders far from water (Conant and Collins 1998). It is most often found on the banks of waterways, where it forages (mostly on fish and amphibians) and basks in low-lying vegetation (Carpenter 1952; Wright and Wright 1957; Ernst and Ernst 2003). Conant and Collins (1998) report that this species is both semi-aquatic and semi-arboreal, however its morphological features would suggest that it is primarily adapted to movement through arboreal habitats.

Husbandry Data on morphology and locomotor performance were gathered from 12 individuals from each of the five previously described species (n = 12/species). Since body size can have a large influence on locomotor performance (see Jayne and Bennett 1990; Wisco et al. 1997), I used individuals across different species that were relatively similar in body length (Table 1). However, because species differ in body shape, interspecific variation in body mass was quite large (Table 1). I chose to use snakes that were more similar in body length rather than mass because a snake’s length is more likely to be a more important determinant of limbless locomotor performance across various modes compared to mass (B.C. Jayne, pers. comm.). It has been suggested that longer snakes relative to mass can use more push points in a given area during most modes of locomotion (Jayne 1985; 1986; Cundall 1987; Gerald et al. 2008). Individuals of all species used in this study were acquired from either a commercial supplier (Glades Herp Inc.: Bushnell, FL, USA) or private collectors except for N. sipedon. All individual N. sipedon were hand collected from Collins Creek in Butler Co., OH, USA (Gerald and Miskell 2007). All snakes were housed in plastic containers (54 x 21 x 9 cm) within a

60 custom-made rack system and provided newspaper or paper towels for bedding and a small water dish at all times. Snakes were kept in an environmental chamber (25 ± 0.5 °C) set to a 12:12 L:D photoperiod. All snakes were fed approximately the same amount of food relative to body mass each week. Three species (E. guttata, E. alleghaniensis, P. catenifer) were fed pre-killed mice (Mus musculus) of the appropriate size (width of the mouse was approximately equal to the maximum width of the snake) once per week. Thamnophis sauritus were fed live fathead minnows (Pimethales promelas) every other day (two fish per individual per feeding) and N. sipedon were fed live goldfish (Carassius auratus) every other day by placing live or dead fish into their water bowls. Because N. sipedon were larger than T. sauritus, they received 4-5 fish per feeding. No trials were conducted 2 days and 1 day following feeding for mouse-eating and fish-eating snakes, respectively. All individuals were held in captivity (i.e. acclimated) for at least three weeks prior to the start of experimental trials.

Morphological measurements Prior to conducting locomotor trials, morphological data were collected for each individual to examine the effect of body size and shape on performance capabilities. Body mass was measured to the nearest 0.1 g and snout-vent length (SVL) and tail length were measured to the nearest 0.1 cm. Cross-sectional area was calculated using measurements of body width and height, while a measure of body condition or shape was determined by dividing total body length by mass. It has been suggested that the number of vertebrae can influence locomotor abilities of different modes of limbless locomotion because vertebrae are important sites for the attachment of epaxial musculature (Jayne 1986; B.C. Jayne, pers. comm.). Therefore, the number of vertebrae was ascertained by counting the number of pre-caudal ventral scales (Mosauer 1935; Auffenberg 1962; Cundall 1987; Arnold and Bennett 1988).

Microhabitat use Before all locomotor trials were performed, the use of arboreal, terrestrial, and aquatic microhabitats was examined for each species to verify that published qualitative accounts of habitat preferences truly reflected preferences of the specimens used in the present study. Each individual was placed in a Neodesha cage (57 x 27 x 26 cm) containing a 6-cm diameter perch wrapped in burlap carpet used to prevent slipping and to facilitate movement (Gerald et al.

61 2008). The perch was positioned horizontally approximately 18 cm above the substrate. The cage also contained a large water dish (24 x 20 cm x 5 cm) placed on the substrate, which consisted of terrarium carpet. The cage was placed in an environmental chamber set at 25 ± 0.5 °C with a 12:12 photoperiod. Although the chamber was lighted with fluorescent bulbs, red incandescent bulbs placed approximately 1 m from the cage were used to illuminate the cage during the “dark” hours for videotaping. Each individual was videotaped for three randomly selected hrs during a 24 hr period. Two of the three hours were selected during the “light” hours while one hour was videotaped during the “dark” hours. Observations of microhabitat use were recorded using a Videolabs Flexcam (Videolabs, Inc.: Minneapolis, MN, USA) connected to a Panasonic VHS system. The amount of time spent on the perch (i.e. arboreal), on the terrarium carpet (i.e. terrestrial), and in the water (i.e. aquatic) was determined for each snake. Snakes could easily move between these three microhabitats within the cage. Snakes remained completely undisturbed for the duration of these experiments.

Locomotor Performance Trials Maximal velocities and stamina were measured in all 12 individuals for all five species. All trials were conducted at room temperature (24 ± 1 °C) for 4 modes of movement: terrestrial lateral undulation, aquatic lateral undulation (i.e. swimming), concertina, and arboreal. For all trials, the dorsal view of the snake was videotaped using a Sony DCR-TRV460 Digital8 (New York, NY, USA). The sequence of individuals and modes tested were randomized in a repeated- measures design.

Terrestrial lateral undulation: A track (3.00 x 0.75 m) containing terrarium carpet was used to gather data on lateral undulatory burst speed (i.e. velocity) and stamina. This substrate contained numerous projections and small bristles needed for force production for this locomotor mode. The track width was adjusted to at least 30 cm to promote the use of lateral undulation. A rectangular center piece set in the middle of the track produced a 7.25-m long raceway around the perimeter of the rectangular track. Burst speed was measured by videotaping snakes moving on a 2.5-m section of the track for three successive runs with the fastest of the three being considered maximal crawling velocity. The protocol used to elicit maximal speed consisted of

62 lightly tapping the tail of the snake with a painter’s brush or with a finger (see Gerald and Claussen 2007). To measure stamina, snakes were stimulated to move at maximal velocity as just described around the perimeter of the rectangular track. Stamina time was measured to the nearest second with a digital stopwatch which concluded when snakes would no longer move forward after at least five attempts to stimulate movement.

Concertina: The same track used for lateral undulation also was used to measure concertina performance. However, to elicit concertina, the width of the track was adjusted to 3x maximum body width for each individual. This width permitted comparisons of concertina performance among snakes differing in body size (Gerald and Claussen 2007). Smooth plexiglas was used as the substrate for concertina trials to promote the usage of the wooden side walls necessary for this mode of locomotion.

Swimming: To assess maximal swimming velocities, I used a linear trough (220 x 20 x 0.15 cm) marked at 0.5-m intervals and filled with aged water equilibrated to room temperature. Snakes were placed at one end of the trough and stimulated to swim the length of the track three successive times to determine maximum swimming velocity. Swimming stamina was determined by forcing snakes to swim continuously around a circular swimming pool (1.3-m diameter). Snakes were prodded to swim around the pool until they remained motionless (typically on the water’s surface) following at least five attempts to encourage swimming.

Arboreal: Arboreal locomotion, which likely is a hybrid between lateral undulation and concertina (Astley and Jayne 2007; Gerald et al. 2008), was assessed on two 6 cm artificial perches wrapped in rough-textured burlap carpet to provide push-off points and positioned together directly across from each other to produce a 2-m long perch (Gerald et al. 2008). Snakes were positioned on one end of the perch and stimulated to move as previously described at maximum speed down the perch three successive times. The measurement of arboreal locomotor stamina was not possible because of the design of the perches.

Statistical analyses

63 The use of each microhabitat was determined by calculating the proportion of time spent relative to the total observed time. Differences in use of each microhabitat (terrestrial versus arboreal versus aquatic) among species was tested using an analysis of variance (ANOVA). The relationship between microhabitat use and locomotor performance was assessed using simple linear regression for each of the three habitat types. Locomotor velocities were determined from video analyses and were calculated as absolute (m • s-1) and body length relative velocities (SVL • s-1). Differences in velocities and stamina were compared across locomotor modes using repeated measures ANOVAs and Bonferroni tests for multiple comparisons. The effect of morphological variables on velocities and stamina of each mode was determined using linear regression models. However, species differences in performance during various modes on movement were analyzed with an analysis of co-variance (ANCOVA) with mass as a covariate. To examine the potential interspecific and intraspecific trade-offs in locomotor performance across the different locomotor modes, Pearson product moment correlations were used among all locomotor modes for all species combined and each species separately. Many investigators have suggested that since species share at least portions of their evolutionary histories, they themselves cannot be regarded as statistically independent (Felsenstein 1985; 1988; Garland et al. 1993). Therefore, I also statistically analyzed the data using independent contrasts to correct for evolutionary relatedness (Felsenstein 1985). Independent contrasts were calculated for morphological, microhabitat use, and locomotor performance data. Simple linear regressions were conducted on the contrasts of performance versus microhabitat use and of performance versus body size. To assess differences in performance among species, I compared the residuals from each group from the relationship between performance and body mass. In this way, differences in performance among species can be illuminated while taking both evolutionary history and body size into account with any differences being assumed to be caused by some intrinsic variation related to the habitat that a particular species prefers. To correct for evolutionary histories among species, information on the phylogeny of the species is needed, particularly the branch lengths of a phylogenetic tree. Since, to my knowledge, phylogenetic information that includes all five species studied is non-existent, I used results from a number of different studies to develop a phylogenetic tree (Vanhooydonck and

64 Van Damme 2003). A tree with branch length estimates was constructed (Fig. 1) using information from several studies that built trees based on both mitochondrial genes and morphology (Keogh 1996; Rodriquez-Robles and De Jesus-Escobar 1999; de Queiroz et al. 2002; Kelly et al. 2003; Hibbits and Fitzgerald 2005; Lawson et al. 2005). All statistical tests were conducted using Minitab (Minitab Inc., State College, PA) and SAS (SAS Institute, Version 9.1).

Results Morphology The species used in this study significantly differed in mass, SVL, cross-sectional area, number of pre-caudal vertebrae, and body condition (ANOVAs: p < 0.001; Table 1). However, snakes were closer in SVL than the other morphological variables measured. Elaphe alleghaniensis, N. sipedon, and P. catenifer were not significantly different in SVL (p = 0.128). Moreover, N. sipedon and P. catenifer were statistically similar to E. guttata (p = 0.84), which was statistically similar to T. sauritus (p = 0.23).

Microhabitat use Statistical differences among species were detected in the amount of time spent in each microhabitat (Fig. 2; Table 2). As predicted, P. catenifer spent more time on the terrestrial substrate than the other species. Nerodia sipedon (northern watersnakes) spent much more time in the water relative to the other species (p < 0.001). The “semi-arboreal” E. alleghaniensis also spent much more time using the arboreal perch than the other species, though not significantly more than T. sauritus, the other “semi-arboreal” species (Fig. 2; Table 2). Therefore, microhabitat use in the enclosures mostly mirrored published accounts of behaviors observed in nature.

Locomotor performance

For all species combined, snakes displayed faster absolute (ANOVA: F3,237 = 236.4; p <

0.0001) and SVL-relative (F3,237 = 183.0; p < 0.0001) velocities during swimming, followed by lateral undulation, concertina, and arboreal locomotion (Fig. 3). However, the differences between concertina and arboreal velocities were not significant (Least-squares mean tests: p >

65 0.20; Fig. 3). Though all snakes averaged longer lateral undulation stamina times compared to

concertina and swimming (Fig. 4), this difference was not quite statistically significant (F2,150 = 2.62; p = 0.076). Locomotor velocities of all modes were, for the most part, significantly related to body size and shape when examining all species (Figs. 5-9). Body mass was positively related to concertina and swimming velocities, but negatively related to arboreal velocity, though not significantly (Fig. 5). I found no significant relationship between mass and velocity of lateral undulation for all species combined. Mass was positively related to stamina of all modes examined, though only significantly to lateral undulation (Table 3). The linear regressions on the independent contrasts were mostly highly non-significant with one notable exception. Contrasts of mass was positively related to contrasts of concertina velocities (β = 0.0006; p = 0.002), which mirrored the results from the relationship between these same variables before the data transformation (Fig. 5c). I also assessed the relationship between SVL and performance. Interestingly, arboreal velocities displayed a highly significant positive relationship with SVL (Fig. 6a), a result opposite to that of body mass. Lateral undulatory velocities again were not significantly related to SVL (Fig. 6b). Velocities of concertina and swimming were, however, significantly positively related to SVL (Figs. 6c and d). Of the three modes in which stamina was examined, only concertina exhibited a significant positive relationship with SVL (Table 3). I found no significant relationships between any of the independent contrasts of SVL and performance measures (p > 0.25). Body condition was not significantly related to arboreal velocities (Fig. 7a). However, it was positively related to velocities of lateral undulation suggesting that snakes that are longer relative to mass exhibit faster velocities (Fig. 7b). Concertina and swimming velocities displayed a highly significant negative relationship with body condition (Figs. 7c and d), thus, indicating that longer snakes relative to mass attain slower velocities than those that are shorter. Body condition was negatively related to stamina of lateral undulation and swimming (Table 3). A snake’s cross-sectional area was not significantly related to arboreal or lateral undulatory velocities (Figs. 8a and b), though it was positively related to concertina and swimming velocities (Figs. 8c and d). However, concertina stamina was not related to cross- sectional area, though lateral undulatory and swimming stamina were positively related (Table

66 3). The number of vertebrae also showed interesting relationships with the modes examined. Arboreal velocities were positively related to number of vertebrae (Fig. 9a). Velocities of the other three modes displayed a negative relationship with the number of vertebrae (Figs. 9b, c, d). The number of vertebrae has a positive and negative relationship with stamina of concertina and swimming, respectively (Table 3).

Species comparisons To assess differences in performance among snake taxa, I compared species using both uncorrected performance data and the residuals from regressions of the relationships between the contrasts of mass and performance. Since the species used in this study differed in body size, which affects velocity and stamina, mass was used as a covariate in the uncorrected analyses. Moreover, comparing the body length-relative velocities will illuminate differences in performance among species that are not related to mass alone and is likely more ecologically relevant (Van Damme and Van Dooren 1999). Consequently, I report differences among species in terms of both absolute and SVL-relative velocities. However, comparisons among independent contrasts were conducted on absolute velocities only. The uncorrected performance comparisons revealed that velocities and stamina differed among species for all modes examined and that the patterns observed were, for the most part, closely related to the microhabitats each species has been noted to use in nature (Figs. 10 and 11; Table 4). For example, the “semi-arboreal” E. alleghaniensis was faster than the other species when moving on the artificial perch and slowest in the water, whereas the “semi-aquatic” N. sipedon exhibited the slowest velocities on the perch but was the best swimmer in terms of both velocity and stamina. The most terrestrial species of the five (P. catenifer) displayed the fastest concertina velocities and the best stamina during lateral undulation and concertina locomotion. Surprisingly, the ribbon snake (T. sauritus) was able to attain relatively fast velocities during all four modes of locomotion. Elaphe guttata, the “generalist”, displayed mostly an intermediate level of performance for all modes for both velocity and stamina (Figs. 10 and 11; Table 4). After correcting for phylogenetic relatedness, differences were apparent between nodes for all performance measures and modes suggesting that the rates of evolutionary changes leading to certain performance phenotypes differed among taxa (Fig. 12). Furthermore, differences in contrasts among nodes indicate that differences among species are not due strictly

67 to similar evolutionary histories. The large residual value for arboreal velocity for contrast 2 indicates that the two sister species (E. alleghaniensis and E. guttata) possess superior arboreal performance capabilities compared to the other taxa that are not associated with relatedness (Fig. 12a). However, this difference is due to the faster arboreal velocities attained by E. alleghaniensis. The low residual value for contrast 1 for arboreal velocity suggests that the two sister species (N. sipedon and T. sauritus) display much slower velocities than would be predicted from phylogenetic relatedness with this result being due to the slow velocities of N. sipedon on the perch. For swimming velocities and stamina, contrast 1 possessed much higher values because of the superior swimming abilities of N. sipedon (Fig. 12b). Likewise, the lower value of contrast 3 for swimming velocity indicates slower values than would be predicted by rates of evolution. Residual values for performance of lateral undulation and concertina for velocity and stamina also mostly reflect comparisons made among species using the uncorrected performance data (Fig. 12).

Microhabitat use vs. performance Although variation in performance, for the most part, did reflect each species’ previously reported microhabitat preferences, the associations between the artificial microhabitats selected and performance of modes used in that microhabitat showed few statistically significant relationships. These relationships were assessed using the independent contrasts of performance and amount of time spent in each microhabitat (Figs 13 and 14) and on the uncorrected data (Table 5). Though not significant, arboreal velocities were positively related to perch use for all individuals. Swimming velocities and stamina were, however, significantly associated with the use of the aquatic microhabitats (Figs. 13 and 14; Table 5).

Trade-offs in performance I found no statistical evidence for trade-offs (negative correlations) in locomotor velocities across locomotor modes (Table 6). In contrast, significant positive correlations were observed between several modes (Table 6). For example, velocities of lateral undulation were positively related to both swimming and concertina velocities. Significant positive correlations also were observed between modes within each species (p < 0.05). Most notably, arboreal velocities were correlated with both lateral undulation and concertina in N. sipedon. Concertina

68 velocities were positively related to lateral undulation in E. guttata, N. sipedon, and T. sauritus. Stamina times of lateral undulation and concertina also were positively correlated (Table 6). Though there does not appear to be a trade-off at the individual level, there do seem to be trade- offs at the species’ level, especially between arboreal and aquatic locomotion (Fig. 15). As mentioned earlier, N. sipedon displayed the fastest swimming velocities and the slowest arboreal velocities whereas E. alleghaniensis displayed the opposite pattern (Fig. 15). However, T. sauritus moved fairly well during both of the aforementioned modes. There were no other trade- offs apparent between other modes using interspecific comparisons. Instead, the species that crawled faster via lateral undulation also were the best swimmers (Fig. 15).

Discussion Morphological effects on locomotion By studying multiple species, my study is the first to show that locomotor abilities across various modes by snakes are strongly associated with both morphology and microhabitat preferences as suggested by Arnold’s (1983) paradigm. These results confirm previous studies that certain morphological traits and body size have a large influence on locomotor performance. Results from most studies indicate that larger (both mass and SVL) snakes perform better than smaller ones during terrestrial lateral undulation (Arnold and Bennett 1988; Plummer 1997) and during swimming (Aubret et al. 2005; Hopkins and Winne 2006), but, to my knowledge, this is the first study to determine the relationship between size and performance of other limbless locomotor modes. In my study, variation in morphology had differential effects on each of the studied modes. Larger snakes were able to attain faster velocities than smaller ones for concertina and swimming. However, the relationship between velocity and mass was weakly positive for lateral undulation and weakly negative for arboreal movement. The reason for this disparity for arboreal locomotion is likely owing to the increasing importance of balance (Gerald et al. 2008). A larger mass is more difficult to support against gravity, especially on narrower branches. It has been suggested that slender bodies are adaptations to arboreal environments because this body form permits snakes to spread out their mass more easily to better negotiate a complex arrangement of branches and discontinuous gaps (Lillywhite and Henderson 1993; Pizzatto et al. 2007; Gerald et al. 2008). Although more slender bodies relative to mass might help snakes move on and between branches without falling, I found no evidence that more

69 slender snakes attained greater velocities compared to thicker snakes (Fig. 7a). Moreover, this study only assessed movement on a cylinder, whereas a more slender body may confer an advantage when moving on a complex arrangement of relatively thin branches containing many projections. A relatively longer body does seem to make it easier to cross larger discontinuous gaps or use cantilever behaviors (Lillywhite et al. 2000; Jayne and Riley 2007). Snakes that engage in aerial locomotion (genus Chrysopelea) to glide from branch to branch also display better performance for those species that are longer compared to mass (Socha and LaBarbera 2005). As expected, arboreal velocities were positively related to SVL (Fig. 6a), which suggests that body length is important when moving on cylindrical perches because of the ability to form more alternating body loops around the branch, which should enhance stability (Gerald et al. 2008). A greater number and size of body loops also should allow snakes to attain higher speeds because more irregularities are available for force production at a given moment in time. Longer snakes did move significantly faster than shorter ones for all modes except lateral undulation. It is unclear why lateral undulation failed to be related to mass and length, though it could be due to the carpet substrate used to elicit lateral undulation. It is possible that an increase in mass does not allow snakes to generate more force on this particular substrate. Snakes with longer epaxial muscles and tendons, which are more easily supported with fewer vertebrae relative to body length, exhibit more rapid undulatory locomotion (Jayne 1988b; Moon and Gans 1998; Moon 2000). Consequently, snakes with fewer vertebrae should attain faster velocities due to the presence of larger (in terms of cross-sectional area) and longer epaxial muscles and tendons that produce strong unilateral muscle activity needed for the lateral bending necessary for rapid movement (Jayne 1988b). My results support this hypothesis in that three of the four modes displayed an inverse relationship between locomotor performance and number of vertebrae (Fig 9). Interestingly, maximal arboreal velocity showed a strong positive relationship with number of vertebrae, which is likely due to the importance of producing alternating body loops around the perch necessary for simultaneous balance and movement. Though an electromyographic study on arboreal snake locomotion would be needed to fully address this question, small-radius lateral bends, which would be more easily produced by shorter epaxial musculature, may be better suited for movement on a perch. If this is the case, this would suggest that a trade-off exists between maximal velocities of arboreal and undulatory (i.e. lateral

70 undulation and swimming) locomotion. Although not statistically significant, arboreal velocities were negatively correlated with swimming velocities and lateral undulation and swimming stamina. This potential trade-off is analogous to the hypothesized trade-off between rapid locomotion and prey constriction, which respectively require long versus shorter epaxial muscles (i.e. few versus more vertebrae) for maximal performance (Gans 1962; Ruben 1977). More vertebrae allow for shorter epaxial muscles, which allow constrictors to produce tighter bends to more quickly kill their prey. Though it should be noted that elongated epaxial muscles (along with fewer vertebrae) seem to be capable of producing the small lateral bends needed for constriction (see Moon 2000), many of the more basal snake lineages (e.g. Boidae) strictly use constriction as a mechanism for prey capture and tend to have more vertebrae and relatively short epaxial muscles and tendons (Underwood 1976). Thus, selection pressures favoring morphological changes to enhance constriction early on in snake evolution might have more easily permitted snakes to move into and begin utilizing arboreal habitats, leading to an adaptive radiation of constrictors with arboreal habits. This idea is corroborated by the fact that many species of boids are arboreal and are excellent constrictors (Ditmars 1933; Lillywhite and Henderson 1993). Future studies should seek to precisely examine the relationship between feeding behaviors and locomotion in snakes. Body condition was positively related to lateral undulatory velocities, but inversely related to velocities of concertina and swimming velocities (Fig. 7). However, examining the relationship between lateral undulatory velocities and body condition with the data for the elongate Thamnophis sauritus removed reveals a strong negative relationship. This suggests that long, thinner snakes relative to body mass move more poorly (speed and stamina) during all four modes of locomotion compared to short, bulkier snakes. These results suggest that although body length is likely important to provide more sites along the lateral portions of the body for force generation and for forming loops to balance and grip during arboreal locomotion, the distribution and mass of epaxial muscles play a greater role in locomotor abilities of all modes. This could be due to the higher cross-sectional area of epaxial musculature associated with bulkier snakes which would allow for more force to be produced at a given point of contact with the substrate (Bonnet et al. 1998). It has been suggested that relatively longer snakes are better at concertina locomotion than thicker ones because of their ability to stretch the body farther during a given cycle of

71 movement (Jayne 1988a). During a study on Nerodia and Elaphe, Jayne (1988a) noted that relatively longer snakes were able to perform concertina at a given tunnel width more easily owing to the fact that more forward progression can be achieved as the angle between bend in the body and tunnel approaches 90°. This would allow snakes to increase the speed of concertina in a way that is analogous to increasing stride length as a mechanism to increase velocity during limbed locomotion. The fact that SVL and body condition were positively and negatively related to velocities of all modes studied, respectively, suggests that the cross- sectional area of the epaxial musculature is a better predictor of performance than is body length. Snake body cross-sectional area showed significant positive associations with velocities of concertina and swimming and slightly positive associations with stamina for all modes measured (Fig. 8). Since the snakes used in this study were very similar in body width and height (p > 0.50), differences in cross-sectional area among species represent relatively equal differences between body width and body height. Therefore, it is probable that the relationships between cross-sectional area and locomotor performance result from differences in body height, because greater height translates into greater lateral surface area and, hence, more epaxial muscle mass contracting to generate force against the environment. Because the terrestrial substrate used to elicit lateral undulation contained many smaller projections, increasing lateral surface area may not improve movement of this mode. However, increasing lateral surface area did enhance performance of concertina and swimming, modes in which the entire lateral surface generates propulsion by pushing against the medium. This suggests that body shape (i.e. width versus height) relative to overall mass may be more important for determining concertina and swimming abilities compared to other modes. This also would explain the strongly positive correlation between body mass and velocities of concertina and swimming. Though it would seem that increased body size, in particular height, would be selected for in species that utilize concertina through tunnels, the intuitive disadvantage of using a large body to burrow into the substrate no doubt selects for smaller body sizes in the vast majority of fossorial species. Morphological adaptations to increase body height are present in fully marine species (i.e. Hydrophiidae), which possess a laterally-compressed, paddle-like tails to generate extra propulsion in water but reduced speed on land (Shine and Shetty 2001; Aubret and Shine 2008b).

Differences in locomotor performance among modes

72 All snakes displayed much faster velocities during swimming compared to the other modes. Previous studies also have found that snakes swim much faster than they can move on land (Scribner and Weatherhead 1995; Finkler and Claussen 1999; Shine et al. 2003; Gerald and Claussen 2007; Isaac and Gregory 2007). Snakes are likely using the same muscles to generate propulsion in water and on land (lateral undulation), however, the functional roles of each epaxial muscle may shift when transitioning from water to land (Gillis and Blob 2001). Therefore, snakes are likely able to achieve faster speeds in water because of the drastic reduction in friction and because more lateral surface area is available to push against the surrounding medium. Studies comparing performance of different modes other than terrestrial lateral undulation and swimming are extremely scarce. This study showed that concertina SVL-relative velocities, which fell in the range of velocities found in previous studies (Gerald and Claussen 2007) were on average 18% of terrestrial lateral undulatory velocities. This is not surprising due to the stop-and-go nature (Jayne and Davis 1991) and high energetic cost (Walton et al. 1990) of concertina locomotion. Concertina involves substantial changes in momentum and high frequencies of movement that constrain performance capabilities of this mode. Due to the kinematic properties of concertina, snakes would be expected to have low stamina for this mode if moving at maximal velocity. This was not the case; concertina stamina was only slightly lower than stamina during lateral undulation and very similar to swimming suggesting that maintaining maximal velocities for each of these modes requires a similar amount of energy. But because maximum attainable velocities differed among modes, snakes were able to travel much farther during swimming, followed by lateral undulation and concertina during stamina trials. One must be cautious when comparing stamina among modes and species from this study for two reasons. First, I observed a large amount of variation in stamina among individuals suggesting that perhaps not all individuals were exhausted at the conclusion of each trial. Second, snakes often reacted differently to tail prodding in different situations. During stamina trials on land, snakes would sometimes coil their body and begin to strike at the stimulus instead of utilizing flight behaviors. It is difficult to say whether or not snakes were exhausted during these situations since shifts in anti-predator strategies can depend on a number of different factors associated with the likelihood of the snake surviving the predation attempt (Hertz et al.

73 1982). Reactions to stimulation during stamina trials also seemed to differ among species. During terrestrial lateral undulation trials, T. sauritus were much more likely to turn and strike than were other species, possibly because of their smaller size. During swimming stamina trials, N. sipedon was the only species that would attempt to strike while in the water. Arboreal locomotion was, by far, the slowest mode of locomotion. It should be noted that this locomotor mode was assessed on a simplified artificial perch (i.e. cylinder) that was used to eliminate confounding factors, such as incline or vegetative projections that likely have a large influence on locomotor ability (Gerald et al. 2008). Arboreal limbless locomotion is complex in that it involves a combination of undulatory and concertina movements while distributing mass to maintain balance at the same time (Astley and Jayne 2007; Gerald et al. 2008). However, the texture of the branch determines whether snakes can use purchase points to generate thrust (i.e. lateral undulation; Gerald et al. 2008) or if they must use body loops to grip the perch for static contact so another portion can be moved (i.e. concertina; Astley and Jayne 2007). Perches used during this study (rough-textured burlap) allowed snakes to undulate and very little concertina-type movements were observed during trials. Even though snakes were using “lateral undulation” to travel down the branch, velocities were roughly 17 times slower than terrestrial lateral undulation. The difference in velocities between terrestrial lateral undulation and arboreal may echo the problems associated with maintaining balance while moving. Snakes made alternating lateral movements combined with forward movements to ensure that body loops were distributed relatively evenly around both sides of the perch to prevent slipping. Although snakes made observably slower movements to prevent falling, these lateral movements resulted in a further decrease in forward velocity. In nature, there is likely to be much variation in arboreal locomotor abilities because so many factors can influence a snake’s performance in the environment. The size of the snake, the diameter and incline of the branch (Astley and Jayne 2007; Gerald et al. 2008), the texture of the branch, the presence of projections, and the proximity of the branch to other branches are some of the major structural features that can limit limbless arboreal performance. When compared to concertina locomotion, snakes were three times slower when moving on the perch. Astley and Jayne (2007) observed concertina velocities through tunnels that were 12 times greater than arboreal velocities when the tunnel width equaled that of the perch. Since concertina speed tends to decrease as tunnel width relative to maximum body width decreases (Jayne and Davis 1991; Gerald and Claussen 2007)

74 and arboreal velocities can be affected by perch diameter (Astley and Jayne 2007), one should be careful when comparing performance of these two modes between different studies.

Interspecific differences in microhabitat use and locomotor performance Microhabitat use measured in the laboratory during this study was similar to published field data on each species. All species spent more time on the terrestrial substrate compared to the water or perch (Fig. 2; Table 2). However, the terrestrial P. catenifer spent the most time (97%) exhibiting terrestrial behaviors, whereas the semi-arboreal E. alleghaniensis spent the least time. The use of the aquatic “microhabitat” corresponded well to predictions based on qualitative data with the semi-aquatic N. sipedon spending the most time compared to others. Use of the perch was dominated by the two semi-arboreal species (E. alleghaniensis and T. sauritus). Nerodia sipedon, E. guttata, and P. catenifer barely used the perch at all during the experiment. Since the microhabitats used in captivity mostly resembled those reported in field studies, I am confident that the microhabitats used were those “preferred” by each species (Vanhooydonck and Van Damme 2003) and that the microhabitat “categories” widely reported in the literature are valid. Species used in this study displayed variable performance capabilities within and among the locomotor modes assessed. As predicted, this variability appears to be closely associated with interspecific variation in microhabitat use rather than simply evolutionary relatedness given that uncorrected species’ comparisons revealed similar patterns to those using phylogenetically- corrected performance data. The semi-arboreal E. alleghaniensis displayed the fastest arboreal velocities and relatively slow swimming velocities. These data correspond well to qualitative field data suggesting that this species climbs and moves well in trees (Conant and Collins 1998) and reflected this species’ use of the perch within the artificial enclosures. The congeneric generalist E. guttata displayed intermediate performance during three of the four modes measured and the slowest velocities for lateral undulation. Pituophis catenifer, the terrestrial species, showed the highest concertina velocities and the best stamina for lateral undulation and concertina which corresponds well with qualitative and observed microhabitat use. The semi-aquatic N. sipedon achieved the fastest speed and best stamina during swimming relative to the other species. These results also mirror those reports from previous studies that this species is a superior swimmer (Finkler and Claussen 1999). Though this species

75 does not possess any obvious morphological features that enhance swimming abilities, it is the bulkiest of those studied and, presumably, can generate a substantial amount of force while undulating through water. Recently, Pattishall and Cundall (2008) observed that N. sipedon has the ability to laterally compress the rear half to two-thirds of the trunk during swimming to generate more force in a manner similar to that of sea snakes. For arboreal locomotion, N. sipedon showed the lowest velocities despite the fact that this species will commonly climb up and bask on vegetation overhanging water. It is unclear, however, why the closely-related semi- arboreal T. sauritus displayed excellent performance abilities relative to size for speed during all modes. This species is typically found near aquatic habitats in or around vegetation (Conant and Collins 1998). Therefore, these data suggest that this species is more of a generalist from both a microhabitat and performance perspective. On another interesting note, both N. sipedon and T. sauritus displayed the fastest velocities of swimming and terrestrial lateral undulation and both have the fewest number of vertebrae, which supports longer epaxial muscles and tendons as previously mentioned. These data mostly support the notion that locomotor abilities parallel behaviors associated with microhabitat use in snakes. Species and individuals that frequently use aquatic microhabitats are better swimmers, whereas those that utilize arboreal microhabitats more often are better at arboreal locomotion. It is impossible to say whether this represents plasticity in performance related to use of that particular mode or an evolutionary adaptation to aquatic and arboreal microhabitats. Aubret and Shine (2008a) showed that snakes can display locomotor plasticity in response to rearing conditions for terrestrial, arboreal, and aquatic locomotion. All individuals of all species except for the semi-aquatic N. sipedon were captive-raised in similar conditions. Therefore, it is possible that, since they were wild caught, N. sipedon displayed superior swimming abilities because they frequently displayed aquatic behaviors in nature before the experiment, whereas the other species’ potential aquatic behaviors were artificially limited. This argument can not be made for the relationship between arboreal use and arboreal locomotion since the superior climbers were all raised in similar environments. Consequently, the superior arboreal performance abilities observed are likely due to morphological and physiological adaptations that enhance efficiency of movement in the arboreal environment. Though performance abilities during each mode for each species mostly reflected qualitative accounts of behavioral habits of each species in nature, significant relationships were not

76 observed between use of the terrestrial microhabitat and either of the terrestrial modes of movement. It is unclear why there was no relationship between terrestrial use and locomotion. A study examining this relationship using a larger number and variety of species might be successful in revealing this potential correlation. In lizards, patterns of locomotor performance have been shown to be linked with variation in microhabitat use (Irschick and Jayne 1998; 1999; Melville and Swain 2000; Irschick and Garland 2001; Vanhooydonck and Van Damme 2003; McElroy et al. 2007), which likely represents selection for traits that improve locomotion in that habitat. An excellent example of slight differences in morphology which is correlated with variation in microhabitat use and locomotor performance in lizards is observed in the genus Anolis (Losos 1990; Higham et al. 2001; Mattingly and Jayne 2004; Spezzano and Jayne 2004) where different types, or ecomorphs have been described that inhabit different microhabitats (Moermond 1979; Williams 1983). Some ecomorphs have shorter limbs which allow for use of narrower perches (i.e. twigs), though shorter limbs result in slower crawling velocities in most situations due to the decreased stride length (Losos 1990; Vanhooydonck et al. 2002; Spezzano and Jayne 2004). The results from my study are somewhat similar to performance of young Australian tiger snakes (Notechis scutatus), a habitat generalist, that moved faster during terrestrial lateral undulation, swimming, and arboreal locomotion following rearing in the respective habitats showing that snakes exhibit phenotypic plasticity in locomotor performance (Aubret and Shine 2008a). This latter study, however, did not assess concertina locomotion and did not measure arboreal locomotion in the same manner as the present and previous studies. They stimulated snakes to move horizontally across “small branches” which, depending on the density of branches used, would likely allow snakes to undulate across an irregular horizontal substrate in a manner that is practically identical to terrestrial lateral undulation, which may or may not be a good representation of arboreal locomotion (Astley and Jayne 2007; Gerald et al. 2008). Few other studies have shown that interspecific variation in locomotor performance matched qualitative differences in ecology. Isaac and Gregory (2007) observed that swimming speeds of semi-aquatic snakes in the genus Natrix were associated with previous knowledge of the use of aquatic habitats. Scribner and Weatherhead (1995) noted that both locomotor speeds and anti- predator behaviors of the closely-related snakes Thamnophis sirtalis, T. sauritus, and N. sipedon corresponded to observations of habitat associations. Bonnet et al. (2005) showed that species of

77 sea-snake that have been reported to come out and move onto land most often are better able to climb up a rocky cliff overhanging the ocean. Intriguingly, Shine et al. (2003) found that the highly marine snake Emydocephalus annulatus actually moved very slowly on both land and water relative to other more amphibious sea snakes. They postulated that this was due to this species feeding on immobile prey, which does not require speed for foraging.

Trade-offs among locomotor modes It has been suggested that snakes will exhibit a trade-off in locomotor performance between terrestrial lateral undulation and swimming because of differences in the kinematic and muscle activity requirements needed to move a limbless body on land versus water (Jayne 1986; Cundall 1987; Shine et al. 2003). In contrast, I found that velocities of lateral undulation and swimming were positively related. Other studies have found that undulatory crawling and swimming are either not related or are positively related in various snake species (Finkler and Claussen 1999; Shine and Shetty 2001; Gerald and Claussen 2007; Isaac and Gregory 2007). The lack of a trade-off could be due to the large amount of inter-individual variation in performance, which would make it difficult to detect trade-offs if they do exist. Contrary to predictions, it appears that snakes actually can simultaneously maximize performance of lateral undulation on land and in water despite the biomechanical differences. There is very little information available on potential trade-offs between other modes of limbless locomotion other than lateral undulation on land versus water. Gerald and Claussen (2007) observed no correlations between concertina and either terrestrial lateral undulation or swimming in S. dekayi. During the present study, I found significant positive correlations between concertina and lateral undulation and between concertina and swimming for all species combined. Within species, I found that arboreal velocities were positively related to concertina (E. guttata and N. sipedon) and to lateral undulation (N. sipedon). Hence, I did not find any statistical evidence of a trade-off between any of the locomotor modes studied here. However, the relationship between arboreal locomotion and swimming should be examined more closely in the future. At the interspecific level, the species that performed the best during arboreal locomotion performed the worst during swimming and vice versa (E. alleghaniensis versus N. sipedon).

78 Conclusions In snakes, locomotor performance across various ecological situations appears to be closely related to both morphology and microhabitat preference. However, these relationships appear to be stronger for some locomotor modes compared to others. It is difficult to tease apart the influence of morphological variables from that of microhabitat use. One of two possibilities exist that could explain the patterns of microhabitat use, morphology, and locomotor abilities among species. First, snakes may have begun to use certain microhabitat niches because of ecological factors (e.g. competition, predation) which, in turn, selected for morphological features to improve locomotor efficiency in that microhabitat. Since performance among locomotor modes appears to be correlated, the other possibility is that snakes with a given morphology were able to take advantage of a new microhabitat which would, over time, cause them to prefer the new microhabitat. In both instances, selection should continue to favor both increased performance and preference for that particular microhabitat resulting in positive feedback that promotes specialization into certain microhabitats over time. The limbless body plan appears to be well built for radiation into a variety of different habitats. Future studies should further investigate factors such as muscular strength and other morphological variables across a wider range of species to further clarify the relationships among morphology, microhabitat use, and performance.

Acknowledgments I would like to thank the Department of Zoology, Miami University for providing funding and support. I also would like to thank A. Collins, M. Gelpi, J. Kotcher, J. Long, C. Miskell, A. Trout, and R. Whynott for their assistance with husbandry and data collection.

Literature Cited Arnold S.J. 1983. Morphology, performance and fitness. American Zoologist 23:347- 361. Arnold S.J. and A.F. Bennett. 1988. Behavioural variation in natural populations. V. morphological correlates of locomotion in the garter snake (Thamnophis radix). Biological Journal of the Linnean Society 34:175-190.

79 Astley H.C. and B.C. Jayne. 2007. Effects of perch diameter and incline on the kinematics, performance and modes of arboreal locomotion of corn snakes (Elaphe guttata). Journal of Experimental Biology 210:3862-3872. Aubret F. and R. Shine. 2008a. Early experience influences both habitat choice and locomotor performance in tiger snakes. American Naturalist 171:524-531. Aubret F. and R. Shine. 2008b. The origin of evolutionary innovations: locomotor consequences of tail shape in aquatic snakes. Functional Ecology 22:317-322. Aubret F., X. Bonnet, and S. Maumelat. 2005. Tail loss, body condition and swimming performances in tiger snakes, Notechis ater occidentalis. Journal of Experimental Zoology 303A:894-903. Aubret F., X. Bonnet, and R. Shine. 2007. The role of adaptive plasticity in a major evolutionary transition: early aquatic experience affects locomotor performance of terrestrial snakes. Functional Ecology 21:1154-1161. Auffenberg W. 1962. A review of the trunk musculature in the limbless land vertebrates. American Zoologist 2:183-190. Bonnet X., I. Ineich, and R. Shine. 2005. Terrestrial locomotion in sea snakes: the effects of sex and species on cliff-climbing ability in sea kraits (Serpentes, Elapidae, Laticauda). Biological Journal of the Linnean Society 85:433-441. Bonnet X., R. Shine, G. Naulleau, and M. Vacher-Vallas. 1998. Sexual dimorphism in snakes: different reproductive roles favour different body plans. Proceedings of the Royal Society of London, B Biology. 265:179-183. Brown G.P. and P.J. Weatherhead. 2000. Thermal ecology and sexual size dimorphism in northern water snakes, Nerodia sipedon. Ecological Monographs 70:311-330. Burbrink F.T. 2001. Systematics of the Eastern Rat Snake (Elaphe obsoleta). Herpetological Monographs 15:1-53. Carpenter C.C. 1952. Comparative ecology of the common garter snake (Thamnophis sirtalis sirtalis), the ribbon snake (Thamnophis sauritus sauritus), and Butler’s garter snake (Thamnophis butleri) in mixed populations. Ecological Monographs 22:235-258. Conant R. and J.T. Collins. 1998. A field guide to reptiles and amphibians: eastern and central North America, 3rd ed., expanded. Houghton Mifflin, Boston.

80 Cundall D. 1987. Functional morphology. In: Snakes: Ecology and Evolutionary Biology (Ed. by R.A. Seigel, J.T. Collins, S.S. Novak). pp. 106-142. McGraw Hill. New York. Ditmars R.L. 1933. Reptiles of the world. MacMillan Company, New York. Ernst C.H. and E.M. Ernst. 2003. Snakes of the United States and Canada. Smithsonian Books. Washington and London. Felsenstein J. 1985. Phylogenies and the comparative method. American Naturalist 125:1-15. Felsenstein J. 1988. Phylogenies and quantitative characters. Annual Reviews in Ecology and Systematics 19:445-471. 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. Gans C. 1962. Terrestrial locomotion without limbs. American Zoologist 2:167-182.

Gans C. 1986. Locomotion of limbless vertebrates: pattern and evolution. Herpetologica 42:33-46. Garland T. Jr. and J.B. Losos. 1994. Ecological morphology of locomotor performance in squamate reptiles. In: Ecological morphology: integrative organismal biology (Ed. by P.C. Wainwright and S.M. Reilly). pp. 240-302. University of Chicago Press. Garland T. Jr., A.W. Dickerson, C.M. Janis, and J.A. Jones. 1993. Phylogenetic analysis of covariance by computer simulation. Systematic Biology 42:265-292. Gerald G.W. and D.L. Claussen. 2007. Thermal dependencies of different modes of locomotion in neonate Brown Snakes, Storeria dekayi. Copeia 2007:577-585. Gerald G.W. and C.A. Miskell. 2007. Small litter sizes and relative clutch mass of Northern Watersnakes (Nerodia sipedon sipedon) in southwestern Ohio. Ohio Journal of Science 107:84-85. Gerald G.W., M.A. Bailey, and J.N. Holmes. 2006. Habitat utilization of Pituophis melanoleucus melanoleucus (Northern Pinensnakes) on Arnold Air Force Base in Middle Tennessee. Southeastern Naturalist 5:253-264.

81 Gerald G.W., M.J. Mackey, and D.L. Claussen. 2008. Effects of temperature and perch diameter on arboreal locomotion in the snake Elaphe guttata. Journal of Experimental Zoology 309A:147-156. Gillis G.B. and R.W. Blob. 2001. How muscles accommodate movement in different physical environments: aquatic vs. terrestrial locomotion in vertebrates. Comparative Biochemistry and Physiology A 131:61-75. Hertz P.E., R.B. Huey, and E. Nevo. 1982. Fight versus flight: body temperature influences defensive responses of lizards. Animal Behaviour 30:676-679. Hibbitts T.J. and L.A. Fitzgerald. 2005. Morphological and ecological convergence in two natricine snakes. Biological Journal of the Linnean Society 85:363-371. Higham T.E., M.S. Davenport, and B.C. Jayne. 2001. Maneuvering in an arboreal habitat: the effects of turning angle on the locomotion of three sympatric ecomorphs of Anolis lizards. Journal of Experimental Biology 204:4141-4155. Hopkins W.A. and C.T. Winne. 2006. Influence of body size on swimming performance of four species of neonatal natricine snakes acutely exposed to a cholinesterase-inhibiting pesticide. Environmental Toxicology and Chemistry 25:1208-1213. Howitz J.L. 1986. Bull snake predation on black-capped chickadee nest. Loon 58:132. Irschick D.J. 2002. Evolutionary approaches for studying functional morphology: examples from studies of performance capacity. Integrative and Comparative Biology. 42:278-290. Irschick D.J. 2003. Studying performance in nature: implications for fitness variation within populations. Integrative and Comparative Biology. 43:36-47. Irschick D.J. and B.C. Jayne. 1998. Effects of incline on speed, acceleration, body posture and hindlimb kinematics in two species of lizard Callisaurus draconoides and Uma scoparia. Journal of Experimental Biology 201:273-287. Irschick D.J. and B.C. Jayne. 1999. A field study of the effects of incline on the escape locomotion of a bipedal lizard, Callisaurus draconoides. Physiological and Biochemical Zoology 72:44-56. Irschick D.J. and T. Garland Jr. 2001. Integrating function and ecology in studies of adaptation: investigations of locomotor capacity as a model system. Annual Review of Ecology and Systematics 32:367-396.

82 Irschick D.J., B. Vanhooydonck, J. Meyers, and A. Herrel. 2005. Intraspecific correlations among morphology, performance, and habitat use within a green anole lizard (Anolis carolinensis) population. Biological Journal of the Linnean Society. 85:211-221. Isaac L.A. and P.T. Gregory. 2007. Aquatic versus terrestrial locomotion: comparative performance of two ecologically contrasting species of European natricine snakes. Journal of Zoology 273:56-62. Jayne B.C. 1985. Swimming in constricting (Elaphe g. guttata) and nonconstricting (Nerodia fasciata pictiventris) colubrid snakes. Copeia 1985:195-208. Jayne B.C. 1986. Kinematics of terrestrial snake locomotion. Copeia 1986:915-927. Jayne B.C. 1988a. Muscular mechanisms of snake locomotion: an electromyographic study of the sidewinding and concertina modes of Crotalus cerastes, Nerodia fasciata and Elaphe obsoleta. Journal of Experimental Biology 140:1-33. Jayne B.C. 1988b. Muscular mechanisms of snake locomotion: an electromyographic study of lateral undulation of the Florida banded water snake (Nerodia fasciata) and the yellow rat snake (Elaphe obsoleta). Journal of Morphology 197:159-181. Jayne B.C. and A.F. Bennett. 1990. Selection on locomotor performance capacity in a natural population of garter snakes. Evolution 44:1204-1229. Jayne B.C. and J.D. Davis. 1991. Kinematics and performance capacity for the concertina locomotion of a snake (Coluber constrictor). Journal of Experimental Biology 156:539-556. Jayne B.C. and M.A. Riley. 2007. Scaling of the axial morphology and gap-bridging ability of the brown tree snake, Boiga irregularis. Journal of Experimental Biology 210:1148-1160. Kelly C.M.R., N.P. Barker, and M.H. Villet. 2003. Phylogenetics of advanced snakes (Caenophidia) based on four mitochondrial genes. Systematic Biology 52:439-459. Keogh J.S. 1996. Evolution of the colubrid snake tribe Lampropeltini: a morphological perspective. Herpetologica 53:406-416. Kohlsdorf T., T. Garland, and C.A. Navas. 2001. Limb and tail lengths in relation to substrate usage in Tropidurus lizards. Journal of Morphology 248:151-164.

83 Kohlsdorf T., R.S. James, J.E. Carvalho, R.S. Wilson, M.D. Pai-Silva, and C.A. Navas. 2004. Locomotor performance of closely related Tropidurus species: relationships with physiological parameters and ecological divergence. Journal of Experimental Biology 207:1183-1192. Lawson R., J.B. Slowinski, B.I. Crother, and F.T. Burbrink. 2005. Phylogeny of the Colubroidea (Serpentes): new evidence from mitochondrial and nuclear genes. Molecular Phylogenetics and Evolution 37:581-601. Lillywhite H.B. and R.W. Henderson. 1993. Behavioral and functional ecology of arboreal snakes. In: Snakes: Ecology and Behavior (Ed. by Seigel R.A. and J.T. Collins). pp. 1-48. McGraw Hill, New York. Lillywhite H.B., J.R. LaFrentz, Y.C. Lin, and M.C. Tu . 2000. The cantilever abilities of snakes. Journal of Herpetology 34:523-528. Losos J.B. 1990. The evolution of form and function: morphology and locomotor performance in West Indian Anolis lizards. Evolution 44:1189-1203. Losos J.B. and B. Sinervo. 1989. The effects of morphology and perch diameter on sprint performance of Anolis lizards. Journal of Experimental Biology 145:23-30. Losos J.B. and D.J. Irschick. 1996. The effect of perch diameter on escape behaviour of Anolis lizards: laboratory predictions and field tests. Animal Behavior 51:593-602. Losos J.B., B.M. Walton, and A.F. Bennett. 1993. Trade-offs between sprinting and clinging ability in Kenyan chameleons. Functional Ecology 7:281-286. Marr N.V. 1985. Gopher snake preys on northern oriole nestlings. Murrelet 66:95-97. Mattingly W.B. and B.C. Jayne. 2004. Resource use in arboreal habitats: structure affects locomotion of four ecomorphs of Anolis lizards. Ecology 85:1111-1124. McElroy E.J., J.J. Meyers, S.M. Reilly, and D.J. Irschick. 2007. Dissecting the effects of behaviour and habitat on the locomotion of a lizard (Urosaurus ornatus). Animal Behaviour 73:359-365. Melville J. and R. Swain. 2000. Evolutionary relationship between morphology, performance and habitat openness in the lizard genus Niveoscincus (Scincidae: Lygosomidae). Biological Journal of the Linnean Society 70:667-683. Moermond T. 1979. Habitat constraints on the behavior, morphology, and community structure of Anolis lizards. Ecology 60:152-164.

84 Moon B.R. 2000. The mechanics and muscular control of constriction in gopher snakes (Pituophis melanoleucus) and a king snake (Lampropeltis getula). Journal of Zoology 252:83-98. Moon B.R. and C. Gans. 1998. Kinematics, muscular activity, and propulsion in gopher snakes. Journal of Experimental Biology 201:2669-2684. Mosauer W. 1935. The myology of the trunk region of snakes and its significance for ophidian taxonomy and phylogeny. Publications of the University of California-Los Angeles, Biology 1:81-120. Mount R.H. 1975. The reptiles and amphibians of Alabama. University of Alabama Press, Tuscaloosa, AL. Palmer W.M. and A.L. Braswell. 1995. Reptiles of North Carolina. University of North Carolina Press, Chapel Hill, NC. Pattishall A. and D. Cundall. 2008. Dynamic changes in body form during swimming in the water snake Nerodia sipedon. Zoology 1:48-61. Pizzatto L., S.M. Almeida-Santos, and R. Shine. 2007. Life-history adaptations to arboreality in snakes. Ecology 88:359-366. Plummer M.V. 1997. Speed and endurance of gravid and nongravid green snakes, Opheodrys aestivus. Copeia 1997:191-194. Pough F.H. 1989. Organismal performance and Darwinian fitness: approaches and interpretations. Physiological Zoology 62:199-236. de Queiroz A., R. Lawson, and J.A. Lemos-Espinal. 2002. Phylogenetic relationships of North American garter snakes (Thamnophis) based on four mitochondrial genes: how much DNA sequence is enough? Molecular Phylogenetics and Evolution 37:581-601. Rodriguez-Robles J.A. 2003. Home ranges of gopher snakes (Pituophis catenifer, Colubridae) in Central California. Copeia 2003:391-396. Rodriguez-Robles J.A. and J.M. De Jesus-Escobar. 1999. Molecular systematics of New World lampropeltine snakes (Colubridae): implications for biogeography and evolution of food habits. Biological Journal of the Linnean Society 68:355-385. Rossman D.A., N.B. Ford, and R.A. Seigel. 1996. The garter snake: evolution and ecology. University of Oklahoma Press, Norman, OK.

85 Ruben J.A. 1977. Morphological correlates of predatory modes in the coachwhip (Masticophis flagellum) and rosy boa (Lichanura roseofusca). Herpetologica 33:1-6. Scribner S.J. and P.J. Weatherhead. 1995. Locomotion and antipredator behaviour in three species of semi-aquatic snakes. Canadian Journal of Zoology 73:321-329. Shine R. and S. Shetty. 2001. Moving in two worlds: aquatic and terrestrial locomotion in sea snakes (Laticauda colubrina, Laticaudidae). Journal of Evolutionary Biology 14:338-346. Shine R., H.G. Cogger, R.R. Reed, S. Shetty, and X. Bonnet. 2003. Aquatic and terrestrial locomotor speeds of amphibious sea-snakes (Serpentes, Laticaudidae). Journal of Zoology (London) 259:261-268. Socha J.J. and M. LaBarbera. 2005. Effects of size and behavior on aerial performance of two species of flying snakes (Chrysopelea). Journal of Experimental Biology 208:1835-1847. Spezzano L.C. Jr. and B.C. Jayne. 2004. The effects of surface diameter and incline on the hindlimb kinematics of an arboreal lizard (Anolis sagrei). Journal of Experimental Biology 207:2115-2131. Tennant A. 1997. A field guide to snakes of Florida. Gulf Publishing Co, Houston, TX. Tennant A. and R.D. Bartlett. 2000. Snakes of North America: eastern and central regions. Gulf Publishing Co., Houston, TX. Underwood G. 1976. A systematic analysis of boid snakes. In: Morphology and Biology of Reptiles (Ed. by A. d`ABeallairs and C.B. Cox). Linn. Soc. Symp. Ser. vol. 3. pp. 151-175. Academic Press, London. Van Damme R. and T.J.M. Van Dooren. 1999. Absolute versus per unit body length speed of prey as an estimator of vulnerability to predation. Animal Behaviour 57:347- 352. Vanhooydonck B. and R. Van Damme. 2003. Relationships between locomotor performance, microhabitat use and antipredator behaviour in lacertid lizards. Functional Ecology 17:160-169. Vanhooydonck B., R. Van Damme, and P. Aerts. 2002. Variation in speed, gait characteristics and microhabitat use in lacertid lizards. Journal of Experimental Biology 205:1037-1046.

86 Walton M., B.C. Jayne, and A.F. Bennett. 1990. The energetic cost of limbless locomotion. Science 249:524-527. Williams E.E. 1983. Ecomorphs, faunas, island size, and diverse end points in island radiations of Anolis. In: Lizard ecology: studies of a model organism (Ed. by Huey R.B., E.R. Pianka, and T.W. Schoener). pp. 326-370. Harvard University Press, Cambridge. Wisco J., H. Matles, and D. Berigan. 1997. Is the scaling of locomotor performance with body size constant? Ecological Entomology 22:483-486. Wright, A.H., and A.A. Wright. 1957. Handbook of snakes of the United States and Canada. Comstock Publishing Associates, Ithaca, New York.

87 Table 1. The reported habits and morphological data for the five snake species used in this study. Numbers represent means with the standard deviation in parentheses. SVL = snout-vent length, Body condition = total length (cm)/mass (g).

Cross- Tail Length Body Condition No. of Species Habits n Mass (g) SVL (cm) sectional area (cm) (cm/g) vertebrae (mm2) Elaphe guttata Generalist 12 28.1 (16.8) 45.0 (6.8) 7.9 (1.8) 108.2 (44.3) 2.2 (0.4) 231.2 (6.8)

Elaphe alleghaniensis Semi-arboreal 12 56.6 (26.6) 61.0 (10.1) 13.7 (3.0) 154.2 (42.3) 1.5 (0.6) 251.2 (20.1)

Pituophis catenifer Terrestrial 12 73.0 (51.4) 52.8 (11.6) 7.9 (2.0) 226.9 (97.7) 1.0 (0.4) 221.3 (6.9)

Thamnophis sauritus Semi-arboreal 12 11.4 (3.3) 37.8 (3.9) 16.6 (4.8) 57.6 (15.9) 5.1 (1.2) 174.9 (3.7)

Nerodia sipedon Semi-aquatic 12 119.1 (47.2) 54.0 (6.8) 15.2 (3.3) 351.4 (134.1) 0.6 (0.2) 141.2 (3.8)

88

Table 2. Statistically significant differences in terrestrial, aquatic, and arboreal microhabitat use between five species of snake (n = 12/species). The order of use is listed from left to right with the species on the left using that particular microhabitat the most and the one on the right using it the least. Superscript letters denote statistical differences. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus.

Order of use Terrestrial Pca Egab Nsabc Tsabc Eac

Aquatic Nsa Tsb Pcb Eab Egb

Arboreal Eaa Tsab Nsb Egb Pcb

89

Table 3. Linear regression parameters for the relationships (y = α + βx) between uncorrected morphology and stamina of three different modes of limbless locomotion in 5 species of snake (n = 12/species). SVL = snout-vent length, Body condition = total length (cm)/mass (g).

Locomotor Mode α β R2 p Mass (g) Lateral undulation 912.5 3.50 0.10 0.02 Concertina 883.1 1.07 0.02 0.32 Swimming 801.6 1.91 0.05 0.12

SVL (cm) Lateral undulation 776.8 6.34 0.02 0.33 Concertina 444.6 9.32 0.09 0.03 Swimming 714.8 3.70 0.01 0.47

Body Lateral undulation 1387.6 -144.2 0.11 0.02 Condition Concertina 1040.8 -48.8 0.03 0.25 (length/mass) Swimming 1059.4 -82.7 0.07 0.07

Cross- Lateral undulation 824.7 1.66 0.14 0.01 sectional Concertina 871.4 0.45 0.02 0.31 area (mm2) Swimming 743.6 0.91 0.08 0.05

No. of Lateral undulation 1381.9 -1.18 0.01 0.54 vertebrae Concertina 433.1 2.49 0.08 0.05 Swimming 1710.5 -3.79 0.16 <0.01

90

Table 4. Statistically significant differences in maximal snout-vent length (SVL)-relative velocities and stamina of multiple modes of limbless locomotion between five species of snake (n = 12/species). The order of use is listed from left to right with the species on the left displaying superior performance and the one on the right displaying poorer performance abilities. Superscript letters denote statistical differences. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus.

Performance Mode Order of use

Arboreal Eaa Tsb Egbc Pcc Nsd

Lateral Undulation Tsa Nsb Eabc Pcc Egc Velocity Concertina Pca Tsb Nsb Eab Egb

Swimming Nsa Tsb Pcb Egb Eab

Lateral undulation Pca Nsab Egb Tsb Eab

Stamina Concertina Psa Egab Tsab Eab Nsb

Swimming Nsa Egb Eab Pcb Tsb

91

Table 5. Linear regression parameters for the relationships (y = α + βx) between uncorrected data on performance during each of four locomotor modes and amount of time spent in each microhabitat type by 5 snake species (n = 12/species).

Locomotor Performance vs. Microhabitat use α β R2 p Mode

Arboreal Velocity vs. Arboreal use 0.04 0.11 0.58 0.13

Lateral Velocity vs. Terrestrial use 1.72 -1.26 0.54 0.16 Undulation Stamina vs. Terrestrial use 240.01 1117.70 0.44 0.23

Velocity vs. Terrestrial use 0.04 0.10 0.12 0.56 Concertina Stamina vs. Terrestrial use 447.16 622.18 0.29 0.35

Velocity vs. Aquatic use 1.06 3.68 0.91 0.01 Swimming Stamina vs. Aquatic use 755.71 3162.12 0.88 0.02

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Table 6. Pearson product-moment correlation coefficients between locomotor performance measures (maximal snout-vent length-relative velocity and stamina) of four modes of limbless locomotion in 5 snake species (n = 12/species). Arb = arboreal, LU = terrestrial lateral undulation, Conc = concertina, Swim = swimming. * = statistical significance at the 0.05 level.

Velocity Stamina

Arb LU Conc Swim LU Conc Swim Velocity Arb 1.000 LU 0.058 1.000 Conc -0.103 0.216* 1.000 Swim -0.117 0.382* 0.326* 1.000 Stamina LU -0.276 -0.213 0.075 -0.071 1.000

Conc 0.053 -0.395* -0.031 -0.374* 0.460* 1.000 Swim -0.181 0.061 0.327* 0.349* -0.011 -0.243 1.000

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Thamnophis sauritus 1 Nerodia sipedon

4 Pituophis catenifer

3 Elaphe alleghaniensis

2 Elaphe guttata

Figure 1. A topology of 5 snake species based on both mitochondrial genes and morphology (Keogh 1996; Rodriquez-Robles and De Jesus-Escobar 1999; de Queiroz et al. 2002; Kelly et al. 2003; Hibbits and Fitzgerald 2005; Lawson et al. 2005) used to determine independent contrasts of morphological measurements, locomotor performance, and microhabitat use. Numbers represent the nodes for which each contrast was calculated.

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1.0

0.8

Terrestrial 0.6 Aquatic Arboreal

0.4 Proportion of time spent time of Proportion 0.2

0.0 Ts Eg Pc Ns Ea

Figure 2. Proportion of time spent in terrestrial, aquatic, and arboreal microhabitats within artificial enclosures for individuals of five species of snake (n = 12/species) during 3 hrs of observations. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus.

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0.8 A

) 0.6 -1

0.4

Maximal velocity (m * s (m velocity Maximal 0.2

0.0 Arb Conc LU Swim 1.6 B 1.4 )

-1 1.2

1.0

0.8

0.6

0.4 Maximalvelocity (SVL * s

0.2

0.0 Arb Conc LU Swim Locomotor mode Figure 3. Average maximal absolute (A) and snout-vent length (SVL)-relative (B) velocities for 5 species of snakes (n = 12/species) combined for arboreal (Arb), terrestrial lateral undulation (LU), concertina (Conc), and aquatic (Swim) locomotion. Both performance measures were statistically different across all modes (p < 0.0001) except for the difference between arboreal and concertina (p = 0.20). Error bars represent ± SE.

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1400

1200

1000

800

600 Stamina (s)

400

200

0 LU Conc Swim

Locomotor Mode

Figure 4. Average stamina times for 5 species of snake (n = 12/species) for terrestrial lateral undulation (LU), concertina (Conc), and aquatic (Swim) locomotion. Differences among modes were not statistically significant (p = 0.076). Error bars represent ± SE.

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Figure 5. Relationship between body mass and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D).

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Figure 6. Relationship between snout-vent length (SVL) and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D).

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Figure 7. Relationship between body condition (cm/g) and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D).

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Figure 8. Relationship between cross-sectional area (mm2) and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D).

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Figure 9. Relationship between number of pre-caudal vertebrae and velocities attained by snakes (five species, n = 12/species) during arboreal locomotion (A), lateral undulation (B), concertina (C), and swimming (D).

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0.20 2.0

Arboreal 0.18 A 1.8 Lateral Undulation B Concertina Swimming 0.16 ) 1.6 -1 0.14 1.4 0.12 1.2 0.10 1.0 0.08 0.8 0.06

Maximal velocity (SVL * s 0.6 0.04

0.02 0.4

0.00 0.2 Eg Ea Pc Ns Ts Eg Ea Pc Ns Ts 0.12 1.0 Arboreal C Lateral Undulation D Concertina Swimming 0.10 0.8 ) -1 0.08 0.6

0.06

0.4 0.04 Maximal velocity (m * s 0.2 0.02

0.00 0.0 Eg Ea Pc Ns Ts Eg Ea Pc Ns Ts Species

Figure 10. A comparison of uncorrected maximal velocities (snout-vent length (SVL)-relative (A,B) and absolute (C,D)) between five species of snake (n = 12/species) for each of four modes of locomotion. Because of differences in scale, average arboreal and concertina velocities are shown together (A,C), while lateral undulation and swimming velocities are shown together (B,D). Note the differences in the y-axis between the panels. Error bars depict ± SE. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus.

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2000 Lateral Undulation 1800 Concertina Swimming

1600

1400

1200

1000 Stamina time (s)

800

600

400 Eg Ea Pc Ns Ts

Species

Figure 11. Mean duration of stamina by five species of snake (n = 12/species) moving during three modes of locomotion. Error bars depict ± SE. Ea = Elaphe alleghaniensis, Eg = Elaphe guttata, Ns = Nerodia sipedon, Pc = Pituophis catenifer, Ts = Thamnophis sauritus.

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Figure 12. Residuals from the relationships between independent contrasts (Felsenstein 1985; Am. Nat. 125:1-15) of locomotor velocities (A) and stamina (B) and the independent contrasts of body mass for four modes (A) and three modes (B) of limbless locomotion in 5 species of snakes.

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Figure 13. Relationships between the independent contrasts (Felsenstein 1985; Am. Nat. 125:1- 15) of locomotor velocities and independent contrasts of microhabitat use for arboreal (A), terrestrial lateral undulation (B), terrestrial concertina (C), and aquatic (D) locomotion and the respective microhabitats in which these modes are used in 5 species of snakes.

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Figure 14. Relationships between the independent contrasts (Felsenstein 1985; Am. Nat. 125:1- 15) of stamina and independent contrasts of microhabitat use for terrestrial lateral undulation (A), terrestrial concertina (B), and aquatic (C) locomotion by 5 snake species and the respective microhabitats in which these modes are used.

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1.2

1.0

0.8

0.6

0.4 % of maximal speed maximal of % Elaphe guttata Elaphe alleghaniensis 0.2 Pituophis catenifer Nerodia sipedon Thamnophis sauritus

0.0 Arb LU Conc Swim

Locomotor mode

Figure 15. Interspecific comparisons of the uncorrected average maximal velocities attained by five species of snake (n = 12/species) during four modes of locomotion. Velocities are represented as a percentage of the fastest average velocity by a particular species within each locomotor mode (i.e. the velocities of the fastest species were set to 1.0) to facilitate comparisons across modes. Arb = arboreal, LU = lateral undulation, Conc = concertina, Swim = swimming.

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General Conclusions

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Though limbless locomotion may be fundamentally different from limbed locomotion, it is effected by similar abiotic and biotic factors because of the underlying physiological (e.g. muscle properties, metabolic capacities) similarities (Bennett 1990; Biewener 2003). Temperature is, arguably, the most important abiotic factor limiting the locomotor abilities of ectothermic animals. However, the precise influence of temperature on limbless locomotion depends heavily on the mode or gait used for movement (Stevenson et al. 1985; Scribner and Weatherhead 1995; Finkler and Claussen 1999). Various locomotor modes appear to be affected by temperature differently, which mirrors the disparity in the physiological, kinematic, and metabolic properties of each mode. These contrasts in thermal dependencies could help explain the distribution and behaviors of snakes in different environments. For example, arboreal limbless locomotion appears to be greatly hindered at lower temperatures due to the large reduction in speed and balance (i.e. increased probability of falling). Because of these two factors, temperature could be a major factor limiting the use of arboreal microhabitats and behaviors by snakes in cooler environments. This is corroborated by the fact that arboreal species represent approximately 50% and 10% of all snake species in tropical versus temperate environments, respectively. Morphology also determines locomotor abilities in different microhabitats with body mass being the primary biotic factor defining locomotion via most modes (except arboreal). Although snakes have a superficially similar body shape, body length relative to mass and number of vertebrae also influence how well snakes move in various situations because these factors are likely related to the size and overall mass of the musculature needed to generate force against the external environment. However, it is more difficult for larger snakes to move on perches relative to smaller ones. This is due to the increasing risk of an injury due to falling off a branch as snakes get larger. Snakes with more vertebrae can achieve faster arboreal velocities because they are capable of producing the stronger small-radius lateral bends needed to loop around and grip a perch. A snake’s body size and shape also is likely determined by selective pressures associated with feeding habits (e.g. constricting snakes tend to have more vertebrae) and locomotion in preferred microhabitats. It is difficult to conclude that interspecific variation in microhabitat preference is guided by variation in morphology or vice versa. Selection for these traits would likely occur simultaneously to allow certain species to become specialists.

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Though many species are specialists in terms of habitat preference (e.g. sea kraits, Laticauda) or diet (queen snakes, Regina), most of the 2300 species of snakes are capable of using multiple microhabitats (Greene 1997) and many are considered generalists in terms of microhabitat usage. For example, all snakes appear to be capable of swimming and even sea snakes are able to crawl on land fairly well (Shine et al. 2003). My results suggest that, although snakes can possess morphological and physiological adaptations for movement in their preferred microhabitat, most of these enhancements do not interfere with, and oftentimes boost, locomotor efficiency in multiple situations. This would permit selection to favor habitat generalist lifestyles more easily in snakes. It should be noted that some modifications, such as the paddle-like extension of the tail of sea snakes to improve swimming, does interfere with movement via other modes (Aubret and Shine 2008). Hypotheses suggesting that snakes cannot simultaneously possess adaptations that optimize performance during more than one locomotor mode (Cundall 1987; Shine and Shetty 2001; Shine et al. 2003) have not been supported by the results of my studies. In contrast, my results indicate that most modes are, in fact, positively related (possible exception between swimming and arboreal locomotion) suggesting that adaptations (e.g. longer epaxial muscles and tendons, increased body length) to enhance performance can do so during multiple modes concurrently despite the kinematic differences. Compared to limbed lizards, snakes seem to perform very well, and in some cases better (e.g. arboreal), in different microhabitat types. Since adaptations seem to improve locomotion in multiple situations, selection should continue to strongly favor these morphological and physiological traits in snakes. Though there are many examples of vertebrate taxa displaying limb reduction or complete limb loss (e.g. salamanders, lizards, mammals), I am not aware of any instances of body shortening and limbs being selected for in limbless animals. However, I should note that it is likely much easier for selection to promote the loss of limbs rather than their secondary development. There also are a few examples of lizards with short, stocky bodies (e.g. Phyrnosomatidae). This suggests that the elongate, limbless body form is, at least from a movement perspective, advantageous over limbed animals in a variety of different microhabitats producing an evolutionary stable strategy for this body form. There is little doubt that snakes, being a monophyletic group, evolved from limbed lizards and began radiating by the mid-to late-Cretaceous (Rage 1987). There has, however,

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been much debate on the factors that gave rise to the origin of snakes. Present-day limbless lizards and those showing various degrees of limb reduction are mostly terrestrial species that are usually found in dense or thick groundcover. Presumably, limbs reduce streamlining when these animals are moving or “swimming” through dense vegetation. This led early researchers to consider “grass swimming” to be the initial factor selecting against the presence of limbs (Camp 1923; Rage 1987). This explanation has mostly been rejected because of a lack of anatomical or fossil evidence. There are three other more likely hypotheses for the origins of snakes. First, snakes evolved from an aquatic ancestor that was closely related to mosasaurs. Many of the fossils related to snakes have been found in marine deposits. Though, limb reduction is found in some aquatic animals (e.g. cetaceans), it has not been observed in aquatic lizards. It also should be noted that both lizards and crocodilians swim by laterally undulating the body with both pairs of limbs tucked closely beside the body to improve streamlining. Another hypothesis is that snakes have a fossorial origin. This is the most widely accepted idea because almost all primitive snakes (blind snakes; Typhlopidae and Leptotyphlopidae) and many limb-reduced lizards exhibit fossorial lifestyles (Rage 1987). Opththalmological data indicate that vision was greatly reduced in early snakes further indicating a burrowing origin (Underwood 1970). However, there are several traits present in snakes that are inconsistent with a fossorial origin (see Rage 1987). The hypothesis put forth by Rage (1987) suggests that early snakes inhabited crevices or tree holes, which would have promoted body elongation, and subsequently showed underground behaviors to select for limb reduction. This idea is inconsistent with data on limb reduction in lizards that indicate that body elongation occurs in concert with limb reduction (Gans 1975). Though my data suggest that snakes swim better than they move on land and that swimming is less affected by temperature, this does not suggest that the limbless body form initially evolved for swimming. Although this cannot be ruled out, snakes likely evolved on land where larger and more elongated snakes could have been selected to assist with movement through tunnels or in dark crevices where limbs would impede movement. In tunnels, snakes move very slowly (i.e. concertina) compared to other modes but they are still able to move relatively easily despite the narrow gap available for force generation. Effective concertina requires tight lateral bends from an elongated body. Speed and endurance of concertina would likely be greatly hindered if legs were present. Although concertina is a very slow and energetically expensive mode (Walton et al. 1990), selection could favor limb

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reduction if concertina is improved to allow snakes to use burrows for finding food or escaping predators. Another idea not previously considered is that limblessness evolved to improve arboreal locomotion. I found that limbless locomotion is less affected by perch diameter compared to arboreal locomotion of a lizard of equal mass. This suggests that one major advantage of an elongate, limbless body form is the ability to move on and utilize smaller branches for a number of different tasks, such as foraging, escaping predators, and thermoregulating. Although there is a lack of fossil evidence to support this claim, snakes are thought to have evolved from varanoid lizards, for which extant forms are known to be excellent climbers with some being strongly arboreal. The snake body form seems to be well suited for movement in a three-dimensional environment and usage of this microhabitat could have been a factor contributing to body elongation and limb reduction in the snake ancestor. The most likely scenario seems to be that once the process of elongation and limb reduction was initiated (perhaps due to the use of crevices or burrows), then the use of additional microhabitats (e.g. aquatic, dense vegetation, burrows, arboreal) could have added further directional selection pressures towards the snake body form. Until additional fossil evidence emerges, it will be difficult to say for certain what the exact pressure was that caused snakes to lose their legs.

Conclusion Snakes have long been thought of as being at a disadvantage because of their limblessness. However, their elongated bodies and associated body mass have allowed them to use an array of habitats because of their ability to change modes depending on the situation. Moreover, the skeletal and muscular modifications related to body elongation promotes the lateral flexion capable of producing a variety of different movements (i.e. modes) that is a “protoadaptation” to movement through a diversity of microhabitats. This has permitted snakes to adapt quickly to, and radiate into, new environments with only certain abiotic factors (i.e. temperature) limiting their distributions and use of particular habitats. Consequently, snakes, because of their limblessness, are a successful group that is more adept at living in many different microhabitats than their limbed counterparts.

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Literature Cited Aubret F. and R. Shine. 2008. The origin of evolutionary innovations: locomotor consequences of tail shape in aquatic snakes. Functional Ecology 22:317-322. Bennett A.F. 1990. Thermal dependence of locomotor capacity. American Journal of Physiology 259:R253-R258. Biewener A.A. 2003. Animal locomotion. Oxford University Press. Camp C.L. 1923. Classification of the lizards. Bulletin of the American Museum of Natural History 48:289-481. Cundall D. 1987. Functional Morphology. In: Snakes: ecology and evolutionary biology (Ed. by Seigel R.A., J.T. Collins, and S.S. Novak). pp. 106-140. Blackburn Press. Gans C. 1975. Tetrapod limblessness: evolution and functional corollaries. American Zoologist 15:455-467. Greene HW. 1997. Snakes: the evolution of mystery in nature. Berkeley, CA. University of California Press. 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. Rage J.C. 1987. Fossil history. In: Snakes: ecology and evolutionary biology (Ed. by Seigel R.A., J.T. Collins, and S.S. Novak). pp. 51-76. Blackburn Press. Scribner S.J. and P.J. Weatherhead. 1995. Locomotion and antipredator behaviour in three species of semi-aquatic snakes. Canadian Journal of Zoology 73:321-329. Shine R. and S. Shetty. 2001. Moving in two worlds: aquatic and terrestrial locomotion in sea snakes (Laticauda colubrina, Laticaudidae). Journal of Evolutionary Biology 14:338-346. Shine R., H.G. Cogger, R.R. Reed, S. Shetty, and X. Bonnet. 2003. Aquatic and terrestrial locomotor speeds of amphibious sea-snakes (Serpentes, Laticaudidae). Journal of Zoology 259:261-268. Stevenson R.D., C.R. Peterson, and J.S. Tsuji. 1985. The thermal dependence of locomotion, tongue flicking, digestion, and oxygen consumption in the wandering garter snake. Physiological Zoology 58:46-57.

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Underwood G. 1970. The eye. In: Biology of the Reptilia, vol. 2 (Ed. by Gans C. and T.S. Pearson). pp. 1-97. Academic Press, London. Walton M., B.C. Jayne, and A.F. Bennett. 1990. The energetic cost of limbless locomotion. Science 249:524-527.

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