Phenotypic Responses to Invasion in the Brown Anole (Anolis sagrei)
Tamara L. Fetters
Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State
University in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
In
Biological Sciences
Joel W. McGlothlin, Chair
William A. Hopkins
Ignacio T. Moore
Kendra B. Sewall
Jeffrey R. Walters
August 9, 2018
Blacksburg, Virginia
Key words: geographic variation, invasive species, life history, thermal physiology,
Anolis sagrei, adaptation, evolution
Phenotypic Responses to Invasion in the Brown Anole (Anolis sagrei)
Tamara L. Fetters
ABSTRACT
Invasive species often encounter climatic conditions that differ significantly from those of their native range. These environmental shifts may trigger phenotypic responses, resulting through some combination of adaptation and plasticity, that enable the invader to persist under novel thermal regimes. In this dissertation, I examine phenotypic changes in a tropical lizard that has successful invaded a cooler temperate climate, specifically examining traits that may promote survival and reproduction in their new range. First, I examined physiological traits, as I predicted greater cold tolerance would be necessary to survival in the invasive range. I found that invasive populations tolerated lower temperatures, exhibited greater maximum sprint speeds, and had higher metabolic rates than native populations. Next, I examined how life-history traits may change in the invasive range in order to facilitate reproduction under shorter breeding and growing seasons. I found that compared to native females, invasive females had shorter interlaying intervals and produced eggs that hatched more quickly. Once I quantified changes physiological and life-history traits that may have aided in successful establishment, I executed a common garden study to determine whether changes were the result of adaptation or plasticity. I found that differences in critical thermal minimum, metabolic rate, interlaying interval, and incubation period were maintained in lab-reared offspring, while measures of sprint speed converged. My results provide evidence that life history
and physiology can evolve rapidly during invasion. These findings are useful to understanding contemporary evolution, and also provide valuable insight on how species respond to environmental shifts, both during invasions and as a result of climate change.
Phenotypic Responses to Invasion in the Brown Anole (Anolis sagrei)
Tamara L. Fetters
GENERAL AUDIENCE ABSTRACT
When species invade a new area, they often face different climates that make can make survival and reproduction challenging. In response, species may alter traits in order to adjust to new temperatures and conditions. In this dissertation, I examine trait changes in a tropical lizard that has successfully invaded a cooler temperate climate, specifically examining traits that may help them to survive and reproduce in their new range. First, I examined physiological traits, as I predicted greater cold tolerance would be necessary to survival in the invasive range. I found that invasive populations tolerated lower temperatures, could sprint faster, and had higher metabolism than native populations.
Next, I examined how reproductive traits may change in the invasive range in order to facilitate reproduction under shorter breeding and growing seasons. I found that compared to native females, invasive females had less time between egg lays and produced eggs that hatched more quickly. Once I assessed how traits may have changed in the new range, I determined whether changes resulted from evolution or not. I found that differences in low temperature tolerance, metabolic rate, the time between egg lays, and incubation period were the result of evolution, while sprint speed did not seem to be the result of evolution. My results provide evidence that traits can evolve rapidly during invasion, allowing invasive species to persist and spread in new areas.
DEDICATION
To Kieran Ari, Asa James, and Molly Virginia
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ACKNOWLEDGEMENTS
First, I want to give my endless thanks to Joel McGlothlin, who is a talented scientist, supportive advisor, and all-around excellent human being. It has been a pleasure to work with you, and I am grateful to have had a mentor who has believed in me and pushed me to become a better scientist.
I would like to thank my committee members, Ignacio Moore, Kendra Sewall,
Bill Hopkins, and Jeff Walters for sharing their advice on matters of both science and life. Your guidance and insight has meant so much to me.
Thank you to members of the McGlothlin Lab and our team of undergraduate researchers and volunteers. There was never a shortage of help, whether with animal care, fieldwork, providing feedback, or just talking science. I’d like to give special thanks to
Eric Wice, Thomas Wood, John Abramyan, Prabh Dhillon, Caitlin McCaughan, Kathryn
Moore, Tyler Miller, and Emily Watts. You all made coming to work fun.
Thank you to the student and faculty members of the Interfaces of Global Change
Community, who have enriched my graduate experience immeasurably. In particular, thank you to Bill Hopkins, Jeff Walters, and Bruce Hull, who have taught me to challenge my views and see the bigger picture. Thanks to you, I will never find myself unable to explain my science to a stranger in an elevator.
Thank you to Virginia Tech and the Department of Biological Sciences. I consider myself lucky to be surrounded by such amazing faculty and graduate students who inspire me each and every day.
To my family for always believing in me, and for showing me through your example the value of hard work and perseverance. vi
To my wife, Ariel Leon: There are no words. Or maybe there are too many words? I’ll limit it to a few: Strong. Confident. Smart. Empowering. Thanks for inspiring me to slay.
Lastly and most importantly, to my husband, Justin Clough: None of this would have been possible without your steadfast dedication to our family and our goals. Thank you for being on my team.
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ATTRIBUTIONS
Dr. Joel W. McGlothlin, Assistant Professor, Department of Biological Sciences,
Virginia Tech, Blacksburg. Dr. McGlothlin was my advisor and advisory committee chair. He assisted in the design, execution, and statistical analyses of all of the studies presented here, and is a co-author on all publications and manuscripts resulting from this dissertation.
Dr. William A. Hopkins, Professor, Department of Fish and Wildlife Conservation,
Virginia Tech, Blacksburg. Dr. Hopkins assisted in the design, execution, and statistical analyses of the studies presented in Chapters II and IV. He is a co-author on the manuscripts resulting from those chapters.
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TABLE OF CONTENTS
Abstract...... ii
General Audience Abstract...... iii
Dedication...... v
Acknowledgments...... vi
Attributions...... viii
Table of Contents...... ix
List of Figures...... xii
List of Tables...... xiii
Chapter I: Introduction...... 1
References...... 9
Chapter II: Thermal physiology differs between native and invasive populations of the brown anole (Anolis sagrei)
Abstract...... 17
Introduction...... 18
Methods...... 21
Results...... 25
Discussion...... 27
Acknowledgments...... 34
References...... 35
Tables...... 43
Figures...... 47
ix
Chapter III: Life histories and invasions: Accelerated laying rate and incubation time in an invasive lizard, Anolis sagrei
Abstract...... 52
Introduction...... 52
Methods...... 55
Results...... 59
Discussion...... 61
Acknowledgments...... 64
References...... 65
Tables...... 68
Figures...... 70
Chapter IV: Evolution of physiological and life-history traits following invasion of a cooler climate
Abstract...... 72
Introduction...... 73
Methods...... 77
Results...... 81
Discussion...... 83
Acknowledgments...... 89
References...... 90
Tables...... 96
Figures...... 97 x
Chapter V: Synthesis...... 102
References...... 108
xi
LIST OF TABLES
Chapter II: Thermal physiology differs between native and invasive populations of the brown anole (Anolis sagrei)
Table 1. Characteristics of study populations...... 42
Table 2. General linear model results for critical thermal minimum...... 43
Table 3. General linear mixed model results for maximum sprint speed...... 44
Table 4 General linear mixed model results for metabolic rate...... 45
Chapter III: Life histories and invasions: Accelerated laying rate and incubation time in an invasive lizard, Anolis sagrei
Table 1. Characteristics of study populations...... 67
Table 2. Results of linear mixed models...... 68
Chapter IV: Evolution of physiological and life-history traits following invasion of a cooler climate
Table 1. Sample sizes and characteristics of offspring at time of testing...... 95
xii
Chapter II: Thermal physiology differs between native and invasive populations of the brown anole (Anolis sagrei)
Figure 1. Temperature data from study populations...... 46
Figure 2. Critical thermal minimum values across populations...... 47
Figure 3. Maximum sprint speeds across temperatures and populations...... 48
Figure 4. Standard Metabolic Rates across temperatures and populations...... 49
Chapter III: Life histories and invasions: Accelerated laying rate and incubation time in an invasive lizard, Anolis sagrei
Figure 1. Individual egg-laying trajectories for females from four populations in a common lab environment...... 69
Figure 2. Interlaying interval, incubation period, egg mass, and hatchling mass across populations...... 70
Chapter IV: Evolution of physiological and life-history traits following invasion of a cooler climate
Figure 1. Critical thermal minimum values for parents and offspring...... 96
Figure 2. Maximum sprint speed for parents and offspring...... 97
Figure 3. Standard Metabolic Rate for parents and offspring...... 98
Figure 4. Interlaying Interval for parents and offspring...... 99
Figure 5. Incubation Period for parents and offspring...... 100
xiii
CHAPTER I: INTRODUCTION
Tamara L. Fetters
General Introduction
The spread of invasive species is a leading threat to biodiversity worldwide
(Wilcove et al. 1998). Invasive species have substantial impacts on the environment, including decreasing the abundance and richness of native species, altering habitats, disrupting ecosystem processes, and introducing disease (McGeoch et al. 2010; Vitousek et al. 1996). In addition to being ecologically damaging, invasions can also adversely impact the economy by necessitating costly prevention, mitigation, and eradication regimes (Kolar and Lodge 2001; Lodge et al. 2006; Pimentel et al. 2005). While invasive species are already ubiquitous, as globalization progresses the occurrence and spread of invasive species is predicted to increase further (Hulme 2009; Mooney and Cleland
2001).
However, along with the challenges posed by invasive species comes the unique opportunity to observe evolutionary and ecological responses to changing environments over relatively large spatial and temporal scales (Baker and Stebbins 1965; Lee 2002).
Invasive species inevitably encounter new biotic and abiotic factors during introduction, such as the loss/gain of predators, prey, and parasites, or altered environmental conditions
(Holway et al. 2002; Keane and Crawley 2002; Prenter et al. 2004). In response, invaders often display rapid changes in phenotypic trait expression that facilitate their survival and persistence (Huey et al. 2000; Losos et al. 2001; Phillips et al. 2006). In most instances, it
1
is unknown whether this phenotypic differentiation is accomplished via plasticity, adaptation, or a combination of the two (Colautti and Lau 2015).
Quantifying the phenotypic changes that occur during invasion and identifying the relative contributions of adaptation and plasticity to these trait changes can greatly benefit our fundamental understanding of evolution. Research on invasive species has helped shape views of local adaption, speciation, reproductive isolation, species distributions, and dispersal (Nosil et al. 2005; Sax et al. 2007), and some of the most prominent and well-documented examples of contemporary evolution have come from studying the colonization of species in new environments (Reznick and Ghalambor 2001; Sakai et al.
2001). Further, insights into applied research questions such as managing and preventing invasions, predicting invasion limits, and modeling responses to global climate change can be gained by examining phenotypic change during invasion (Suarez and Tsutsui
2008).
Phenotypes and Invasion
Several model frameworks have been proposed for understanding the progression of invasions (Blackburn et al. 2011; Richardson et al. 2000; Williamson and Fitter 1996)
While each model varies slightly, there is consensus among the models that there are three sequential stages species pass through on their path to becoming invasive. During the first stage, introduction, the species enters a new habitat. Next, during establishment, the species survives and reproduces in the new habitat. Lastly, during spread, the species disperses further into secondary locations (Blackburn et al. 2011; Sakai et al. 2001). Each 2
stage acts as a selective filter, posing barriers that prevent the vast majority of species from proceeding from one stage to the next (Zenni and Nuñez 2013). For the purposes of this dissertation, I will focus primarily on the establishment stage.
During the establishment phase, species must both survive and reproduce in their new environment. Failure during this stage can occur if the species is incapable of surviving in the invaded area, or if they survive but are incapable of reproducing
(Blackburn et al. 2011). During establishment, initial threats to survival and reproduction are often posed by abiotic factors, such as changes in temperature (Gerhardt and Collinge
2007; Kelley 2014; Moyle and Light 1996). Ectotherms are particularly affected by novel climatic regimes, as temperature influences various aspect of their ecology, including development, behavior, movement, immune function, sensory processing, foraging ability, digestion, and reproduction (Angilletta et al. 2002; Bennett 1980; Huey and
Stevenson 1979; Stevenson et al. 1985). We therefore expect survival and fitness to be strongly affected by changes in ambient temperature and climate via alterations in temperature-dependent traits (Huey and Kingsolver 1989). Successful establishment thus depends on adaptive trait responses that enable the species to conform to the newly encountered climate (Campbell-Staton et al. 2016; Huang et al. 2006).
All ectotherms have some capacity to modify their behavioral and physiological responses to match current climatic conditions (Angilletta 2009; Seebacher and Franklin
2011). While behavioral mechanisms for thermoregulation can partially shield terrestrial ectotherms from unfavorable environments, there are limits to this manipulation, such as during the nighttime when they are less able to escape the physical environment (Muñoz et al. 2014). As the invaded environment becomes more disparate from that of their 3
native range, invasive species may become less able to compensate for temperature changes via behavioral thermoregulation. Thus, alterations to thermophysiological traits that allow for the survival of an invader in its new range may play an important role during establishment.
Traditionally, thermophysiological traits in ectotherms have been viewed as highly canalized. Subsequently ectotherms are not predicted to respond favorably to changing climatic conditions (Huey et al. 2012; Janzen 1967) and ectothermic invaders, particularly reptiles, have been relatively less studied compared to other taxonomic groups (Pyšek et al. 2008). However, emerging studies have demonstrated that ectotherms do remodel thermophysiological responses in response to new environments
(Kolbe et al. 2012; Leal and Gunderson 2012; Wilson and Franklin 2002), and quantifying physiological changes during ectothermic invasions represents a burgeoning research interest.
A number of thermophysiological traits may be involved in coordinating an adaptive response to the newly encountered environment, among them temperature tolerances, metabolism, and locomotive performance. In ectotherms, the temperatures at which survival is possible is referred to as the tolerance zone (Huey and Kingsolver
1989). The range of this zone is proportional to the range of environmental temperatures experienced by an individual (Deutsch et al. 2008). Even brief periods of exposure to extreme heat or cold can affect the measured parameters of the tolerance zone (Angilletta
2009), and thus traits such as highest temperature tolerated and lowest temperature tolerated may adjust to reflect the climate of the invaded range. Performance for traits such as locomotion also varies with temperature (Huey and Stevenson 1979). When 4
maximum locomotive performance aligns with the experienced temperature of an ectotherm, survival is maximized because the risk of predation is lowered, foraging capability is increased, and energy expenditure is decreased (Irschick and Losos 1998;
Logan et al. 2014). Invaders may show increased locomotive performance at new temperatures experienced in their environment in order to optimize survival and fitness.
Metabolism may also be affected by changes in climate. In particular, the metabolic rate measured at any given temperature is predicted to be lower for ectotherms in areas with stable environmental conditions, and higher in areas with greater temperature fluctuations
(Clarke 1993). This occurs because in areas with greater environmental variation, increased metabolic rate can compensate for the physiological depression that occurs during periods of suboptimal conditions (Hochachka and Somero 1984).
As previously mentioned, successful establishment depends on both survival and reproduction. While the aforementioned physiological traits may facilitate survival, ectothermic invaders cannot persist if they cannot adequately reproduce in the new environment as well. Thus, changes in life-history traits may also be expected for successful establishment to occur (Allen et al. 2017; Capellini et al. 2015). Changes to climate can influence a myriad of reproductive characteristics in ectotherms, among them the timing and duration of the breeding season, clutch size, laying patterns, and rates of incubation (Adolph and Porter 1993). Breeding seasons tend to be longer in more stable environments and shorter in more variable environments; thus, rapid reproduction may be favored in variable environments to compensate for short breeding seasons. Clutch size and laying patterns can also differ based on breeding season length; clutches may be larger or spaced more closely temporally in areas with shorter breeding seasons. Rates of 5
incubation also tend to increase in areas with shorter breeding seasons to compensate for shorter periods of optimal conditions for growth and development (While et al. 2015).
Adaptation and plasticity
Once we identify traits that may have aided in successful establishment, it is important to understand the mechanisms underlying those traits. Historically, adaptive evolution has been viewed in a narrow context, with changes in phenotypes necessarily resulting from changes in genotypes. However, in some cases phenotypic variation may actually (1) precede and facilitate an adaptive genetic response, or (2) buffer the genotype from selection and render a subsequent change in genotype unnecessary (Ghalambor et al. 2007). Trait divergence between populations is thus by itself is not indicative of adaptation through selection. Rather, variation likely occurs through some combination of evolutionary adaptation and phenotypic plasticity, with the relative contribution of each likely varying over the course of the invasion.
Plasticity occurs when a genotype produces differing phenotypes under different environmental conditions. These changes are often reversible, presuming that the trait is not fixed during development (West-Eberhard 2003). Phenotypic plasticity can enable a population to persist under novel environmental conditions by allowing the phenotype the flexibility to conform to the encountered environment (Ghalambor et al. 2007). Plasticity may be particularly important during the initial stages of invasion because of the speed at which it allows organisms to cope to immediate environmental challenges (Sakai et al.
2001). Depending on how close the plastic response is to the new phenotypic optimum 6
and how costly plasticity is for the organism, plasticity may eventually facilitate adaptive evolution through directional selection (Price et al. 2003).
Genetic adaptation occurs when selection favors genotypes that perform well under new environmental conditions. The novel selective pressures encountered during invasions can promote evolutionary change (Mooney and Cleland 2001), and these genetic changes can occur rapidly, often within tens of generations (Carroll et al. 2007).
Genetic adaptation can continue past establishment, enabling for further range expansion
(Broennimann et al. 2007). While genetic adaptation is often implicated as a causal mechanism for the trait changes recorded between native and invasive populations, much of this work has been observational. Experimental studies that combine observations of phenotypic trait changes during invasion with explicit tests for genetic adaptation are needed.
Current study
The brown anole (Anolis sagrei) is a highly invasive species native to Cuba and
The Bahamas. A. sagrei was first detected in southern Florida in the early 1900s, and has since spread into Georgia, Louisiana, and Texas (Kolbe et al. 2004). Climatic models indicate that a thermal niche shift has occurred between the native Caribbean range and introduced southeastern range, with populations in the introduced range experiencing lower seasonal temperatures and humidity (Angetter et al. 2011). In this dissertation, I used the invasion of the brown anole to quantify trait changes that may have contributed
7
to its successful establishment and to further assess the mechanisms underlying these changes.
Brown anoles were collected from two populations in the native range and four populations in the invasive range that span a wide geographic and climatic range. In
Chapter II, I examine how thermophysiological traits vary across native and invasive populations of the brown anole by comparing measures of the lowest temperature tolerated, maximum sprint speed, and metabolic rate from individuals of each population.
In Chapter III, I examine how life-history traits vary across native and invasive brown anoles by comparing egg laying rate and incubation time across populations. Chapter IV explores if differences in thermophysiological and life-history traits result from genetic adaptation by performing a controlled common garden experiment and comparing trait measures of lab-reared offspring across populations.
8
REFERENCES
Adolph, S. C., and W. P. Porter. 1993. Temperature, activity, and lizard life histories. The
American Naturalist 142:273-295.
Allen, W. L., S. E. Street, and I. Capellini. 2017. Fast life history traits promote invasion
success in amphibians and reptiles. Ecology Letters 20:222-230.
Angetter, L.-S., S. Loetters, and D. Roedder. 2011. Climate niche shift in invasive
species: the case of the brown anole. Biological Journal of the Linnean Society
104:943-954.
Angilletta, M. J. 2009, Thermal adaptation: a theoretical and empirical synthesis, Oxford
University Press.
Angilletta, M. J., P. H. Niewiarowski, and C. A. Navas. 2002. The evolution of thermal
physiology in ectotherms. Journal of thermal Biology 27:249-268.
Baker, H. G., and G. L. Stebbins. 1965, Genetics of colonizing species, proceedings
International Union of Biological Sciences Symposia on General Biology 1964:
Asilomar, Calif.).
Bennett, A. F. 1980. The thermal dependence of lizard behaviour. Animal Behaviour
28:752-762.
Blackburn, T. M., P. Pyšek, S. Bacher, J. T. Carlton, R. P. Duncan, V. Jarošík, J. R.
Wilson et al. 2011. A proposed unified framework for biological invasions.
Trends in ecology & evolution 26:333-339.
Broennimann, O., U. A. Treier, H. Müller‐Schärer, W. Thuiller, A. Peterson, and A.
Guisan. 2007. Evidence of climatic niche shift during biological invasion.
Ecology letters 10:701-709. 9
Campbell-Staton, S. C., S. V. Edwards, and J. B. Losos. 2016. Climate‐mediated
adaptation after mainland colonization of an ancestrally subtropical island lizard,
Anolis carolinensis. Journal of evolutionary biology 29:2168-2180.
Capellini, I., J. Baker, W. L. Allen, S. E. Street, and C. Venditti. 2015. The role of life
history traits in mammalian invasion success. Ecology Letters 18:1099-1107.
Carroll, S. P., A. P. Hendry, D. N. Reznick, and C. W. Fox. 2007. Evolution on
ecological time‐scales. Functional Ecology 21:387-393.
Clarke, A. 1993. Seasonal acclimatization and latitudinal compensation in metabolism:
do they exist? Functional Ecology 7:139-149.
Colautti, R. I., and J. A. Lau. 2015. Contemporary evolution during invasion: evidence
for differentiation, natural selection, and local adaptation. Molecular Ecology
24:1999-2017.
Deutsch, C. A., J. J. Tewksbury, R. B. Huey, K. S. Sheldon, C. K. Ghalambor, D. C.
Haak, and P. R. Martin. 2008. Impacts of climate warming on terrestrial
ectotherms across latitude. Proceedings of the National Academy of Sciences
105:6668-6672.
Gerhardt, F., and S. K. Collinge. 2007. Abiotic constraints eclipse biotic resistance in
determining invasibility along experimental vernal pool gradients. Ecological
Applications 17:922-933.
Hochachka, P. W., and G. N. Somero. 1984, Biochemical adaptation, Princeton
University Press.
10
Holway, D. A., A. V. Suarez, and T. J. Case. 2002. Role of abiotic factors in governing
susceptibility to invasion: a test with Argentine ants. Ecology 83:1610-1619.
Huang, S.-P., Y. Hsu, and M.-C. Tu. 2006. Thermal tolerance and altitudinal distribution
of two Sphenomorphus lizards in Taiwan. Journal of Thermal Biology 31:378-
385.
Huey, R. B., G. W. Gilchrist, M. L. Carlson, D. Berrigan, and L. s. Serra. 2000. Rapid
evolution of a geographic cline in size in an introduced fly. Science 287:308-309.
Huey, R. B., M. R. Kearney, A. Krockenberger, J. A. Holtum, M. Jess, and S. E.
Williams. 2012. Predicting organismal vulnerability to climate warming: roles of
behaviour, physiology and adaptation. Phil. Trans. R. Soc. B 367:1665-1679.
Huey, R. B., and J. G. Kingsolver. 1989. Evolution of thermal sensitivity of ectotherm
performance. Trends in Ecology & Evolution 4:131-135.
Huey, R. B., and R. Stevenson. 1979. Integrating thermal physiology and ecology of
ectotherms: a discussion of approaches. American Zoologist 19:357-366.
Hulme, P. E. 2009. Trade, transport and trouble: managing invasive species pathways in
an era of globalization. Journal of applied ecology 46:10-18.
Irschick, D. J., and J. B. Losos. 1998. A comparative analysis of the ecological
significance of maximal locomotor performance in Caribbean Anolis lizards.
Evolution 52:219-226.
Janzen, D. H. 1967. Why mountain passes are higher in the tropics. The American
Naturalist 101:233-249.
Keane, R. M., and M. J. Crawley. 2002. Exotic plant invasions and the enemy release
hypothesis. Trends in ecology & evolution 17:164-170. 11
Kelley, A. L. 2014. The role thermal physiology plays in species invasion. Conservation
physiology 2.
Kolar, C. S., and D. M. Lodge. 2001. Progress in invasion biology: predicting invaders.
Trends in Ecology & Evolution 16:199-204.
Kolbe, J. J., R. E. Glor, L. Rodriguez Schettino, A. C. Lara, A. Larson, and J. B. Losos.
2004. Genetic variation increases during biological invasion by a Cuban lizard.
Nature 431:177-181.
Kolbe, J. J., P. S. VanMiddlesworth, N. Losin, N. Dappen, and J. B. Losos. 2012.
Climatic niche shift predicts thermal trait response in one but not both
introductions of the Puerto Rican lizard Anolis cristatellus to Miami, Florida,
USA. Ecology and evolution 2:1503-1516.
Leal, M., and A. R. Gunderson. 2012. Rapid change in the thermal tolerance of a tropical
lizard. The American Naturalist 180:815-822.
Lee, C. E. 2002. Evolutionary genetics of invasive species. Trends in ecology &
evolution 17:386-391.
Lodge, D. M., S. Williams, H. J. MacIsaac, K. R. Hayes, B. Leung, S. Reichard, R. N.
Mack et al. 2006. Biological invasions: recommendations for US policy and
management. Ecological applications 16:2035-2054.
Logan, M. L., R. M. Cox, and R. Calsbeek. 2014. Natural selection on thermal
performance in a novel thermal environment. Proceedings of the National
Academy of Sciences 111:14165-14169.
12
Losos, J. B., T. W. Schoener, K. I. Warheit, and D. Creer. 2001. Experimental studies of
adaptive differentiation in Bahamian Anolis lizards, Pages 399-415
Microevolution Rate, Pattern, Process, Springer.
McGeoch, M. A., S. H. Butchart, D. Spear, E. Marais, E. J. Kleynhans, A. Symes, J.
Chanson et al. 2010. Global indicators of biological invasion: species numbers,
biodiversity impact and policy responses. Diversity and Distributions 16:95-108.
Mooney, H. A., and E. E. Cleland. 2001. The evolutionary impact of invasive species.
Proceedings of the National Academy of Sciences 98:5446-5451.
Moyle, P. B., and T. Light. 1996. Fish invasions in California: do abiotic factors
determine success? Ecology 77:1666-1670.
Muñoz, M. M., M. A. Stimola, A. C. Algar, A. Conover, A. J. Rodriguez, M. A.
Landestoy, G. S. Bakken et al. 2014. Evolutionary stasis and lability in thermal
physiology in a group of tropical lizards. Proceedings of the Royal Society of
London B: Biological Sciences 281:20132433.
Nosil, P., T. H. Vines, and D. J. Funk. 2005. Perspective: reproductive isolation caused
by natural selection against immigrants from divergent habitats. Evolution
59:705-719.
Phillips, B. L., G. P. Brown, J. K. Webb, and R. Shine. 2006. Invasion and the evolution
of speed in toads. Nature 439:803.
Pimentel, D., R. Zuniga, and D. Morrison. 2005. Update on the environmental and
economic costs associated with alien-invasive species in the United States.
Ecological economics 52:273-288.
13
Prenter, J., C. MacNeil, J. T. Dick, and A. M. Dunn. 2004. Roles of parasites in animal
invasions. Trends in ecology & evolution 19:385-390.
Price, T. D., A. Qvarnström, and D. E. Irwin. 2003. The role of phenotypic plasticity in
driving genetic evolution. Proceedings of the Royal Society of London B:
Biological Sciences 270:1433-1440.
Pyšek, P., D. M. Richardson, J. Pergl, V. Jarošík, Z. Sixtova, and E. Weber. 2008.
Geographical and taxonomic biases in invasion ecology. Trends in Ecology &
Evolution 23:237-244.
Reznick, D. N., and C. K. Ghalambor. 2001. The population ecology of contemporary
adaptations: what empirical studies reveal about the conditions that promote
adaptive evolution, Pages 183-198 Microevolution Rate, Pattern, Process,
Springer.
Richardson, D. M., P. Pyšek, M. Rejmánek, M. G. Barbour, F. D. Panetta, and C. J. West.
2000. Naturalization and invasion of alien plants: concepts and definitions.
Diversity and distributions 6:93-107.
Sakai, A. K., F. W. Allendorf, J. S. Holt, D. M. Lodge, J. Molofsky, K. A. With, S.
Baughman et al. 2001. The population biology of invasive species. Annual review
of ecology and systematics 32:305-332.
Sax, D. F., J. J. Stachowicz, J. H. Brown, J. F. Bruno, M. N. Dawson, S. D. Gaines, R. K.
Grosberg et al. 2007. Ecological and evolutionary insights from species invasions.
Trends in ecology & evolution 22:465-471.
14
Seebacher, F., and C. E. Franklin. 2011. Physiology of invasion: cane toads are
constrained by thermal effects on physiological mechanisms that support
locomotor performance. Journal of Experimental Biology 214:1437-1444.
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.
Suarez, A. V., and N. D. Tsutsui. 2008. The evolutionary consequences of biological
invasions. Molecular Ecology 17:351-360.
Vitousek, P. M., C. M. Antonio, L. L. Loope, and R. Westbrooks. 1996. Biological
invasions as global environmental change. American scientist 84:468.
West-Eberhard, M. J. 2003, Developmental plasticity and evolution, Oxford University
Press.
While, G. M., J. Williamson, G. Prescott, T. Horváthová, B. Fresnillo, N. J. Beeton, B.
Halliwell et al. 2015. Adaptive responses to cool climate promotes persistence of
a non-native lizard. Proceedings of the Royal Society of London B: Biological
Sciences 282:20142638.
Wilcove, D. S., D. Rothstein, J. Dubow, A. Phillips, and E. Losos. 1998. Quantifying
threats to imperiled species in the United States. BioScience 48:607-615.
Williamson, M., and A. Fitter. 1996. The varying success of invaders. Ecology 77:1661-
1666.
Wilson, R. S., and C. E. Franklin. 2002. Testing the beneficial acclimation hypothesis.
Trends in Ecology & Evolution 17:66-70.
15
Zenni, R. D., and M. A. Nuñez. 2013. The elephant in the room: the role of failed
invasions in understanding invasion biology. Oikos 122:801-815.
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CHAPTER II: THERMAL PHYSIOLOGY DIFFERS BETWEEN NATIVE AND
INVASIVE POPULATIONS OF THE BROWN ANOLE (ANOLIS SAGREI)
Tamara L. Fetters, William A. Hopkins, and Joel W. McGlothlin
ABSTRACT
Invasive species often experience climatic conditions that differ from those in their native range. Invasions are commonly characterized by phenotypic trait changes that allow invaders to adapt to novel climatic regimes, such as changes in temperature and seasonality. Here we compare measures of thermal physiology between native Bahamian populations of the brown anole (Anolis sagrei) and invasive populations spanning a broad latitudinal gradient in the southeastern United States. Because both daily minimum and mean annual temperatures are lower in the invasive range, we predicted that invasive populations would exhibit greater cold tolerance than native populations and that cold tolerance would increase with latitude. We found that invasive populations tolerated lower temperatures, exhibited greater maximum sprint speeds, and had higher metabolic rates than native populations over a range of temperatures. While we found some suggestive evidence for local adaptation in the highest latitude population studied, these results were not significant, and trait measures tended to differ mainly between the native and invasive ranges rather than across latitudes. The observed changes in thermal physiology have likely facilitated the survival, persistence, and spread of the brown anole despite thermal challenges posed by their new range.
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INTRODUCTION
Invasive species often encounter novel biotic and abiotic conditions in their new range (Blackburn et al. 2011; Cox 2004; Sakai et al. 2001). Phenotypic trait changes frequently occur during invasions that allow invaders to adjust to these novel conditions, subsequently facilitating their successful establishment and spread (Flores-Moreno et al.
2015; Hänfling and Kollmann 2002; Huey et al. 2000). One common axis along which native and invasive populations tend to differ is climate, and changes in the range and variability of experienced temperatures can pose significant challenges to the survival and reproduction of the invader in their new range (Broennimann et al. 2007; Gerhardt and Collinge 2007; Kelley 2014; Moyle and Light 1996). This is particularly true in ectotherms, as performance across a number of traits, such as digestion, metabolism, behavior, locomotion, immune function, sensory output, foraging ability, and reproduction, are sensitive to changes in temperature (Angilletta et al. 2002; Bennett
1980; Huey and Kingsolver 1989; Huey and Stevenson 1979; Seebacher 2005; Stevenson et al. 1985).
Because ectotherms are strongly affected by changes in temperature, cross- latitude ectothermic invasions serve as an ideal model system to evaluate phenotypic responses to rapidly changing environmental conditions over relatively short time scales
(Angilletta 2009; Baker and Stebbins 1965; Lee 2002). Thermal environments vary substantially with changes in latitude; in particular, increases in latitude are correlated with greater fluctuation in daily and annual temperatures (Ghalambor et al. 2006; Janzen
1967). Thermal differences across latitudes are driven largely by the cooler winter and nighttime temperatures experienced at higher latitudes, as daily summer temperatures 18
tend to show less significant latitudinal variation (Ghalambor et al. 2006). Therefore, successful invaders moving from lower to higher latitudes during invasion should exhibit physiological responses that allow them to tolerate colder temperatures in their new range
(Addo-Bediako et al. 2000; Deutsch et al. 2008; Ghalambor et al. 2006).
Here we use the invasion of the brown anole (Anolis sagrei) from its native
Caribbean range to its invasive range in the southeastern United States in order to assess responses of thermal traits to colder temperatures encountered during invasion. The brown anole is native to Cuba and The Bahamas; it was first detected in southern Florida in the late nineteenth century, and has since spread into Georgia, Louisiana, and Texas
(Kolbe et al. 2004). Climatic models indicate that a thermal niche shift has occurred between the native Caribbean range and invasive southeastern range (Angetter et al.
2011), with populations in the invasive range experiencing both lower mean annual temperatures and lower minimum daily temperatures (Figure 1).
Previous work in Anolis lizards has shown differences in thermal traits across latitudes. Several studies have described increases in thermal tolerance in higher-latitude populations of Anolis carolinensis, a species native to the southeastern United States
(Campbell-Staton et al. 2018; Wilson and Echternacht 1987). Physiological trait differences have previously been examined across populations of Anolis sagrei in their invasive range in the southeastern United States (Kolbe et al. 2014). However, this prior study did not include physiological measures from native populations, and thus precludes trait comparisons across invasive and native ranges. The handful of studies that have compared thermal traits between native and invasive populations of anoline lizards have only examined differences in one thermal trait (lowest temperature tolerated) across a 19
relatively small number of populations (Anolis cristatellus in Puerto Rico versus Miami and Key Biscayne, Florida) (Kolbe et al. 2012; Leal and Gunderson 2012).
In this study, we compared two native and four invasive populations spanning a latitudinal transect of ~7°. This allowed for comparisons of thermal trait measures both between native and invasive populations and across a latitudinal gradient. We focused specifically on a suite of traits that we predicted would be affected by the colder temperatures experienced at higher latitudes in the invasive range, including lowest temperature tolerated (measured as critical thermal minimum, CTmin), locomotory performance (measured as maximum sprint speed across a range of temperatures), and metabolic rate (measured as rate of oxygen consumption across a range of temperatures).
In ectotherms, the range of experienced environmental temperatures is correlated with the range of temperatures an individual can tolerate, and even brief periods of exposure to extreme heat or cold can influence upper and lower tolerance limits
(Angilletta 2009; Deutsch et al. 2008). Because mean annual temperature and lowest minimum temperature decrease at higher latitudes, we predicted that the lowest temperature tolerated (CTmin) would be lower in more northern populations. We also predicted that because individuals from higher-latitude populations remain active at lower temperatures, sprint speeds would be faster in more northern invasive populations, particularly at low temperatures. Lastly, in accordance with the thermal compensation hypothesis (Clarke 1991; Hochachka and Somero 1984), which predicts that individuals from colder environments will compensate for temperature-induced physiological depression by increasing their metabolic rate, we hypothesized that higher-latitude
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populations would have higher metabolic rates than lower-latitude populations. Further, we predicted that this trend would be enhanced at lower environmental temperatures.
METHODS
We sampled from 6 populations of Anolis sagrei spanning a wide geographic and climatic range (Table 1). Adult males and females were collected from San Salvador, The
Bahamas in May 2014, from Andros, The Bahamas and Clearwater, Florida in March
2015, from New Orleans, Louisiana in June 2015, and from Valdosta, Georgia and
Naples, Florida in September 2015. Lizards were transported to our laboratory at Virginia
Tech upon capture, and were housed individually at 28/25°C (day/night), a 13L:11D photoperiod, and ~60% relative humidity. Lizards were fed crickets (Gryllodes sigillatus;
Ghann’s Crickets, Augusta, GA) dusted with vitamins and minerals three times per week and watered twice daily. Lizards were allowed an average of one week to acclimate to laboratory conditions before experiments began; all experiments were completed within 8 weeks from the initial date of capture. The order of tests for critical thermal minimum and maximum sprint speed were randomized, and metabolic rate was measured last for all populations.
Critical Thermal Minimum
Critical thermal minimum is a common measure that approximates the temperature at which an ectotherm loses motor capabilities (Cowles and Bogert 1944). In the laboratory, critical thermal minimum (hereafter referred to as CTmin), is measured as the lowest temperature at which a lizard can no longer right itself when placed on its back 21
(Huey and Stevenson 1979). To measure CTmin, we inserted a 30-gauge thermocouple probe connected to an Omega digital thermocouple thermometer (model HH508, sensitivity ±0.1°C) approximately 5 mm into the cloaca of the lizard. The probe was attached to the lizard’s underside using a small square of surgical tape to allow for continuous monitoring of body temperature. Before testing, we allowed lizards to equilibrate to room temperature (20-22°C). They were then placed in a plastic container that was partially submerged in an ice bath in order to cool body temperature. Once body temperature reached 14°C, we tested for righting ability by flipping the lizard on its back.
We attempted to induce a righting response by gently stimulating the hind limbs with tweezers. If the lizard did not right itself within 30 seconds, we recorded its body temperature as its CTmin. If the lizard did right itself, we cooled the lizard by an additional
1.0°C and again tested for righting ability, repeating this procedure until the lizard reached a temperature at which it could no longer right itself.
We used a generalized linear model to examine the effects of population, sex, and mass on CTmin. Tukey’s HSD post hoc tests were used to evaluate significant differences between populations.
Maximum Sprint Speed
We measured maximum sprint speed for each lizard three times across six temperatures: 12°C, 18°C, 24°C, 30°C, 36°C, and 41°C. The order of temperatures was randomized across individuals. Lizards were run at two temperatures on each test day.
Trials for the two temperatures were separated by at least two hours, with at least 48 hours between consecutive test days. 22
Lizards were kept in an incubator set to the target temperature for one hour prior to each trial. We verified that lizards reached each desired temperature with a cloacal thermometer for a subset of individuals. Lizards were placed on a 70 cm long, 3-cm diameter wooden dowel inclined at 20° to discourage jumping. We motivated lizards to run by stimulating the hind legs with a small probe. Each lizard was run three times at each temperature. We excluded a run from the dataset if lizard did not run within 30 s, if it did not remain on the vertical surface of the wooden dowel during the run, or if it could not move at least 10 consecutive cm during a run. A Kodak PlaySport video camera was mounted approximately one meter above the wooden dowel and angled so the dowel was parallel in the field of view. Trials were filmed at a rate of 30 frames per second. We calculated the maximum sprint speed for each trial using motion analysis software,
Kinovea 0.8.15 (www.kinovea.org). Prior to developing this protocol, we attempted to estimate maximum sprint speed for the San Salvador population using an automated racetrack but were unable to obtain accurate measurements from the equipment.
Therefore, this population was excluded from the analysis.
We used a general linear mixed model to examine the effects of population, temperature, sex, snout-vent length (SVL), and run number (first, second, or third) on maximum sprint speed. SVL was used because it is a linear measure of body size and speed was calculated using linear distance. Individual ID was included as a random factor.
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Metabolic Rate
We measured the rate of oxygen consumption for individuals from each population across four temperatures: 15°C, 20°C, 26°C, and 32°C. The order of temperatures was randomized across lizards. Each lizard underwent two respiration trials.
During each trial, the rate of oxygen consumption was tested at two consecutive randomized temperatures for 24 hours per temperature (48 hours total per trial). Trials were separated by at least seven days to allow for recovery. Lizards were massed prior to each trial. To limit oxygen consumption resulting from activity and digestion, we fasted animals for 48 hours prior to each trial and ran animals in total darkness. For quality assurance and control, each respiratory trial also included an empty chamber and a chamber including a medical battery that consumed a known amount of oxygen per minute, which together provided continual confirmation of respirometer performance.
We used a computer-controlled, closed-flow respirometer (Microoxymax;
Columbus Instruments, Columbus, OH, USA) connected to a large environmental chamber to allow for temperature control. Each individual was kept in a glass respirometry chamber for the duration of the trial. Trials began between 1000 and 1200 at a randomized temperature; 24 hours after the trial began, the environmental chamber was set to a second randomized temperature and run for an additional 24 hours. Oxygen
−1 consumption rates (mL h ; VO2) were measured ∼45 min after the start of the trial and every 69 min thereafter, resulting in 19 measurements of VO2 per individual per temperature and 38 measurements of VO2 per trial. Incurrent air was run through columns of anhydrous CaSO4 (Drierite) to absorb moisture before passing into individual respirometry chambers. Air leaving respirometry chambers was dried again before 24
passing into the oxygen sensor. Rates of oxygen consumption and CO2 production were simultaneously measured, and oxygen consumption values were corrected for CO2 concentrations using MicroOxymax software. Chambers were refreshed every five measurements.
We excluded the first three measurements from each temperature treatment to allow metabolic rates to stabilize. We ensured that metabolic rate had stabilized by checking the data graphically. We then estimated standard metabolic rate (SMR) by calculating the lowest quartile value for the remaining 16 measures of oxygen consumption for each temperature. This method removes peaks in oxygen consumption due to activity and circadian rhythms and has been used in previous studies (Homyack et al. 2010; Hopkins et al. 2004). Both body mass and the lowest quartile value for the rate of oxygen consumption were log10-transformed to linearize their relationship.
We used a general linear mixed model to examine the effects of population, temperature, log10 mass, and sex on the log10 lowest quartile value for the rate of oxygen consumption. Both log10 mass and sex were included in our model to account for potential variation in metabolism stemming from differences in reproductive effort between the sexes. Lizard ID was included as a random factor. Tukey’s HSD post hoc tests were used to determine significant differences between groups.
RESULTS
Critical Thermal Minimum
Anoles from all invasive populations tolerated lower temperatures than did anoles from native populations (Table 2, Figure 2, population effect: F5, 208 = 25.27, P < 0.0001). 25
Within the invasive range, lizards from Valdosta had the lowest values for CTmin, lizards from New Orleans had the highest values, and lizards from Naples and Clearwater had intermediate values. CTmin was not predicted by sex (F1,208 = 0.33, P = 0.56) or mass
(F1,208 = 2.14, P = 0.15).
Maximum Sprint Speed
The thermal sensitivity of maximum sprint speed differed among populations
(Table 3, F4,204.6 = 4.68, P = 0.0012). Across temperatures, Valdosta lizards were significantly faster than Andros and Clearwater lizards, while Naples and New Orleans exhibited intermediate speeds (Figure 3). Lizards ran faster with increasing temperatures between 18°C and 36°C, but speed did not differ significantly at temperatures both below and above this range (F5,2279 = 426.50, P < 0.0001). Females generally had faster maximum sprint speeds than males (F1,205.4 = 11.97, P = 0.0007). Smaller individuals generally had faster maximum sprint speeds (F1,205.7 = 4.47, P = 0.0356) except for at
12°C, where maximum sprint speed showed a slight positive correlation with SVL (F5,
2286 = 2.89, P = 0.011). Sprint speed decreased with each consecutive run for females, while males had a higher average maximum sprint speed the second run than their first or third runs (F2,2254 = 4.94, P = 0.0072).
Metabolic Rate
Invasive populations generally had higher rates of oxygen consumption than native populations (Table 4, Figure 4, population effect: F5,151.1 = 7.73, P <0.0001).
Additionally, there was a non-significant trend for oxygen consumption to increase with 26
increasing latitude. Oxygen consumption rate increased significantly with temperature across all four temperature treatments (F3,413.6 = 5843.11, P <0.0001). Oxygen consumption also increased with mass (F1,177.6 = 70.82, P <0.0001), and males had higher rates of oxygen consumption than females (F1,176.4 = 45.41, P <0.0001). We observed greater differences in oxygen consumption between males and females from Valdosta,
Naples, and New Orleans than from Clearwater, Andros, and San Salvador, which was reflected in significant interactions between both sex and population (F1,172.2 = 5.99, P <
0.0001) and log10 mass and population (F5,177.2 = 4.59, P = 0.0006).
DISCUSSION
Our results show that invasive brown anole populations in the southeastern United
States are more cold tolerant than are native Caribbean populations. In general, invasive populations tolerated lower temperatures better than did native populations. Invasive populations also exhibited greater maximum sprint speeds and metabolic rates across temperatures. These phenotypic shifts in thermal traits are likely the result of higher latitude invasive populations experiencing colder environmental temperatures than their native counterparts, and may be attributable to adaptation, phenotypic plasticity, or some combination of the two. There were no significant differences among populations within the invasive range. However, the northernmost population was consistently the most cold tolerant, suggesting the possibility of local adaptation to colder temperatures in higher- latitude invasives.
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Critical Thermal Minimum
Critical thermal minimum was lower across all invasive populations relative to the native populations. This result is consistent with previous work in A. cristatellus, which demonstrated significantly lower CTmin in invasive populations in Miami, Florida when compared to native populations in Puerto Rico (Kolbe et al. 2012; Leal and
Gunderson 2012). We also found a nonsignificant trend for CTmin to decrease in higher latitudes. Kolbe et al. (2014) found a similar pattern in invasive A. sagrei in the southeastern United States, with lizards from Tifton, Georgia tolerating lower temperatures than those from two locations in Florida. Taken together, these two studies suggest that the northern edge of the brown anole invasion is likely more cold tolerant.
Similar trends have also been documented in A. carolinensis occupying similar ranges in the southeastern United States (Wilson and Echternacht 1987) and more broadly across ectotherms (Sunday et al. 2011).
Decreases in survival at low temperatures may be a direct result of cold exposure or may result indirectly from the inability to escape predation, thermoregulate, and/or forage efficiently. Selection pressure is predicted to be stronger for lower thermal limits than for upper ones, because lizards cannot effectively shield against cooler winter and night-time temperatures via behavioral thermoregulation (Muñoz et al. 2014). Being able to tolerate lower temperatures should thus confer a selective advantage in the invasive range, particularly by increasing survival rates during cold winter and nighttime conditions.
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Maximum Sprint Speed
Higher-latitude invasive populations generally achieved greater maximum sprint speeds across all temperatures than lower latitude native populations. A previous study of brown anoles in the southeastern United States found that maximum sprint speed did not differ within the invasive range (Kolbe et al. 2014). In our study, we also found invasive populations generally did not differ significantly in maximum sprint speed. However, we did find significant differences between native and invasive populations. Specifically, the most northern invasive population of Valdosta was significantly faster than the native population of Andros across all tested temperatures. A study examining maximum sprint speed in lab-reared brown anoles originating from thermally contrasting environments in the native Bahamian range also found a similar patterns of consistent higher speeds across all temperatures in lizards from a cooler versus a warmer environment (Logan et al. 2018).
There may be several benefits to increased locomotory capabilities in high- latitude invasive populations. First, since brown anoles are active during the winter, the ability to achieve higher sprint speeds, especially at low temperatures, can translate into increased survival through more effective thermoregulatory behavior, greater foraging success, and decreased rates of predation (Irschick and Losos 1999; Logan et al. 2014).
Although there is no direct evidence of a relationship between sprint speed and survival in our study populations, previous work in other species indicates that faster individuals tend to have higher survival (Miles 2004; Warner and Andrews 2002). In addition, work in Bahamian populations of brown anoles has shown evidence of selection on parameters of the thermal performance curve (Logan et al. 2014). Second, during the continuous 29
range expansion experienced during invasion, traits that increase dispersal ability tend to be favored (Alford et al. 2009; Phillips et al. 2010; Travis and Dytham 2002). Higher maximum sprint speeds could potentially be attributable to a correlated response for selection on dispersal, as is the case in invasive cane toads in Australia (Phillips et al.
2006). Assessing how faster sprint speeds affect both fitness and dispersal would be useful in determining the adaptive significance of faster sprint speeds in more northern populations.
Metabolic Rate
Invasive populations in our study generally had higher rates of oxygen consumption than native populations, and there was a non-significant trend for oxygen consumption to increase with latitude. Kolbe et al. (2014) also found that metabolism differed across the invasive range of the brown anole, but in ways that did not correspond with predictions based on climate and latitude. In that study, the highest metabolism was found in an their intermediate latitude population (Orlando, Florida), followed by their lowest latitude (Miami, Florida) and highest latitude (Tifton, Georgia) populations respectively (Kolbe et al. 2014). In our study, we found the effect of population on metabolism was driven by the inclusion of native populations, with native populations generally having lower metabolic rates than all invasive populations.
Similar metabolic increases in populations from cooler, more variable climates have been observed across ectothermic groups, such as lizards (Tsuji 1988), snakes
(Lourdais et al. 2013), and insects (Addo-Bediako et al. 2002; Berrigan and Partridge
1997). Metabolic rates may increase in populations inhabiting cooler environments in 30
order to maintain similar rates for temperature-dependent physiological processes across environments, effectively offsetting the slowing effect of decreased temperatures
(Hochachka and Somero 1984). Higher metabolic rates may also allow for faster growth and reproduction, which can be beneficial in locations where breeding and growing seasons are seasonally limited (Bozinovic et al. 2011; Clarke 1993; Gotthard et al. 2000).
Invasive brown anole populations exhibit faster reproduction than native populations (Fetters and McGlothlin 2017). Combined with the higher SMR in invasive populations shown here, these results are consistent with the idea that metabolic rate sets the pace of life (Brown et al. 2004; Glazier 2015), and lends support for the expected positive correlation between metabolic rate and reproductive output. While metabolism and reproduction have been shown to covary across taxa (Boratyński et al. 2013; Le Lann et al. 2011) and an evolutionary link between the two has been suggested (Auer et al.
2018), it is unclear whether increases in metabolism in invasive populations drive increases in reproductive rate or vice versa, or alternatively whether some unknown third factor could drive increases in both.
Comparisons Across Ranges and Latitudes
We consistently found that measures for CTmin, sprint speed, and metabolic rate showed the greatest differences between the native and invasive ranges, with relatively less trait variation occurring among populations within each range. For example, while
Andros (native) and Naples (invasive) are separated by fewer than two degrees of latitude, they exhibit CTmin values that differ by ~2°C. Meanwhile, Naples (invasive) and
Valdosta (invasive) are separated by nearly five degrees of latitude, yet their CTmin values 31
differ by less than 1°C. The difference in cold tolerance across invasive populations is seemingly less than what would be predicted from the available climate data, providing no clear evidence of localized cold adaptation within the invasive range.
This apparent lack of local adaptation among invasive populations could be due to several potential factors. First, gene flow between populations may limit further phenotypic differentiation. Second, many northern populations are recently established, and there may not have been sufficient time for local adaptation to occur. Finally, temperature regimes may not be different enough across invasive populations to select for significant changes among them. Because the genetic variance within the invasive range of the brown anole has been shown to exceed that found in the native range due to multiple introductions, it is unlikely that the lack of local adaptation results from insufficient genetic variance (Kolbe et al. 2004).
A clear question raised by our findings is whether differences in cold adaptation across native and invasive populations are driven by plasticity, genetic adaption, or a combination of the two. Previous work has shown that both critical thermal minimum and metabolic rate acclimate in the lab in response to cold exposure across several species of
Anolis lizards, suggesting some level of plasticity for cold adaptive traits (Kolbe et al.
2014; Leal and Gunderson 2012; Ragland et al. 1981; Wilson and Echternacht 1987).
Additionally, common garden studies in A. carolinensis and A. sagrei provide support for a genetic basis for population differences in critical thermal minimum (Campbell-Staton et al. 2016) and maximum sprint speed (Clarke 1991; Logan et al. 2018). Future work incorporating acclimation and common garden experiments is needed to elucidate the specific mechanisms underpinning trait variation across native and invasive ranges. 32
Regardless of the underlying mechanism, our results demonstrate clear population differences in thermal traits between native and invasive brown anoles, suggesting that the ability of invasive populations to adapt to colder conditions may have enabled the brown anole to survive and spread under novel climatic regimes.
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ACKNOWLEDGEMENTS
We thank Ignacio Moore, Julie Wiemerslage, Eric Wice, Thomas Wood, Prabh Dhillon,
Kathryn Moore, John Abramyan, Iva Veselá and our undergraduate volunteers for their help collecting and caring for animals and recording data; Angela Hornsby, Kerry
Gendreau and other McGlothlin Lab members for their manuscript comments; and Forfar
Field Station in North Andros and Gerace Research Centre in San Salvador for their field accommodations.
34
REFERENCES
Addo-Bediako, A., S. Chown, and K. Gaston. 2002. Metabolic cold adaptation in insects:
a large‐scale perspective. Functional Ecology 16:332-338.
Addo-Bediako, A., S. L. Chown, and K. J. Gaston. 2000. Thermal tolerance, climatic
variability and latitude. Proceedings of the Royal Society of London B: Biological
Sciences 267:739-745.
Alford, R. A., G. P. Brown, L. Schwarzkopf, B. L. Phillips, and R. Shine. 2009.
Comparisons through time and space suggest rapid evolution of dispersal
behaviour in an invasive species. Wildlife Research 36:23-28.
Angetter, L.-S., S. Loetters, and D. Roedder. 2011. Climate niche shift in invasive
species: the case of the brown anole. Biological Journal of the Linnean Society
104:943-954.
Angilletta, M. J. 2009, Thermal adaptation: a theoretical and empirical synthesis, Oxford
University Press.
Angilletta, M. J., P. H. Niewiarowski, and C. A. Navas. 2002. The evolution of thermal
physiology in ectotherms. Journal of thermal Biology 27:249-268.
Auer, S. K., C. A. Dick, N. B. Metcalfe, and D. N. Reznick. 2018. Metabolic rate evolves
rapidly and in parallel with the pace of life history. Nature communications 9:14.
Baker, H. G., and G. L. Stebbins. 1965, Genetics of colonizing species, proceedings
International Union of Biological Sciences Symposia on General Biology 1964:
Asilomar, Calif.).
Bennett, A. F. 1980. The thermal dependence of lizard behaviour. Animal Behaviour
28:752-762. 35
Berrigan, D., and L. Partridge. 1997. Influence of temperature and activity on the
metabolic rate of adult Drosophila melanogaster. Comparative Biochemistry and
Physiology Part A: Physiology 118:1301-1307.
Blackburn, T. M., P. Pyšek, S. Bacher, J. T. Carlton, R. P. Duncan, V. Jarošík, J. R.
Wilson et al. 2011. A proposed unified framework for biological invasions.
Trends in ecology & evolution 26:333-339.
Boratyński, Z., E. Koskela, T. Mappes, and E. Schroderus. 2013. Quantitative genetics
and fitness effects of basal metabolism. Evolutionary Ecology 27:301-314.
Bozinovic, F., P. Calosi, and J. I. Spicer. 2011. Physiological correlates of geographic
range in animals. Annual Review of Ecology, Evolution, and Systematics 42:155-
179.
Broennimann, O., U. A. Treier, H. Müller‐Schärer, W. Thuiller, A. Peterson, and A.
Guisan. 2007. Evidence of climatic niche shift during biological invasion.
Ecology letters 10:701-709.
Brown, J. H., J. F. Gillooly, A. P. Allen, V. M. Savage, and G. B. West. 2004. Toward a
metabolic theory of ecology. Ecology 85:1771-1789.
Campbell-Staton, S. C., A. Bare, J. B. Losos, S. V. Edwards, and Z. A. Cheviron. 2018.
Physiological and regulatory underpinnings of geographic variation in reptilian
cold tolerance across a latitudinal cline. Molecular ecology.
Campbell-Staton, S. C., S. V. Edwards, and J. B. Losos. 2016. Climate‐mediated
adaptation after mainland colonization of an ancestrally subtropical island lizard,
Anolis carolinensis. Journal of evolutionary biology 29:2168-2180.
36
Clarke, A. 1991. Cold adaptation. Journal of Zoology 225:691-699.
—. 1993. Seasonal acclimatization and latitudinal compensation in metabolism: do they
exist? Functional Ecology 7:139-149.
Cox, G. W. 2004, Alien species and evolution: the evolutionary ecology of exotic plants,
animals, microbes, and interacting native species, Island Press.
Deutsch, C. A., J. J. Tewksbury, R. B. Huey, K. S. Sheldon, C. K. Ghalambor, D. C.
Haak, and P. R. Martin. 2008. Impacts of climate warming on terrestrial
ectotherms across latitude. Proceedings of the National Academy of Sciences
105:6668-6672.
Fetters, T. L., and J. W. McGlothlin. 2017. Life histories and invasions: accelerated
laying rate and incubation time in an invasive lizard, Anolis sagrei. Biological
Journal of the Linnean Society 122:635-642.
Flores-Moreno, H., E. S. García-Treviño, A. D. Letten, and A. T. Moles. 2015. In the
beginning: phenotypic change in three invasive species through their first two
centuries since introduction. Biological invasions 17:1215-1225.
Gerhardt, F., and S. K. Collinge. 2007. Abiotic constraints eclipse biotic resistance in
determining invasibility along experimental vernal pool gradients. Ecological
Applications 17:922-933.
Ghalambor, C. K., R. B. Huey, P. R. Martin, J. J. Tewksbury, and G. Wang. 2006. Are
mountain passes higher in the tropics? Janzen's hypothesis revisited. Integrative
and comparative biology 46:5-17.
Glazier, D. S. 2015. Is metabolic rate a universal ‘pacemaker’for biological processes?
Biological Reviews 90:377-407. 37
Gotthard, K., S. Nylin, and C. Wiklund. 2000. Individual state controls temperature
dependence in a butterfly (Lasiommata maera). Proceedings of the Royal Society
of London B: Biological Sciences 267:589-593.
Hänfling, B., and J. Kollmann. 2002. An evolutionary perspective of biological invasions.
Trends in Ecology & Evolution 17:545-546.
Hochachka, P. W., and G. N. Somero. 1984, Biochemical adaptation, Princeton
University Press.
Homyack, J. A., C. A. Haas, and W. A. Hopkins. 2010. Influence of temperature and
body mass on standard metabolic rate of eastern red-backed salamanders
(Plethodon cinereus). Journal of Thermal Biology 35:143-146.
Hopkins, W., J. Roe, T. Philippi, and J. Congdon. 2004. Standard and digestive
metabolism in the banded water snake, Nerodia fasciata fasciata. Comparative
Biochemistry and Physiology Part A: Molecular & Integrative Physiology
137:141-149.
Huey, R. B., G. W. Gilchrist, M. L. Carlson, D. Berrigan, and L. s. Serra. 2000. Rapid
evolution of a geographic cline in size in an introduced fly. Science 287:308-309.
Huey, R. B., and J. G. Kingsolver. 1989. Evolution of thermal sensitivity of ectotherm
performance. Trends in Ecology & Evolution 4:131-135.
Huey, R. B., and R. Stevenson. 1979. Integrating thermal physiology and ecology of
ectotherms: a discussion of approaches. American Zoologist 19:357-366.
Irschick, D. J., and J. B. Losos. 1999. Do lizards avoid habitats in which performance is
submaximal? The relationship between sprinting capabilities and structural habitat
use in Caribbean anoles. The American Naturalist 154:293-305. 38
Janzen, D. H. 1967. Why mountain passes are higher in the tropics. The American
Naturalist 101:233-249.
Kelley, A. L. 2014. The role thermal physiology plays in species invasion. Conservation
physiology 2.
Kolbe, J. J., J. C. Ehrenberger, H. A. Moniz, and M. J. Angilletta, Jr. 2014. Physiological
variation among invasive populations of the brown anole (Anolis sagrei).
Physiological and Biochemical Zoology 87:92-104.
Kolbe, J. J., R. E. Glor, L. Rodriguez Schettino, A. C. Lara, A. Larson, and J. B. Losos.
2004. Genetic variation increases during biological invasion by a Cuban lizard.
Nature 431:177-181.
Kolbe, J. J., P. S. VanMiddlesworth, N. Losin, N. Dappen, and J. B. Losos. 2012.
Climatic niche shift predicts thermal trait response in one but not both
introductions of the Puerto Rican lizard Anolis cristatellus to Miami, Florida,
USA. Ecology and evolution 2:1503-1516.
Le Lann, C., T. Wardziak, J. Van Baaren, and J. J. van Alphen. 2011. Thermal plasticity
of metabolic rates linked to life‐history traits and foraging behaviour in a
parasitic wasp. Functional Ecology 25:641-651.
Leal, M., and A. R. Gunderson. 2012. Rapid change in the thermal tolerance of a tropical
lizard. The American Naturalist 180:815-822.
Lee, C. E. 2002. Evolutionary genetics of invasive species. Trends in ecology &
evolution 17:386-391.
39
Logan, M. L., R. M. Cox, and R. Calsbeek. 2014. Natural selection on thermal
performance in a novel thermal environment. Proceedings of the National
Academy of Sciences 111:14165-14169.
Logan, M. L., J. D. Curlis, A. L. Gilbert, D. B. Miles, A. K. Chung, J. W. McGlothlin,
and R. M. Cox. 2018. Thermal physiology and thermoregulatory behaviour
exhibit low heritability despite genetic divergence between lizard populations.
Proc. R. Soc. B 285:20180697.
Lourdais, O., M. Guillon, D. DeNardo, and G. Blouin-Demers. 2013. Cold climate
specialization: adaptive covariation between metabolic rate and thermoregulation
in pregnant vipers. Physiology & behavior 119:149-155.
Miles, D. B. 2004. The race goes to the swift: fitness consequences of variation in sprint
performance in juvenile lizards. Evolutionary Ecology Research 6:63-75.
Moyle, P. B., and T. Light. 1996. Fish invasions in California: do abiotic factors
determine success? Ecology 77:1666-1670.
Muñoz, M. M., M. A. Stimola, A. C. Algar, A. Conover, A. J. Rodriguez, M. A.
Landestoy, G. S. Bakken et al. 2014. Evolutionary stasis and lability in thermal
physiology in a group of tropical lizards. Proceedings of the Royal Society of
London B: Biological Sciences 281:20132433.
Phillips, B. L., G. P. Brown, and R. Shine. 2010. Life‐history evolution in range‐
shifting populations. Ecology 91:1617-1627.
Phillips, B. L., G. P. Brown, J. K. Webb, and R. Shine. 2006. Invasion and the evolution
of speed in toads. Nature 439:803.
40
Ragland, I. M., L. C. Wit, and J. C. Sellers. 1981. Temperature acclimation in the lizards
Cnemidophorus sexlineatus and Anolis carolinensis. Comparative Biochemistry
and Physiology Part A: Physiology 70:33-36.
Sakai, A. K., F. W. Allendorf, J. S. Holt, D. M. Lodge, J. Molofsky, K. A. With, S.
Baughman et al. 2001. The population biology of invasive species. Annual review
of ecology and systematics 32:305-332.
Seebacher, F. 2005. A review of thermoregulation and physiological performance in
reptiles: what is the role of phenotypic flexibility? Journal of Comparative
Physiology B 175:453-461.
Shaklee, R. V. 1996, Weather and climate, San Salvador Island. The Bahamian Field
Station Limited: San Salvador, Bahamas.
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.
Sunday, J. M., A. E. Bates, and N. K. Dulvy. 2011. Global analysis of thermal tolerance
and latitude in ectotherms. Proceedings of the Royal Society of London B:
Biological Sciences 278:1823-1830.
Travis, J. M., and C. Dytham. 2002. Dispersal evolution during invasions. Evolutionary
Ecology Research 4:1119-1129.
Tsuji, J. S. 1988. Seasonal profiles of standard metabolic rate of lizards (Sceloporus
occidentalis) in relation to latitude. Physiological Zoology 61:230-240.
41
Warner, D. A., and R. M. Andrews. 2002. Laboratory and field experiments identify
sources of variation in phenotypes and survival of hatchling lizards. Biological
Journal of the Linnean Society 76:105-124.
Wilson, M. A., and A. C. Echternacht. 1987. Geographic variation in the critical thermal
minimum of the green anole, Anolis carolinensis (Sauria: Iguanidae), along a
latitudinal gradient. Comparative Biochemistry and Physiology Part A:
Physiology 87:757-760.
42
Table 1. Characteristics of study populations.
Location Range Coordinates Males Mass Females Mass (Abbreviation) (g) (g) San Salvador, The native 24.07° N, 74.27° 20 4.42 19 1.66 Bahamas (S) W North Andros, The native 24.70° N, 78.02° 20 3.30 21 1.26 Bahamas (A) W Naples, Florida (P) invasive 26.08° N, 81.47° 17 4.19 12 1.94 W Clearwater, FL (C) invasive 27.97° N, 82.80° 24 5.10 23 2.12 W New Orleans, LA invasive 29.95° N, 90.07° 21 5.69 20 2.38 (N) W Valdosta, GA (V) invasive 30.83° N, 83.28° 15 4.40 10 2.07 W
43
Table 2. General linear model results for critical thermal minimum.
Fixed Effects F df P Population 25.27 5, 202 <0.0001 Sex 0.33 1, 202 0.5641 Mass 2.14 1, 202 0.1451 Population × sex 1.09 5, 202 0.3651
44
Table 3. General linear mixed model results for maximum sprint speed.
Random Effect % Var P Lizard ID 36.96 <0.0001
Fixed Effects F df P Population 4.68 4, 204.6 0.0012 Temperature 426.50 5, 2279 <0.0001 Sex 11.97 1, 205.4 0.0007 SVL 4.47 1, 205.7 0.0356 Run number 2.53 2, 2259 0.0801 Population × temperature 2.61 20, 2281 0.0001 Sex × population 2.26 4, 204.7 0.0642 Sex × temperature 5.49 5, 2287 <0.0001 Sex × run number 4.94 2, 2254 0.0072 Population × run number 1.85 8, 2254 0.0645 Temperature × run number 1.26 10, 2255 0.2495 Temperature × SVL 2.98 5, 2286 0.0111 Sex × population × temperature 2.48 20, 2280 0.0003
45
Table 4. General linear mixed model results for metabolic rate.
Random Effect % Var P Lizard ID 42.95 <0.0001
Fixed Effects F df P Population 7.73 5, 151.1 <0.0001 Temperature 5843.11 3, 413.6 <0.0001 Sex 45.41 1, 176.4 <0.0001 Log mass 70.82 1, 177.6 <0.0001 Population × temperature 1.23 15, 413.6 0.2437 Population × log mass 4.59 5, 177.2 0.0006 Population × sex 5.99 5, 172.2 <0.0001 Temperature × sex 0.64 3, 413.6 0.5922 Log mass × sex 3.42 1, 164.7 0.0661 Population × temperature × sex 0.75 15, 413.2 0.7335
46
Figure 1. Temperature data from study populations. Circles represent mean annual temperatures. Native populations are depicted as closed circles and invasive populations as open circles. Bars represent averages for lowest and highest recorded temperature for each year included in the dataset. Temperature data for San Salvador are from 1955-1980 (Shaklee 1996). For all other populations, data are from 1981-2015. Data for North Andros was obtained from the National Climatic Data Center. Data for Naples, Clearwater, New Orleans, and Valdosta was obtained from the PRISM Climate Group (2017).
47
Figure 2. Critical thermal minimum values across populations. Invasive populations (open circles) had lower critical thermal minimum values than native populations (closed circles). Populations are listed in order of increasing latitude (S=San Salvador, The Bahamas; A=North Andros, The Bahamas; P=Naples, Florida; C=Clearwater, Florida; N=New Orleans, Louisiana; V=Valdosta Georgia). Circles represent the average value for each population; bars represent standard error. Letters indicate significantly different groups (Tukey’s HSD).
48
Figure 3. Maximum sprint speeds across temperatures and populations. Maximum sprint speed increased with temperature. Across temperatures, sprint speeds were generally higher for the most northern invasive population (V) than the native population (A). Populations are delineated as in Figure 1.
49
0.11 15°C 0.10
0.09
0.08
0.07
0.06 S A P C N V 0.18 20°C
0.16 /hr) 2
0.14
0.12
0.10 S A P C N V
0.32 26°C
0.30
0.28
0.26
0.24
0.22 S A P C N V Standard Metabolic Rate (ml O 0.55 32°C
0.50
0.45
0.40
0.35 S A P C N V
50
Figure 4. Standard Metabolic rates across temperatures and populations. Invasive populations generally had higher rates of oxygen consumption than native populations. Populations are delineated as in Figure 1. Circles represent the backtransformed least squares mean value for each population, which was used to adjust for model covariates such as mass and sex. Bars represent back-transformed standard errors. Letters indicate significantly different groups (Tukey’s HSD). Note that the scale for standard metabolic rate changes with temperature.
51
CHAPTER III: LIFE HISTORIES AND INVASIONS: ACCELERATED LAYING
RATE AND INCUBATION TIME IN AN INVASIVE LIZARD, ANOLIS SAGREI
Tamara L. Fetters and Joel W. McGlothlin
This article has been published as:
Fetters, T.L., and McGlothlin, J.W. 2017. Life histories and invasions: accelerated
laying rate and incubation time in an invasive lizard, Anolis sagrei. Biological
Journal of the Linnean Society 122:635-642.
ABSTRACT
Faster life histories are correlated with greater invasion success across taxa. However, comparisons of life-history traits across native and invasive ranges are rare, and thus it is unknown whether invasions lead directly to evolutionary shifts in life histories. Here we compare life history traits of three invasive populations of brown anoles (Anolis sagrei) to a representative native population. In a common garden, we measured a number of reproductive traits including egg-laying rate and incubation period. We hypothesized that invasive populations would exhibit faster reproduction because fast life histories are favoured both by the invasion itself and by shorter breeding seasons in the invasive range. Compared to native females, invasive females had shorter interlaying intervals and produced eggs that hatched more quickly. Invasive and native populations did not differ consistently in egg size, hatching success, or hatchling size. Our results indicate that life
52
history traits have rapidly diverged during the brown anole invasion, potentially facilitating the successful establishment and expansion of the species range.
INTRODUCTION
Across taxa, successful invaders often display fast life histories with rapid reproductive rates that help facilitate their establishment in new environments (Allen, Street &
Capellini, 2017; Capellini et al., 2015; Richardson & Rejmanek, 2004). For example, phylogenetic comparisons show that invasive amphibians and reptiles that have large or frequent clutches are more likely to become established and subsequently spread, while successful mammalian invaders typically exhibit large litter sizes and a long reproductive lifespan (Allen et al., 2017; Capellini et al., 2015). Studies that compare the life histories of the same species across native and invasive ranges are rare, however, and it is unclear whether successful invaders possess fast life history traits prior to invasion or whether life history traits tend to change during invasion events.
Changes in life history traits in non-native populations may be favoured by several factors. First, invasion itself may exert pressure for faster reproduction. During the early stages of invasion, populations experience rapid growth, and individuals that reproduce either faster or earlier are at a selective advantage (Dobson, 2007). Faster reproduction can also offset high mortality rates that are often observed in novel environments (Tayeh et al., 2015). Second, novel environmental conditions experienced by invaders may exert pressure for faster reproduction. This is particularly likely when ectotherms invade cooler climates, due to shorter breeding seasons (Goldberg, 1974; Lee et al., 1989; Morrison & Hero, 2003) and reduced access to conditions optimal for 53
offspring growth and development (Qualls & Shine, 2000; Warner & Shine, 2007).
Invaders may respond to such challenges by laying larger or more frequent clutches or producing faster-hatching eggs (Adolph & Porter, 1993).
The brown anole (Anolis sagrei) is a small lizard that has been introduced into the south-eastern United States within the past 150 years from a mixture of native populations in Cuba and The Bahamas (Kolbe et al., 2004). While initially limited to southern Florida, populations began to expand rapidly in the mid-twentieth century, now reaching the current range of as far north as Georgia and as far west as Texas. The exponential range expansion and high population densities achieved by the brown anole characterize conditions under which selection for faster reproduction is predicted to occur
(Burton, Phillips & Travis, 2010; Kolbe et al., 2004; Lee, 2010; Schoener & Schoener,
1980).
Additionally, invasive populations of brown anoles experience significantly lower temperatures (Angetter, Lotters & Rodder, 2017) and more pronounced seasonality relative to their native range. This should lead to selection for faster reproduction to compensate for shortened breeding and growing seasons. Anoles have an unusual system of reproduction: instead of producing multiple-egg clutches like most lizards, females are constrained to lay a single egg at a time (Losos 2009). Female anoles produce eggs more or less continuously throughout a lengthy breeding season (Lee et al., 1989). The inability to adjust clutch size may cause anoles to be especially sensitive to selection favouring faster reproduction because more of their eggs will be exposed to the suboptimal conditions that occur early and late in the breeding season. Such selection may result in either increased egg-laying rate or alterations of egg characteristics to 54
facilitate hatching success and/or survival of hatchlings. Because seasonality increases with distance from the equator, selection may be more intense in higher latitudes, leading to latitudinal gradients in reproductive traits.
Here we test for increases in reproductive rate during an invasion by comparing two traits associated with quickened reproduction, rate of egg laying and egg incubation period, across native and invasive populations of the brown anole in a common laboratory environment. We hypothesized that because both invasion and increased seasonality favour faster life histories, invasive females should have faster egg-laying rates and produce faster-hatching eggs than native females, with reproductive rates increasing in higher latitudes. Additionally, because changes to life history traits are often associated with some cost (Stearns, 1992), we measured egg mass, hatchling mass, and hatching success as potential trade-offs with reproductive speed.
METHODS
Between March and September 2015, we collected adult A. sagrei from one native population and three invasive populations spanning a broad latitudinal and climatic range
(Table 1). We chose North Andros as a representative population from the native range for two reasons. First, as previously noted, the invasion of North America derives from a mixture of Bahamian and Cuban populations. We could not sample from Cuba due to permit restrictions, but Andros serves as the best Bahamian approximation for Cuba.
Andros is the largest of the Bahamian islands, and is geographically proximal to Cuba; the Great Bahamas bank it occupies was separated from Cuba by ~15 km during the last ice age (Losos, 2009). Brown anoles from Andros are more closely related to populations 55
from West-Central Cuba than to many other Bahamian populations (Kolbe et al., 2004).
Second, as shown below, North Andros females exhibit reproductive traits characteristic of native populations other Bahamian islands.
After capture, lizards were housed individually in a laboratory colony at 28/25°C
(day/night), a 13L:11D photoperiod, and ~60% relative humidity. Lizards were fed crickets dusted with vitamins and minerals and watered regularly. We paired lizards in
June 2016 (Andros, n = 22 pairs; Clearwater, n = 10 pairs; New Orleans, n = 12 pairs;
Valdosta, n = 8 pairs). Each male was housed with a single female for 90 d in a 37 cm ×
22 cm × 25 cm cage (Lee’s Kritter Keeper). Females were provided with a small container of potting soil for depositing eggs. We began to check for eggs three weeks after pairing, and then continued to check for eggs 5 times per week throughout the course of the experiment. Eggs (n = 612) were transferred to a container with a 1:1 mixture of vermiculite and water and placed in a 28°C incubator, which was checked twice daily for hatchlings until all eggs had either hatched (n = 458) or failed to hatch
(within 8 weeks, n = 154). Five females and one male died during the course of the experiment. The first female did not die until 68 d after pairing. The male died 35 d after pairing, but the female continued to lay eggs regularly using stored sperm. Therefore, we retained laying data from all females in the dataset.
We compared reproductive rates across populations using two traits, interlaying interval (the duration between consecutive egg lays) and incubation period (number of days until hatching). For each egg (excluding the first egg found), we calculated interlaying interval as the number of days elapsed since the last egg was found. If more than egg was found on the same day, one egg was assigned a value in the normal way, 56
while the remaining eggs were assigned a value of zero. Of the 72 eggs receiving a value of zero, 59 were found in a female’s first egg check. This occurred in 29 out of the 50 females in the experiment (range = 2 – 7 eggs, average = 3.03). For eggs that successfully hatched, incubation period was calculated as the number of days between egg discovery and hatching. Analyses of incubation period included only hatchlings from eggs with known lay dates (i.e., from eggs found the day after an egg check had been performed; n
= 195).
We measured egg mass, hatchling mass, and hatching success as potential trade- offs with reproductive rate. Eggs and hatchlings were weighed immediately after being found using a digital balance (± 0.01 g). Hatching success for each egg was scored as either 1 (hatched) or 0 (did not hatch). Analyses of these three variables used only eggs/hatchlings for which the exact lay date was known (n = 256 eggs, 195 hatchlings).
We used linear mixed models to test for differences in interlaying intervals, incubation period, egg mass, and hatching mass among populations. Each model included population as a fixed factor, number of days since pairing as a covariate, and female ID as a random factor. Analysis of interlaying interval also included two additional fixed factors indicating whether an the lay dates of the focal egg and the previously laid egg were exactly known, as well as all possible interactions between fixed factors. To analyse population differences in hatching success, we used a generalized linear mixed model with binomial errors including the same fixed and random effects. Tukey’s HSD post hoc tests were used when appropriate to evaluate significant differences between specific population pairs. As noted in the results, we explored alternative models including various covariates (female mass, which was measured at the end of the experiment, egg 57
mass, and incubation period, as appropriate) as potential explanations for population differences. Population × covariate interactions were initially included, but were removed from the model if P > 0.1.
To ensure that North Andros was representative of other populations in the native range, we also estimated interlaying interval and incubation period from previous studies of brown anoles bred using identical methods. These data derive from studies of
Bahamian island populations, listed here in order of increasing latitude: Great Exuma
(23.50 N, 75.78 W, 83 pairings in 2012 producing 361 eggs, R. M. Cox and J. W.
McGlothlin, unpublished data), San Salvador (24.11 N, 74.46 W, 82 pairings in 2014-
2016 producing 285 eggs, J. W. McGlothlin, unpublished data), Eleuthera (24.70 N,
76.20 W, 71 pairings in 2012 producing 341 eggs, R. M. Cox and J. W. McGlothlin, unpublished data), North Andros (24.70 N, 78.02 W, 79 pairings in 2015-2016 producing
450 eggs, J. W. McGlothlin unpublished data), and South Bimini (25.70 N, 79.28 W, 101 pairings in 2006-2009 producing 465 eggs, J. W. McGlothlin, J. B. Losos, and E. D.
Brodie III, unpublished data). In all studies, a single female was paired with a male for between 13 and 130 days and then placed in an individual cage where she was allowed to lay eggs using stored sperm into a small container of potting soil. We included eggs found within the first 90 days after pairing in our data set. In these studies, we collected eggs weekly instead of daily, which should cause interlaying interval to be estimated with greater error and incubation period to be biased downward because eggs will have incubated in a female’s cage for an average of 3.5 days before being found. For this reason, we added 3.5 days to the observed incubation period for each egg. Interlaying interval and incubation period were estimated for each population as least-squares means 58
from mixed models including population as a fixed factor, days since pairing as a covariate, a population × days since pairing interaction, and female as a random effect.
RESULTS
Interlaying Interval
Females from invasive populations laid eggs more rapidly than did females from the native population, with average interlaying intervals that were shorter by ~2 d (Figs. 1,
2A; Table 2A). Knowledge of the exact laying date of the previous egg, but not the focal egg, significantly affected our estimate of interlaying interval (increase of 0.6 d if exact date was known, Table 2A). In addition, egg production slowed somewhat over the course of the experiment, with interlaying intervals lengthening at a rate of 0.069 d per diem (Fig. 1, Table 2A). This effect remained significant (P = 0.03) even when only exact estimates of interlaying interval were retained in the model (n = 77), suggesting it was not driven by eggs found during the first egg check. Adding female mass as a covariate did not produce a significant effect (Table 2A) or alter the results of the model, even though invasive females were on average ~30% (0.6 g) heavier than native females (ANOVA,
F3, 41 = 4.52, P < 0.0001; invasive populations did not significantly differ from one another in mass, Tukey’s HSD).
Incubation Period
Eggs from all invasive populations hatched ~5 d faster than eggs from the native population (Fig. 2B; Table 2A). There was also a significant interaction between days since pairing and population, with later-laid eggs hatching slightly faster in the native 59
population (-0.039 d d-1) but not in the invasive populations (Table 2A). Neither egg mass nor female mass affected incubation time when added as covariates (Table 2A).
Potential Fitness Costs of Increased Reproductive Rate
Egg mass did not differ significantly among populations, although eggs from New
Orleans and North Andros showed a non-significant trend toward being slightly smaller
(Fig. 2C, Table 2B). Eggs became significantly lighter at laying over the course of the experiment in all populations, but this decrease was very small (0.0004 g d-1; Table 2B).
Female mass had no effect when added as a covariate (Table 2B).
Populations did not differ in overall hatching success, but there was a significant population × days since pairing interaction (Table 2B). Specifically, later-laid eggs were less likely to hatch in the native population but not in the invasive populations. This effect remained when female mass and egg mass (which did not significantly affect hatching success, Table 2B) were included in the model as covariates.
Populations differed in hatchling mass, with hatchlings from New Orleans significantly smaller than the two southernmost populations, and Valdosta hatchlings intermediate (Fig. 2D; Table 2B). Later hatchlings were significantly smaller than earlier hatchlings (0.0004 g d-1; Table 2B). Including covariates (female mass, egg mass, and incubation time) in this model removed the population effect (F3, 36.21 = 1.74, P = 0.18) and revealed that both egg mass (0.70 g g-1) and incubation period (0.004 g d-1) affected hatchling mass (Table 2B). Specifically, smaller, faster-hatching eggs produced smaller hatchlings, suggesting a potential fitness cost of rapid reproduction.
60
Reproductive Rate Across the Native Range
Using data from previous studies, we found that North Andros females have a reproductive rate comparable to that of other populations in the native range. We found significant population differences in both interlaying interval and incubation period (P <
0.0001). In this dataset, North Andros had an interlaying interval (least-squares mean ±
S.E., 9.08 ± 0.422 d) indistinguishable from that of the nearby South Bimini (9.29 ±
0.437 d), but significantly faster than the other three islands (San Salvador, 11.98 ± 0.540 d, Eleuthera 12.57 ± 0.478 d, Great Exuma, 13.05 ± 0.468 d; Tukey HSD). The incubation period of North Andros (35.26 ± 0.19) was indistinguishable from both South
Bimini (35.25 ± 0.17) and San Salvador (34.45 ± 0.23) but significantly faster than
Eleuthera (37.02 ± 0.24) and Great Exuma (37.24 ± 0.21).
DISCUSSION
Our results show that invasive populations of A. sagrei have quickened reproduction compared to their native range by significantly reducing both interlaying interval and egg incubation period. This finding corroborates comparative studies showing that successful invasion is correlated with more rapid life history traits and provides an example of reproductive traits becoming faster in a species during a single invasion event (Allen et al., 2017; Capellini et al., 2015; Richardson & Rejmanek, 2004). The differences in both egg incubation time and interlaying interval between the native and invasive ranges were striking in their magnitude, with interlaying interval decreasing by over 30% and egg incubation time decreasing by over 15% in invasive populations. A decrease in incubation period has been previously observed in wall lizards that have invaded cooler 61
climates (While et al., 2015); however, to our knowledge, this represents the first observed increase in egg-laying rate during a reptile invasion.
While data from our previous breeding experiments show that there is some variation in reproductive traits across Bahamian islands, when compared to our invasive populations, we found that invasive populations are consistently faster in their interlaying interval and incubation time than all native populations measured. Because reproductive traits were measured in a common environment, differences between native and invasive populations cannot have arisen from immediate environmental effects. Population differences may be attributable to some combination of three potential sources: a founder effect, adaptive evolution, or developmental plasticity. A founder effect is unlikely, as admixture of individuals from multiple genetically distinct introductions has increased genetic variation in invasive populations relative to source populations (Kolbe et al.,
2008). We have not yet tested for population differences in reproductive traits of lab- reared offspring, and thus we cannot distinguish between adaptive evolution and developmental plasticity as a source of population differences. If the observed differences are indeed a response to selection during the invasion, this would represent rather rapid evolution, as the observed shift would have occurred within the past 150 years.
We found no differences in reproductive rate within the invasive range, suggesting that reproductive rate has not yet diverged further following invasion.
Previous studies have shown latitudinal variation in incubation period in various lizard genera across large geographic ranges (Du et al., 2010; Goodman, 2008); however, these studies have focused primarily on native lizards. Most of the brown anole’s range expansion has happened over the last half century, and newer edge populations may not 62
have been established long enough for local adaptation to have occurred. Alternatively, gene flow, a lack of genetic variation, or lack of selection for a further increase in reproductive rate in northern populations may explain the lack of differentiation.
We found no evidence that populations with accelerated reproductive rates consistently suffer a fitness cost in terms of egg mass or hatching success. While we found no relationship between female mass and egg-laying rate, invasive females tended to be larger, potentially alleviating some of the energetic costs of laying eggs more rapidly. In contrast, faster-hatching eggs produced smaller hatchlings, particularly in our most northern populations, suggesting a potential trade-off and the possibility that expansion at the northern edge of the range may be limited by reduced survival of smaller juveniles.
Of the small portion of invasions that result in the establishment of a non-native population, even fewer go on to proliferate and spread, and understanding what distinguishes successful invaders is a longstanding pursuit of invasion biology (Kolar &
Lodge, 2001; Mack et al., 2000). Our results suggest that life-history traits can change rapidly during an invasion and potentially facilitate its success. The shift in invasive populations to shorter incubation periods and interlaying intervals has likely contributed to the establishment, persistence, and range expansion of the brown anole in its invasive range, particularly into areas with novel climatic conditions. Future studies examining changes in life-history during invasions are needed for a more general understanding of how evolution contributes to the success of invasions.
63
ACKNOWLEDGMENTS
We thank Tyler Miller, Ignacio Moore, Emily Watts, Thomas Wood, Eric Wice, Julie
Wiemerslage, and numerous undergraduate volunteers for assistance with collecting animals, animal care, and data collection; Edmund Brodie III and Robert Cox for providing access to reproductive data; Sarah Foltz, Angela Hornsby, and other
McGlothlin lab members for manuscript comments; Robin Andrews and Jonathan Losos for helpful discussions; and Forfar Field Station for accommodations while in North
Andros. We also thank Daniel Mesquita and two anonymous reviewers for their helpful comments on the manuscript. T.L. Fetters was supported by a NSF Graduate Research
Fellowship (DGE-1651272). Research was supported by a Rosemary Grant Award from the Society for the Study of Evolution and Virginia Tech. All procedures were approved by the Virginia Tech Institutional Animal Care and Use Committee (protocol 13-105), collections were made under permits from The Bahamas Ministry of Agriculture and
BEST Commission, and import of animals was approved by the United States Fish and
Wildlife Service.
64
REFERENCES
Adolph SC, Porter WP. 1993. Temperature, activity, and lizard life histories. The
American Naturalist 142: 273-295.
Allen WL, Street SE, Capellini I. 2017. Fast life history traits promote invasion success
in amphibians and reptiles. Ecology Letters 20: 222-230.
Angetter LS, Lotters S, Rodder D. 2017. Climate niche shift in invasive species: the case
of the brown anole. Biological Journal of the Linnean Society 104: 943-954.
Burton OJ, Phillips BL, Travis JM. 2010. Trade-offs and the evolution of life-histories
during range expansion. Ecology Letters 13: 1210-1220.
Capellini I, Baker J, Allen WL, Street SE, Venditti C. 2015. The role of life history traits
in mammalian invasion success. Ecology Letters 18: 1099-1107.
Dobson FS. 2007. A lifestyle view of life-history evolution. Proceedings of the National
Academy of Sciences USA 104: 17565-17566.
Du W-G, Warner DA, Langkilde T, Robbins T, Shine R. 2010. The physiological basis of
geographic variation in rates of embryonic development within a widespread
lizard species. The American Naturalist 176: 522-528.
Goldberg SR. 1974. Reproduction in mountain and lowland populations of the lizard
Sceloporus occidentalis. Copeia: 176-182.
Goodman RM. 2008. Latent effects of egg incubation temperature on growth in the lizard
Anolis carolinensis. Journal of Experimental Zoology Part A: Ecological
Genetics and Physiology 309: 525-533.
Kolar CS, Lodge DM. 2001. Progress in invasion biology: predicting invaders. Trends in
Ecology & Evolution 16: 199-204. 65
Kolbe JJ, Glor RE, Rodriguez Schettino L, Lara AC, Larson A, Losos JB. 2004. Genetic
variation increases during biological invasion by a Cuban lizard. Nature 431: 177-
181.
Kolbe JJ, Larson A, Losos JB, de Queiroz K. 2008. Admixture determines genetic
diversity and population differentiation in the biological invasion of a lizard
species. Biology Letters 4: 434-437.
Lee CE. 2010. Evolution of invasive populations. Proceedings of the National Academy
of Sciences USA 104: 15793-15798.
Lee JC, Clayton D, Eisenstein S, Perez I. 1989. The reproductive cycle of Anolis sagrei
in Southern Florida. Copeia: 930-937.
Losos J. 2009. Lizards in an evolutionary tree: Ecology and adaptive radiation of anoles.
University of California Press: Berkeley, California.
Mack RN, Simberloff D, Lonsdale WM, Evans H, Clout M, Bazzaz FA. 2000. Biotic
invasions: Causes, epidemiology, global consequences, and control. Ecological
Applications 10: 689-710.
Morrison C, Hero JM. 2003. Geographic variation in life-history characteristics of
amphibians: a review. Journal of Animal Ecology 72: 270-279.
Qualls FJ, Shine R. 2000. Post-hatching environment contributes greatly to phenotypic
variation between two populations of the Australian garden skink, Lampropholis
guichenoti. Biological Journal of the Linnean Society 71: 315-341.
Richardson DM, Rejmanek M. 2004. Conifers as invasive aliens: a global survey and
predictive framework. Diversity and Distributions 10: 321-331.
66
Schoener TW, Schoener A. 1980. Densities, sex ratios, and population structure in four
species of Bahamian Anolis lizards. The Journal of Animal Ecology: 19-53.
Stearns SC. 1992. The evolution of life histories. Oxford University Press Oxford.
Tayeh A, Hufbauer RA, Estoup A, Ravigne V, Frachon L, Facon B. 2015. Biological
invasion and biological control select for different life histories. Nature
Communications 6: 7268.
Warner DA, Shine R. 2007. Fitness of juvenile lizards depends on seasonal timing of
hatching, not offspring body size. Oecologia 154: 65-73.
While GM, Williamson J, Prescott G, Horváthová T, Fresnillo B, Beeton NJ, Halliwell B,
Michaelides S, Uller T. 2015. Adaptive responses to cool climate promotes
persistence of a non-native lizard. Proceedings of the Royal Society of London B:
Biological Sciences 282: 20142638.
67
Table 1. Characteristics of study populations.
Annual temperature (°C)
Location Range Coordinates Mean Min Max (Abbreviation) Source North Andros, The native 24.70° N, 78.02° W 25.08 10.63 34.67 a Bahamas (NA) Clearwater, FL (C) invasive 27.97° N, 82.80° W 23.04 0.51 34.48 b New Orleans, LA (NO) invasive 29.95° N, 90.07° W 21.23 -3.09 36.15 b Valdosta, GA (V) invasive 30.83° N, 83.28° W 19.56 -6.71 36.77 b
Temperature measures reflect averages of mean annual temperature and daily maximum and minimum temperature for each year from 1981- 2015. aNational Climatic Data Center. bPRISM Climate Group Oregon State University, http://prism.oregonstate.edu, created 19 Jan 2017.
68
Table 2. Results of linear mixed models.
A. Reproductive rates of native and invasive populations
Interlaying interval Incubation period
Random effect % Var P %Var P
Female ID 5.95 0.17 11.1 0.16
Fixed effects F df P F df P Population 8.00 3, 25.49 0.0006 108.4 3, 35.4 <0.0001 Exact lay date of focal egg known 1.64 1, 530.5 0.20 ------Exact lay date of previous egg known 10.86 1, 527.2 0.001 ------Population × focal 0.43 3, 531.9 0.73 ------Population × previous 0.40 3, 527.4 0.75 ------Focal × previous 1.24 1, 543 0.27 ------Population × focal × previous 1.22 3, 541.6 0.30 ------Days since pairing 65.42 1, 540.9 <0.0001 1.29 1, 176.9 0.26 Population × days ------3.88 3, 177.1 0.01 Female mass* 1.97 1, 21.71 0.18 1.66 1, 25.11 0.21 Egg mass* ------0.20 1, 128.7 0.66
B. Potential trade-offs of increased reproductive rate
Egg mass Hatching success† Hatchling mass
Random effect % Var P % Var P %Var P
Female ID 23.2 0.008 30.5 0.234 13.2 0.08
Fixed effects F df P F df P F df P Population 2.31 3, 39.12 0.09 1.88 3, 39.6 0.145 4.34 3, 39.68 0.01 Days since pairing 26.1 1, 232.4 <0.0001 0.09 1, 248.0 0.757 5.78 1, 178.9 0.02 Population × days ------4.20 3, 248.0 0.006
Female mass* 0.85 1, 36.48 0.36 3.00 1, 36.9 0.092 0.07 1, 27.72 0.79 Egg mass* ------1.14 1, 219.0 0.289 36.02 1, 118.7 <0.0001 Incubation period* ------4.79 1, 160.9 0.03
*These covariates were tested in a separate model from other listed effects. †Binomial error distribution.
69
22 North Andros (native) Clearwater (invasive) 20 18 16 14 12 10 8 6 4 2
22 New Orleans (invasive) Valdosta (invasive)
Eggs laid Eggs 20 18 16 14 12 10 8 6 4 2 0 20 30 40 50 60 70 80 20 30 40 50 60 70 80 Days since pairing
Figure 1. Individual egg-laying trajectories for females from four populations in a common lab environment. Invasive females produced eggs more rapidly than native females. Lines are spline fits for individual females.
70
A B 8 A B B B 38 A B B B ) ) d d
( 36 (
l
6 d a v o i r 34 r e t e n p i
4 32 n g o n i i t y a 30 a b l r 2 u e c t n
n 28 I I 0 26 NA C NO V NA C NO V C D 0.21 A A A A 0.24 A A B AB )
0.20 g (
) 0.22 s g ( s
a s 0.19 s m
a 0.20 g m n
i
0.18 l g h g c t E
a 0.18
0.17 H
0.16 0.16 NA C NO V NA C NO V
Figure 2. Interlaying interval, incubation period, egg mass, and hatchling mass across populations. Invasive populations (C, NO, V) have shorter (A) interlaying intervals and (B) incubation periods than the native population (NA). Populations did not differ in egg mass (C), but hatchlings were heaviest in the two southernmost populations. Population abbreviations are given in Table 1. Circles represent averages for individual females; bars represent standard errors. Letters indicate significantly different groups (Tukey’s HSD).
71
CHAPTER IV: EVOLUTION OF PHYSIOLOGICAL AND LIFE-HISTORY TRAITS
FOLLOWING INVASION OF A COOLER CLIMATE
Tamara L. Fetters, William A. Hopkins, and Joel W. McGlothlin
ABSTRACT
Invasive species often change phenotypically in response to novel environmental conditions in their new range. Assessing how genetic adaptation and phenotypic plasticity contribute to such changes can provide valuable insight into how species respond to environmental shifts in general. Invasive populations of brown anoles (Anolis sagrei) in the southeastern United States show increases in cold tolerance, maximum sprint speed, and metabolic rate in response to temperatures that are lower and more variable relative to their native range in the Caribbean. Additionally, invasive populations reproduce more quickly, with shorter durations between egg lays and faster incubation periods. We tested whether these trait changes resulted from genetic adaptation by rearing offspring from one native and one invasive population under identical laboratory conditions. We found that population differences in critical thermal minimum, metabolic rate, and reproductive rate were maintained in lab-reared offspring, suggesting genetic evolutionary change. In contrast, sprint speed differences were not maintained, suggesting the importance of plasticity. While it has been hypothesized that tropical ectotherms have limited capacity to evolve in response to new thermal environments, leading to predictions that they may not fare well as invaders, our results suggest that both physiology and life-history traits can and do evolve rapidly. 72
INTRODUCTION
Invasive species often encounter novel climates that pose significant challenges to survival and reproduction in their new range (Blackburn et al. 2011; Sakai et al. 2001).
While phenotypic adjustments that allow invaders to persist under novel thermal regimes are well documented (Cox 2004; Huey et al. 2000; Lee 2010), relatively few studies have examined whether phenotypic differentiation between native and invasive populations is achieved through genetic adaptation, phenotypic plasticity, or some combination of the two (Colautti and Lau 2015). Models of invasion typically view invaders as having fixed phenotypes and often exclude an invasive population’s capacity for evolutionary change
(Whitney and Gabler 2008). However, recent studies have shown that species facing novel selection pressures from the environment are capable of genetic adaptation over relatively short periods of time (Carroll et al. 2007; Moran and Alexander 2014). Further, species that evolve in response to new environments are more likely to become established, and exhibit greater spread both spatially and temporally (Szűcs et al. 2017).
How trait changes arise can have important implications for how invasions will progress, yet studies that explicitly test for evolution of traits during invasion remain uncommon, particularly for vertebrates (Pyšek et al. 2008). In order to better understand which phenotypes are likely to change during invasion and how, studies that both quantify trait changes and identify the underlying causal mechanisms are needed.
The first challenges faced by invasive species during establishment are often posed by abiotic factors, such as changes in temperature (Gerhardt and Collinge 2007;
Kelley 2014; Moyle and Light 1996). Ectotherms are particularly sensitive to changes in 73
thermal environment, as their performance and fitness are highly dependent on body temperature (Angilletta et al. 2002; Bennett 1980; Huey and Stevenson 1979; Stevenson et al. 1985). Successful establishment depends on trait responses that enable the species to conform to the newly encountered climate (Campbell-Staton et al. 2016; Huang et al.
2006). Specifically, invaders must be able to both survive and reproduce in their new environment (Blackburn et al. 2011), which may involve coordinated responses across a number of traits.
Ectotherms are often able to survive under different environmental conditions through physiological adjustments that may be plastic or adaptive in nature (Angilletta
2009; Seebacher and Franklin 2011). Traditionally, lizard thermal physiology has been thought to be resistant to directional selection, and several lines evidence support this
‘static’ view. First, behavioral thermoregulation may shield ectotherms against selection on thermal physiology (Bogert 1949; Huey et al. 2003). Alterations in habitat use (Muñoz and Losos 2018), basking frequency (Adolph 1990), and timing of activity (Hertz and
Huey 1981) can protect individuals from exposure to suboptimal temperatures. Second, thermal traits are highly conserved across diverse lizard taxa (Hertz et al. 1983) and have been shown to correlate more with phylogeny than location, suggesting a relatively low potential for local adaptation across thermal gradients (Grigg and Buckley 2013).
Conversely, the ‘labile’ view predicts that thermal physiology does vary with environmental temperature. Recent work has shown that while behavioral mechanisms for thermoregulation can partially shield terrestrial ectotherms from exposure to suboptimal temperatures, there are limits to this manipulation such as during the nighttime when there are less opportunities to thermoregulate (Muñoz et al. 2014). 74
Further, as the invaded environment becomes more disparate from that of their native range, invasive species may become less able to compensate for temperature changes via behavioral thermoregulation and adaptive changes in physiology may become necessary
(Gunderson and Stillman 2015).
While changes in physiological traits likely facilitate the survival of ectothermic invaders in their new environment, successful establishment also depends on the ability to reproduce under novel thermal regimes. Changes in reproduction and life-history traits during invasion have received considerably less attention. Just as with physiological traits, behavioral manipulations may preclude selection on reproductive traits. For example, females can choose thermally suitable habitats during oviposition (Angilletta et al. 2009), thus limiting thermal selection on eggs and embryos. Recent work has shown that traits such as incubation period and egg-laying patterns can rapidly change in invasive populations (Fetters and McGlothlin 2017; While et al. 2015). However, it is unclear whether such changes result from plasticity or adaptation.
Introductions of tropical lizards into more temperate areas serve as natural experiments to assess how traits necessary for survival and reproduction respond to cooler, more variable climates. Results from empirical studies support the hypothesis that thermal traits in lizards can shift during invasions to match local environmental conditions in the new range (Fetters and McGlothlin 2017; Kolbe et al. 2014; Kolbe et al.
2012; Leal and Gunderson 2012); however, the mechanisms underpinning trait divergence between populations remain unclear. While acclimation experiments suggest that traits such as cold tolerance can decrease in response to prolonged cold exposure, a lack of convergence in thermal traits across populations even after prolonged cold 75
exposure suggest that there is also some genetic component to trait changes (Kolbe et al.
2012). To date, no explicit tests for genetic adaptation in physiological or life history traits across native and invasive populations of these tropical lizards have been performed, and whether the observed responses to shifts in environmental conditions are the result of rapid evolution is unknown.
Here we use a common garden study of an ectothermic invader, the brown anole
(Anolis sagrei), in order to test for genetic adaptation of phenotypic traits across native and invasive populations. The brown anole is a highly invasive lizard native to the
Caribbean that has invaded the southeastern United States within the last century.
Previous work has shown that invasive populations of the brown anole have diverged from native populations across a variety of physiological traits, with invasive anoles exhibiting faster sprint speeds, higher metabolic rates, and increased cold tolerance relative to native anoles (Fetters et al., in prep). Additionally, differences in life-history traits have been observed across the two ranges. Specifically, invasive females have shorter interlaying intervals and produce faster-hatching eggs than do native females
(Fetters and McGlothlin 2017). In this study, we bred wild-caught individuals from a low-latitude native population and a high-latitude invasive population and compared physiological and life-history traits measured in offspring reared under identical laboratory conditions. Convergence of trait measures in offspring would suggest that differences in wild-caught parents result from plasticity or acclimation as the ‘static’ view predicts, while maintained differences in offspring trait measures would support the
‘labile’ view and provide evidence for recent genetic adaptation in the invasive range.
76
METHODS
Parental Trait Measures
We measured critical thermal minimum, sprint speed, and metabolic rate in wild- caught adult males and females from two native and four invasive populations of brown anoles. Methods and results for this experiment can be found in Fetters et al. (chapter 2).
Interlaying interval and incubation period were measured in one native and three invasive populations. Methods and results for this experiment can be found in Fetters and
McGlothin (2017). Due to space constraints, we chose to compare offspring measures in only the highest (Valdosta) and lowest (North Andros) latitude populations for which we measured all traits, as differences in trait measures were the greatest in magnitude across these two populations in our previous studies. Wild-caught lizards from these populations were found to differ significantly in measures of critical thermal minimum (Fig. 1a), maximum sprint speed (Fig 2a), metabolic rate (Fig 3a), interlaying interval (Fig. 4a), and incubation period (Fig 5a).
Common Garden Experiment
Males and females each population (North Andros, The Bahamas, n = 22 pairs;
Valdosta, Georgia, n = 8 pairs) were paired in June 2016. During pairing, each female was housed with a single male in a 37 cm × 22 cm × 25 cm cage (Lee’s Kritter Keeper).
Females were provided with a small container of potting soil for depositing eggs, and eggs were collected a minimum of five times per week. Additionally, we collected eggs laid by females caught from the same population in Valdosta, Georgia in May 2016 that were gravid at the time of capture (n = 15). These females were individually housed in a 77
30 cm x 20 cm x 20 cm cage (Lee’s Kritter Keeper), and we collected eggs in the same manner as for paired females. Eggs were collected until October 2016.
Eggs were transferred to a container with a 1:1 mixture of vermiculite and water, and placed in a 28 °C incubator until hatching. All eggs had hatched by November 2016.
Hatchlings were housed individually at 25/22°C (day/night), a 13L:11D photoperiod, and
~60% relative humidity. We reared 46 offspring from Andros (female, n = 17; male, n =
29) and 55 offspring from Valdosta (female, n = 29; male n = 26). Until they reached 3 months of age, hatchlings were fed pinhead crickets (Acheta domestica) dusted with vitamins and minerals (Rep-Cal, Los Gatos, CA) daily. Pinhead crickets were produced in the lab from adults supplied by Fluker’s Cricket Farm (Port Allen, LA). Between 3 and
12 months of age all lizards were fed two to three 1/4 inch crickets (Gryllodes sigillatus;
Ghann’s Cricket Farm, Augusta, GA) dusted with vitamins and minerals (Rep-Cal, Los
Gatos, CA) three times per week. Past 12 months of age, we fed females three to five 1/2 inch crickets and males five to seven 3/8 inch crickets dusted with vitamins and minerals three times per week.
Trait Measures
We measured critical thermal minimum, sprint speed, metabolic rate, interlaying interval, and incubation period in lab-reared offspring (Table 1). When each lizard reached 270 days of age, we measured critical thermal minimum and sprint speed, randomizing the order of the two tests. Afterward, we measured each lizard’s metabolic rate. Metabolic rate was always measured last because lizards are exposed to cold temperatures for up to 48 hours during testing, and we wanted to ensure other thermal 78
trait measures did not acclimate to lower temperatures. For each test, we followed the same methods found in Fetters et al. (chapter 2), which are described in brief below.
Critical thermal minimum was measured as the lowest temperature at which a lizard can no longer right itself when placed on its back. We cooled lizards in a container submerged in an ice bath while continuously monitoring body temperature. We tested for righting ability at 14°C and decreasing increments of 1.0°C until righting ability was lost.
Maximum sprint speed was measured at six temperatures: 12°C, 18°C, 24°C,
30°C, 36°C, and 41°C. Each lizard was run three times at each temperature, and the order of temperatures was randomized. Lizards were filmed running on a 70-cm long, 3-cm diameter wooden dowel inclined at 20°. Maximum sprint speed was calculated for each trial using motion analysis software, Kinovea 0.8.15 (www.kinovea.org). We excluded trials in which the lizard did not run within 30 s of the start of the trial, did not remain on the vertical surface of the wooden dowel during the trial, or did not move at least 10 consecutive cm during the trial.
Metabolic rate was measured as the rate of oxygen consumption across four randomized temperatures: 15°C, 20°C, 26°C, and 32°C. Lizards were placed in glass chambers in an incubator connected to a computer-controlled, closed-flow respirometer
(Microoxymax; Columbus Instruments, Columbus, OH, USA). Lizards were fasted for 48 hours prior to each trial and were held in darkness to minimize oxygen consumption due to digestion and activity. For each temperature, 19 measurements of oxygen consumption
−1 rates (mL h ; VO2) were taken over 24 hours. The first three measurements from each temperature treatment were excluded to allow metabolic rates to stabilize, and the remaining 16 measures were used to calculate the lowest quartile value for the rate of 79
oxygen consumption. Both body mass and the lowest quartile value for the rate of oxygen consumption were log10-transformed to linearize their relationship.
After all thermal traits had been measured, offspring were moved into a larger animal room held at 28/25°C (day/night), a 13L:11D photoperiod, and ~60% relative humidity in August 2017. Offspring from each population were paired for 10 weeks between February and April 2018 to measure life history traits (interlaying interval and incubation period). Full methods for assessing interlaying interval and incubation period
(Fetters and McGlothlin 2017) are described elsewhere. In brief, male and female offspring from each population (Andros, n = 14 pairs; Valdosta, n = 24 pairs) were paired for 10 weeks under the same conditions as during breeding in our common garden experiment. The number of pairs was lower for Andros due to an uneven sex ratio in the offspring that survived until pairing (male, n = 25; female, n = 14). We ensured males and females were not related prior to pairing. After pairing, we checked for eggs no less than 6 times per week. Eggs (n = 283) were massed, placed in a container with a 1:1 mixture of vermiculite and water, and incubated at 28°C. We checked twice daily for hatchlings until all eggs had either hatched (n = 147) or failed to hatch (within 8 weeks, n
= 136). For each individual egg, we calculated interlaying interval as the number of days elapsed since the last egg was found and incubation period as the number of days between egg discovery and hatching.
Statistical Analysis
For all trait measures, we performed statistical analyses using general linear models. For physiological measures, we examined the effects of: sex, population, and 80
mass on critical thermal minimum; population, temperature, sex, snout-vent length
(SVL), and run number on maximum sprint speed; and population, temperature, log10 mass, and sex on the log10 lowest quartile value for the rate of oxygen consumption.
Lizard ID was included as a random factor for maximum sprint speed and rate of oxygen consumption.
Analyses of incubation period included only hatchlings from eggs with known lay dates (i.e., from eggs found the day after an egg check had been performed; n = 108). We examined the effects of population, number of days since pairing, whether the exact lay date of the focal egg was known, whether the exact lay date of the previously laid egg was known, and female mass on interlaying interval. For incubation period, population, days since pairing, egg mass, and female mass were included as fixed effects. Female ID was included as a random factor in both analyses.
RESULTS
Critical Thermal Minimum
Lab-reared offspring from our invasive population (Valdosta) tolerated lower temperatures than did those from our native population (Andros) (Fig 1b, F1,84 = 12.19, P
= 0.0008). While CTmin values were lower in the invasive population for both sexes, females showed a greater difference in CTmin values between populations than males, leading to a significant interaction of sex and population (F1,84 = 7.90, P = 0.0061). Mass
(F1,84 = 2.97, P =0.09) and sex (F1,84 = 0.16, P = 0.69) were not significant predictors of
CTmin.
81
Sprint Speed
Maximum sprint speed did not differ by population (Fig 2b , F1,97.41 = 1.14, P =
0.2882). Sprint speed significantly increased with increasing temperature between 12°C and 30°C, but did not change at temperatures above 30°C (F5, 800.6 = 79.72, P < 0.0001).
Females generally achieved greater maximum sprint speeds than males (F1,87.67 =5.06, P
= 0.0269). There was a significant interaction between SVL and population, with sprint speed tending to increase with SVL in Valdosta lizards, and decrease with SVL in
Andros lizards (F1,66.32 = 5.94, P = 0.0175). Maximum sprint speed also increased more with SVL at higher temperatures (F5,797.4 = 2.25, P = 0.477).
Metabolic Rate
Offspring from Valdosta exhibited higher rates of oxygen consumption than offspring from Andros (Figure 3b , F1,47.09 = 5.98, P = 0.0183). Oxygen consumption rates increased with temperature and differed significantly across all four temperatures measured (F3, 131.8 = 4651.89, P <0.0001). Oxygen consumption increased with log10 mass (F1,61.44 = 66.06, P <0.0001), and males had higher rates of oxygen consumption than females (F1,51.83 = 7.51, P = 0.0084). Oxygen consumption rate differed more between males and females from Andros than from Valdosta (F1,47.22 = 6.76, P = 0.0124); this was likely due to there being greater mass differences between the sexes for offspring from Andros.
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Interlaying Interval
Females from the invasive population had a shorter duration between consecutive egg lays than females from the native population (Fig 4b, F1,21.14 = 5.53, P = 0.0285).
Female mass (F1,18.2 = 2.07, P = 0.1672) and number of days since pairing (F1, 228.5 =
0.0019, P =0.9650) were not significant predictors of interlaying interval.
Incubation Period
Female offspring from the invasive population laid eggs that hatched significantly faster than those from the native population (Fig 5b, F1,16.92 = 89.16, P <0.0001). The incubation period increased with days since pairing (F1,84.49 = 19.67, P <0.0001), and this trend was more pronounced for Andros than Valdosta (F1,85.83 = 5.0739, P = 0.0268).
DISCUSSION
Our results strongly suggest that there is a genetic basis to differences in thermal and life-history traits between native and invasive populations of the brown anole.
Although our design cannot definitively exclude pre-laying maternal effects as a cause, standardization of incubation and rearing conditions effectively removed any variation that may arise from population differences in post-laying maternal effects. Offspring reared in a common garden maintained similar phenotypes as their wild-caught parents, with invasive offspring having lower critical thermal minima, higher metabolic rates, faster interlaying intervals, and faster incubation periods than native offspring. The only trait which for which we did not find evidence of a genetic component was sprint speed, which converged in our common garden. Our data suggest that thermophysiological and 83
life-history traits have evolved rapidly in response to novel environmental conditions encountered during invasion, likely facilitating the successful establishment and spread of the brown anole in the southeastern United States.
While most of the traits examined here appear to be labile and respond readily to selection, some traits may be shielded from selection, and thus remain static across differing environments. Behavioral thermoregulation can buffer traits from selection, but this manipulation is limited by the available thermal environments. During the day, solar radiation creates ample thermal heterogeneity (Adolph 1990); selection for traits that are relevant during daytime activity , such as sprint speed, may be relatively weak as lizards may be more readily able to obtain adequate temperatures for performance. Conversely, night-time body temperatures are largely governed by substrate and ambient temperatures, and the capacity for behavioral thermoregulation is limited (Adolph and
Porter 1993). Selection on traits such as critical thermal minimum and metabolic rate may be stronger, as lizards cannot behaviorally buffer themselves against night-time cold exposure and must adapt physiologically (Muñoz et al. 2014). Traits may also experience varying selection pressure as they differ in their thermal sensitivities. For example, locomotion has a large performance breadth, and lizards may be able to reach adequate speeds across a broad range of temperatures. Subsequently, locomotion is not as sensitive to changes in temperature as other traits, which can dampen thermal selection on locomotory performance (Angilletta et al. 2002).
Selection has also evidently altered life-history traits in invasive brown anoles, and the faster reproduction observed in the invasive range likely results from key differences in both climate and population dynamics between the two ranges. The 84
breeding and growing season is shortened in higher-latitude invasive populations due to increased seasonality and temperature variation the new range (Lee et al. 1989). By quickening both the interlaying interval and incubation period, invasive lizards presumably optimize the number of offspring produced and lengthen their access to a favorable growing environment. Additionally, the rapid population expansion characteristic of recent invasions can drive changes in life-history traits as individuals that produce more offspring will quickly obtain higher allele frequencies in exponentially growing populations (Dobson 2007; Phillips et al. 2010).
While genetic adaptation appears to account for a majority of the observed trait differences across native and invasive populations of the brown anole, there is some evidence that plasticity plays a complementary role. Previous work in lizards has found that pre-and post-hatching thermal environments can have both reversible and permanent effects on offspring phenotype (Booth 2006; Qualls and Shine 2000). It is unlikely that pre-hatching conditions differ between the native and invasive range, as invasive populations should only be reproductively active during portions of the year when environmental conditions converge between the ranges, and should cease reproduction when temperatures diverge (Lee et al. 1989). However, based on their size upon capture and our knowledge of growth rates, wild-caught individuals likely hatched during the previous year. Post-hatching environments may differ markedly across ranges, with lizards from invasive populations presumably experiencing extreme temperature fluctuations during the winter following hatching that native populations do not. If differing environments experienced during development cause plastic differences in sprint speed in wild-caught populations, this could explain why sprint speed converges in 85
lab-reared offspring. Future studies exploring the effects of developmental environment on phenotypes would be beneficial.
Our results suggest that there may also be a plastic component to population differences in critical thermal minima. Critical thermal minimum values were lower for laboratory born native offspring relative to measures of their wild-caught parents.
Previous studies show that critical thermal minimum values lower in response to exposure to cooler temperatures in captivity (Kolbe et al. 2012). Native offspring may have lowered their critical thermal minima in response to the mean temperature in the laboratory environment being cooler than the mean temperature experienced in the native range. Critical thermal minimum values were consistent between wild-caught and laboratory born invasive offspring, which may reflect rearing conditions being closer to the mean annual temperature in the invasive range.
Previous empirical and theoretical work in lizards has focused on how either thermal traits (Kolbe et al. 2012; Leal and Gunderson 2012) or life-history traits (While et al. 2015) change in response to novel climates, but less is known about the potential linkage between the two. Higher rates of reproduction generally correspond with higher metabolism on the slow-fast spectrum of life-histories (Brown et al. 2004; Ricklefs and
Wikelski 2002). Recent work across diverse taxa such as fish (Auer et al. 2018), mammals (Sadowska et al. 2009), and insects (Le Lann et al. 2011) have shown that experimental manipulations of either metabolism or life-history lead to correlated responses in the other, providing evidence the two may be evolutionarily coupled.
While we found increases in both metabolism and reproductive rate in invasive populations, in a native congener, Anolis carolinensis, which occupies a similar range in 86
the southeastern United States, temperature and metabolism shown an opposite pattern of correlation where cooler environmental temperatures are associated with lower metabolic rate (Campbell-Staton et al. 2018). This suggests that on a macroevolutionary timescale, lower metabolic rates may actually be selected for in response to cooler conditions. This calls into question whether both reproduction and metabolism are responding to the same selective agent, or perhaps whether selection for one trait causes evolution in both because of a functional linkage between metabolism and life-history. For example, it is possible that the increases in metabolic rate observed in invasive populations are not selected for, but are simply the result of correlated selection on faster reproduction.
Regardless of the mechanism, this study is one of the first to document changes in both reproduction and metabolism in the same species during invasions, and further work examining the links between trait changes is warranted.
Understanding how ectothermic populations respond to rapid environmental change is proving critical to forming accurate predictions of how species will fare both during invasions and under current estimates of global climate change (Moran and
Alexander 2014). Populations that evolve during invasions have been shown to both spread faster and obtain larger ranges (Szűcs et al. 2017). However, models of future range limits often ignore adaptive potential and can lead to overly optimistic predictions
(Sinclair et al. 2012). Similarly, current forecasts for ectotherms undergoing climate warming assume rapid adaptive responses will be unlikely to protect vulnerable populations (Huey et al. 2009; Sinervo et al. 2010). Our results suggest that we should not view ectotherms as unable to adapt to rapid changes in climate as the static view
87
suggests, but rather should incorporate knowledge of evolutionary responses of thermal and life-history traits to better predict outcomes for invasions and global change.
88
ACKNOWLEDGEMENTS
We thank Ignacio Moore, Julie Wiemerslage, Caitlin McCaughan, Eric Wice,
Thomas Wood, Prabh Dhillon, John Abramyan, Iva Veselá, Kathryn Moore, Tyler
Miller, Emily Watts, and our undergraduate volunteers for their help with animal collection and care and experimental assistance.
89
REFERENCES
Adolph, S. C. 1990. Influence of behavioral thermoregulation on microhabitat use by two
Sceloporus lizards. Ecology 71:315-327.
Adolph, S. C., and W. P. Porter. 1993. Temperature, activity, and lizard life histories. The
American Naturalist 142:273-295.
Angilletta, M. J., P. H. Niewiarowski, and C. A. Navas. 2002. The evolution of thermal
physiology in ectotherms. Journal of thermal Biology 27:249-268.
Auer, S. K., C. A. Dick, N. B. Metcalfe, and D. N. Reznick. 2018. Metabolic rate evolves
rapidly and in parallel with the pace of life history. Nature communications 9:14.
Bennett, A. F. 1980. The thermal dependence of lizard behaviour. Animal Behaviour
28:752-762.
Blackburn, T. M., P. Pyšek, S. Bacher, J. T. Carlton, R. P. Duncan, V. Jarošík, J. R.
Wilson et al. 2011. A proposed unified framework for biological invasions.
Trends in ecology & evolution 26:333-339.
Bogert, C. M. 1949. Thermoregulation in reptiles, a factor in evolution. Evolution 3:195-
211.
Booth, D. T. 2006. Influence of incubation temperature on hatchling phenotype in
reptiles. Physiological and Biochemical Zoology 79:274-281.
Brown, J. H., J. F. Gillooly, A. P. Allen, V. M. Savage, and G. B. West. 2004. Toward a
metabolic theory of ecology. Ecology 85:1771-1789.
Campbell-Staton, S. C., A. Bare, J. B. Losos, S. V. Edwards, and Z. A. Cheviron. 2018.
Physiological and regulatory underpinnings of geographic variation in reptilian
cold tolerance across a latitudinal cline. Molecular ecology. 90
Campbell-Staton, S. C., S. V. Edwards, and J. B. Losos. 2016. Climate‐mediated
adaptation after mainland colonization of an ancestrally subtropical island lizard,
Anolis carolinensis. Journal of evolutionary biology 29:2168-2180.
Carroll, S. P., A. P. Hendry, D. N. Reznick, and C. W. Fox. 2007. Evolution on
ecological time‐scales. Functional Ecology 21:387-393.
Colautti, R. I., and J. A. Lau. 2015. Contemporary evolution during invasion: evidence
for differentiation, natural selection, and local adaptation. Molecular Ecology
24:1999-2017.
Cox, G. W. 2004, Alien species and evolution: the evolutionary ecology of exotic plants,
animals, microbes, and interacting native species, Island Press.
Dobson, F. S. 2007. A lifestyle view of life-history evolution. Proceedings of the
National Academy of Sciences USA 104:17565-17566.
Fetters, T. L., and J. W. McGlothlin. 2017. Life histories and invasions: accelerated
laying rate and incubation time in an invasive lizard, Anolis sagrei. Biological
Journal of the Linnean Society 122:635-642.
Grigg, J. W., and L. B. Buckley. 2013. Conservatism of lizard thermal tolerances and
body temperatures across evolutionary history and geography. Biology letters
9:20121056.
Gunderson, A. R., and J. H. Stillman. 2015. Plasticity in thermal tolerance has limited
potential to buffer ectotherms from global warming. Proc. R. Soc. B
282:20150401.
91
Hertz, P. E., and R. B. Huey. 1981. Compensation for altitudinal changes in the thermal
environment by some Anolis lizards on Hispaniola. Ecology 62:515-521.
Hertz, P. E., R. B. Huey, and E. Nevo. 1983. Homage to Santa Anita: thermal sensitivity
of sprint speed in agamid lizards. Evolution 37:1075-1084.
Huang, S.-P., Y. Hsu, and M.-C. Tu. 2006. Thermal tolerance and altitudinal distribution
of two Sphenomorphus lizards in Taiwan. Journal of Thermal Biology 31:378-
385.
Huey, R. B., C. A. Deutsch, J. J. Tewksbury, L. J. Vitt, P. E. Hertz, H. J. Á. Pérez, and T.
Garland. 2009. Why tropical forest lizards are vulnerable to climate warming.
Proceedings of the Royal Society of London B: Biological Sciences 276:1939-
1948.
Huey, R. B., G. W. Gilchrist, M. L. Carlson, D. Berrigan, and L. s. Serra. 2000. Rapid
evolution of a geographic cline in size in an introduced fly. Science 287:308-309.
Huey, R. B., P. E. Hertz, and B. Sinervo. 2003. Behavioral drive versus behavioral inertia
in evolution: a null model approach. The American Naturalist 161:357-366.
Huey, R. B., and R. Stevenson. 1979. Integrating thermal physiology and ecology of
ectotherms: a discussion of approaches. American Zoologist 19:357-366.
Kolbe, J. J., J. C. Ehrenberger, H. A. Moniz, and M. J. Angilletta, Jr. 2014. Physiological
variation among invasive populations of the brown anole (Anolis sagrei).
Physiological and Biochemical Zoology 87:92-104.
Kolbe, J. J., P. S. VanMiddlesworth, N. Losin, N. Dappen, and J. B. Losos. 2012.
Climatic niche shift predicts thermal trait response in one but not both
92
introductions of the Puerto Rican lizard Anolis cristatellus to Miami, Florida,
USA. Ecology and evolution 2:1503-1516.
Le Lann, C., T. Wardziak, J. Van Baaren, and J. J. van Alphen. 2011. Thermal plasticity
of metabolic rates linked to life‐history traits and foraging behaviour in a
parasitic wasp. Functional Ecology 25:641-651.
Leal, M., and A. R. Gunderson. 2012. Rapid change in the thermal tolerance of a tropical
lizard. The American Naturalist 180:815-822.
Lee, C. E. 2010. Evolution of invasive populations. Proceedings of the National
Academy of Sciences USA 104:15793-15798.
Lee, J. C., D. Clayton, S. Eisenstein, and I. Perez. 1989. The reproductive cycle of Anolis
sagrei in Southern Florida. Copeia:930-937.
Moran, E. V., and J. M. Alexander. 2014. Evolutionary responses to global change:
lessons from invasive species. Ecology Letters 17:637-649.
Muñoz, M. M., and J. B. Losos. 2018. Thermoregulatory Behavior Simultaneously
Promotes and Forestalls Evolution in a Tropical Lizard. The American Naturalist
191:E15-E26.
Muñoz, M. M., M. A. Stimola, A. C. Algar, A. Conover, A. J. Rodriguez, M. A.
Landestoy, G. S. Bakken et al. 2014. Evolutionary stasis and lability in thermal
physiology in a group of tropical lizards. Proceedings of the Royal Society of
London B: Biological Sciences 281:20132433.
Phillips, B. L., G. P. Brown, and R. Shine. 2010. Life‐history evolution in range‐
shifting populations. Ecology 91:1617-1627.
93
Pyšek, P., D. M. Richardson, J. Pergl, V. Jarošík, Z. Sixtova, and E. Weber. 2008.
Geographical and taxonomic biases in invasion ecology. Trends in Ecology &
Evolution 23:237-244.
Qualls, F. J., and R. Shine. 2000. Post-hatching environment contributes greatly to
phenotypic variation between two populations of the Australian garden skink,
Lampropholis guichenoti. Biological Journal of the Linnean Society 71:315-341.
Ricklefs, R. E., and M. Wikelski. 2002. The physiology/life-history nexus. Trends in
Ecology & Evolution 17:462-468.
Sadowska, E. T., K. Baliga‐Klimczyk, M. K. Labocha, and P. Koteja. 2009. Genetic
correlations in a wild rodent: grass‐eaters and fast‐growers evolve high basal
metabolic rates. Evolution: International Journal of Organic Evolution 63:1530-
1539.
Sakai, A. K., F. W. Allendorf, J. S. Holt, D. M. Lodge, J. Molofsky, K. A. With, S.
Baughman et al. 2001. The population biology of invasive species. Annual review
of ecology and systematics 32:305-332.
Sinclair, B. J., C. M. Williams, and J. S. Terblanche. 2012. Variation in thermal
performance among insect populations. Physiological and Biochemical Zoology
85:594-606.
Sinervo, B., F. Mendez-De-La-Cruz, D. B. Miles, B. Heulin, E. Bastiaans, M. Villagrán-
Santa Cruz, R. Lara-Resendiz et al. 2010. Erosion of lizard diversity by climate
change and altered thermal niches. Science 328:894-899.
94
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.
Szűcs, M., M. Vahsen, B. Melbourne, C. Hoover, C. Weiss-Lehman, and R. Hufbauer.
2017. Rapid adaptive evolution in novel environments acts as an architect of
population range expansion. Proceedings of the National Academy of Sciences
114:13501-13506.
While, G. M., J. Williamson, G. Prescott, T. Horváthová, B. Fresnillo, N. J. Beeton, B.
Halliwell et al. 2015. Adaptive responses to cool climate promotes persistence of
a non-native lizard. Proceedings of the Royal Society of London B: Biological
Sciences 282:20142638.
Whitney, K. D., and C. A. Gabler. 2008. Rapid evolution in introduced species,‘invasive
traits’ and recipient communities: challenges for predicting invasive potential.
Diversity and Distributions 14:569-580.
95
Table 1. Sample sizes and characteristics of offspring at time of testing.
Andros, The Bahamas Valdosta, Georgia Age Male Mass Female Mass Male Mass Female Mass (days) n (g) n (g) n (g) n (g) CTmin 284 28 1.76 16 1.38 20 1.88 25 1.61 Sprint speed 281 30 1.77 17 1.36 18 1.89 21 1.60 Metabolism 303 18 1.87 11 1.44 12 1.94 16 1.68 Interlaying Interval 515 14 3.90 14 2.06 24 4.99 24 2.74 & Incubation Period
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Figure 1. Critical thermal minimum values for parents and offspring. (a) Wild-caught lizards from Valdosta (V) had significantly lower critical thermal minimum values than native lizards from North Andros (A) (data replotted from Chapter 2). (b) This pattern was maintained in offspring reared in a common garden. Circles represent the least squares mean value for each population; bars represent standard errors.
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Figure 2. Maximum sprint speed for parents and offspring. (a) In wild-caught parents, invasive lizards from Valdosta were faster across all temperatures than native lizards from Andros. (b) These differences were not maintained in a common garden, and lab-reared native and invasive offspring did not differ in maximum sprint speed across temperatures. Circles represent the least squares mean value for each population; bars represent standard errors.
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Figure 3. Standard Metabolic Rate for parents and offspring. In both (a) wild-caught parents and (b) lab-reared offspring, invasive lizards had higher standard metabolic rates than native lizards. Circles represent the backtransformed least squares mean value for each population, which was used to adjust for model covariates such as mass and sex. Bars represent back-transformed standard errors. Note that the scale for standard metabolic rate differs between offspring and parents as offspring were smaller in mass than parents at the time of testing.
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Figure 4. Interlaying Interval for parents and offspring. In both (a) wild-caught parents and (b) lab-reared offspring, invasive lizards had shorter durations between consecutive egg lays than native lizards. Circles represent the least squares mean value for each population; bars represent standard errors.
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Figure 5. Incubation Period for parents and offspring. In both (a) lab-reared offspring and (b) wild-caught parents, invasive lizards had shorter incubation periods than native lizards. Circles represent the least squares mean value for each population; bars represent standard error.
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CHAPTER V: SYNTHESIS
Tamara L. Fetters
In order to become established, invaders must be capable of both surviving and reproducing in their new range, often under novel climatic conditions (Blackburn et al.
2011; Sakai et al. 2001). To do so, invaders often display rapid phenotypic changes that allow them to better match local environments, ultimately facilitating their persistence and spread (Huey et al. 2000; Losos et al. 2001; Phillips et al. 2006). The work presented in this dissertation examines phenotypic change across tropical native and temperate invasive populations of the brown anole (Anolis sagrei) that differ significantly in climate
(Angetter et al. 2011). I identified changes to physiology and life history that promote survival and reproduction in the new range, and showed that many of these changes were the result of genetic adaptation. Findings from this study further our basic understanding of contemporary evolution and provide insight into how to best manage and prevent invasions.
Physiology and Invasion
In Chapter II, I examined the effects of climatic differences between the native and invasive range of the brown anole on temperature-dependent physiological traits.
Seasonality is more pronounced in the southeastern United States than in The Bahamas, and subsequently invasive lizards experience lower daily minimum and mean annual 102
temperatures than their native counterparts (Angetter et al. 2011). I predicted that invasive lizards would respond to these temperature differences by increasing cold tolerance. The results supported this hypothesis, and lizards from invasive populations were found to tolerate lower temperatures and exhibit faster maximum sprint speeds and higher metabolic rates than those from native populations. These phenotypic alterations likely increase survival and fitness in the invasive range. Increases in critical thermal minimum may increasing overwintering rates, while increased sprint speeds and metabolic rates may enhance the ability of lizards to escape predation, thermoregulate, and/or forage, particularly during harsh winter conditions (Clarke 1991; Irschick and
Losos 1999; Logan et al. 2014).
Life History and Invasion
Though the changes in physiology likely promote persistence in the new range, successful establishment also depends on invaders being capable of reproduction in their new environment. The brown anole likely faces significant challenges to reproduction in the invasive range; greater temperature variation and seasonality both shorten the length of the breeding season (Goldberg 1974; Lee et al. 1989; Morrison and Hero 2003) and truncate optimal periods for offspring growth and development (Qualls and Shine 2000;
Warner and Shine 2007). In Chapter III, I examined how these changes may have affected life-history traits and reproduction in the brown anole, focusing specifically on egg interlaying intervals and incubation periods. I found that invasive females had shorter durations between consecutive egg lays and produced faster hatching eggs. There was no 103
evidence that invasive populations suffered a fitness cost in terms of egg size, hatchling size, or hatching success. Together, these changes quicken reproduction and may allow the brown anole to compensate for the shortened breeding season in the invasive range
(Adolph and Porter 1993). This study provides one of the first examples in reptiles of life history traits changing during invasion.
Effect of Latitude on Trait Change
For both physiological and life-history traits, phenotypes showed the greatest divergence between populations in the native versus the invasive range. Because the brown anole’s range in the southeastern United States spans a large latitudinal range, and both mean annual and minimum daily temperatures decrease with latitude, we expected to see a correlation between trait measures and latitude. While there was a non-significant trend for the most northern population to be the most cold tolerant across physiological traits, generally phenotypic measures did not seem to vary by latitude. Previous work on brown anoles in their invasive range in the southeastern United States did find that critical thermal minimum values were significantly lower for the most northern population of Tifton, Georgia, which is farther north than our northernmost population of
Valdosta, Georgia, versus two Florida populations (Kolbe et al. 2014). It could be possible that local adaptation is only apparent at the invasion front. Although these edge populations tend to be smaller and more isolated leading to difficulties in obtaining large sample sizes, measuring traits from even more northern populations for comparison would be useful. Additionally, the native populations sampled in this study were all lower 104
in latitude than the invasive populations, and it is therefore difficult to separate the effects of latitude from the effects of invasion on phenotypic trait change. Future work comparing measures from native and invasive populations that occupy the same latitude, such as the Abaco Islands in The Bahamas and Naples, Florida, would help separate the effects of latitude and invasion status.
Adaptation or Plasticity?
Based on the findings of Chapter II and Chapter III that brown anoles differ across numerous measures of physiology and life history, a natural next step was to investigate whether trait divergence results from phenotypic plasticity, genetic adaptation, or some combination of the two. In Chapter IV, I measured physiological and life-history traits in offspring reared under identical laboratory conditions from one native and one invasive population. The majority of offspring trait measures were reflective of their wild-caught parents, providing evidence that phenotypic changes in the invasive range are largely the result of genetic adaptation. Traits such as interlaying interval and incubation period were nearly identical between parents and offspring, suggesting strong genetic control. Other traits seemed to be affected by rearing conditions as well as parentage. For example, measures of critical thermal minima were lower for native lab- reared offspring than for their parents; this was likely due to conditions being cooler in the common garden than they are in the wild. The only trait for which we found no evidence of a genetic component was maximum sprint speed. Differences in locomotor performance across wild-caught populations may result from developmental or reversible 105
plasticity, and future work addressing these possibilities would be beneficial. Overall, our results support that genetic adaptation is the main contributor to the observed trait changes in physiological and life-history traits, with plasticity playing a complementary role.
Selection in the Invasive Range
While it is likely that the increased cold tolerance and faster reproduction exhibited by invasive populations facilitate the survival and reproduction of the brown anole in its new range, this work does not include measures of fitness in the wild, and thus it is unknown whether these changes are ultimately adaptive or non-adaptive.
Generally, it is expected that faster metabolism and reproduction should bear some associated cost, such as shorter lifespans or reduced growth (Stearns 1992). Measuring potential trade-offs in wild populations would help elucidate whether there are costs associated changes in phenotype. Additionally, some changes may not be under direct selection, but rather be indirectly selected for through selection on other traits. For example, metabolism and life-history traits have been shown to be evolutionarily linked
(Auer et al. 2018), and selection for faster reproduction in the invasive range may bring increases in metabolism along with it or vice versa.
While traits certainly face strong selection pressure from the cooler temperatures in the invasive range, it is likely that the process of invasion itself also exerts some additional level of selection on most traits. For instance, during the early stages of invasion when populations are undergoing exponential growth, individuals that can 106
reproduce at faster rates are at a selective advantage and will rapidly obtain higher allele frequencies than slower reproducers (Dobson 2007; Phillips et al. 2010). Invasion can also select for faster dispersers (Alford et al. 2009; Phillips et al. 2010; Travis and
Dytham 2002), and higher maximum sprint speeds may also be partially attributable to selection on dispersal. A possible future direction of this work is to assess fitness and selection gradients across ranges to better understand how selection is acting on traits, and whether the observed shifts are adaptive.
General Conclusions
The predominant view has been that tropical ectotherms are limited in their ability to respond to selection on temperature-dependent traits (Bogert 1949; Hertz et al. 1983).
Subsequently, their ability to respond to novel environmental conditions has been considerably underestimated, both in terms of how they will fare as invaders and in response to global climate change (Huey et al. 2009; Sinclair et al. 2012; Sinervo et al.
2010). The work presented in this dissertation provides evidence that tropical ecotherms are more labile than is currently thought, and are capable of rapid evolutionary change across both physiological and life-history traits when facing novel thermal regimes. It is my hope that this work encourages the inclusion of evolutionary potential when considering outcomes for lizards in terms of both invasion and climate change.
107
Adolph, S. C., and W. P. Porter. 1993. Temperature, activity, and lizard life histories. The
American Naturalist 142:273-295.
Alford, R. A., G. P. Brown, L. Schwarzkopf, B. L. Phillips, and R. Shine. 2009.
Comparisons through time and space suggest rapid evolution of dispersal
behaviour in an invasive species. Wildlife Research 36:23-28.
Angetter, L.-S., S. Loetters, and D. Roedder. 2011. Climate niche shift in invasive
species: the case of the brown anole. Biological Journal of the Linnean Society
104:943-954.
Auer, S. K., C. A. Dick, N. B. Metcalfe, and D. N. Reznick. 2018. Metabolic rate evolves
rapidly and in parallel with the pace of life history. Nature communications 9:14.
Blackburn, T. M., P. Pyšek, S. Bacher, J. T. Carlton, R. P. Duncan, V. Jarošík, J. R.
Wilson et al. 2011. A proposed unified framework for biological invasions.
Trends in ecology & evolution 26:333-339.
Bogert, C. M. 1949. Thermoregulation in reptiles, a factor in evolution. Evolution 3:195-
211.
Clarke, A. 1991. Cold adaptation. Journal of Zoology 225:691-699.
Dobson, F. S. 2007. A lifestyle view of life-history evolution. Proceedings of the
National Academy of Sciences USA 104:17565-17566.
Goldberg, S. R. 1974. Reproduction in mountain and lowland populations of the lizard
Sceloporus occidentalis. Copeia:176-182.
Hertz, P. E., R. B. Huey, and E. Nevo. 1983. Homage to Santa Anita: thermal sensitivity
of sprint speed in agamid lizards. Evolution 37:1075-1084.
108
Huey, R. B., C. A. Deutsch, J. J. Tewksbury, L. J. Vitt, P. E. Hertz, H. J. Á. Pérez, and T.
Garland. 2009. Why tropical forest lizards are vulnerable to climate warming.
Proceedings of the Royal Society of London B: Biological Sciences 276:1939-
1948.
Huey, R. B., G. W. Gilchrist, M. L. Carlson, D. Berrigan, and L. s. Serra. 2000. Rapid
evolution of a geographic cline in size in an introduced fly. Science 287:308-309.
Irschick, D. J., and J. B. Losos. 1999. Do lizards avoid habitats in which performance is
submaximal? The relationship between sprinting capabilities and structural habitat
use in Caribbean anoles. The American Naturalist 154:293-305.
Kolbe, J. J., J. C. Ehrenberger, H. A. Moniz, and M. J. Angilletta, Jr. 2014. Physiological
variation among invasive populations of the brown anole (Anolis sagrei).
Physiological and Biochemical Zoology 87:92-104.
Lee, J. C., D. Clayton, S. Eisenstein, and I. Perez. 1989. The reproductive cycle of Anolis
sagrei in Southern Florida. Copeia:930-937.
Logan, M. L., R. M. Cox, and R. Calsbeek. 2014. Natural selection on thermal
performance in a novel thermal environment. Proceedings of the National
Academy of Sciences 111:14165-14169.
Losos, J. B., T. W. Schoener, K. I. Warheit, and D. Creer. 2001. Experimental studies of
adaptive differentiation in Bahamian Anolis lizards, Pages 399-415
Microevolution Rate, Pattern, Process, Springer.
Morrison, C., and J. M. Hero. 2003. Geographic variation in life-history characteristics of
amphibians: a review. Journal of Animal Ecology 72:270-279.
109
Phillips, B. L., G. P. Brown, and R. Shine. 2010. Life‐history evolution in range‐
shifting populations. Ecology 91:1617-1627.
Phillips, B. L., G. P. Brown, J. K. Webb, and R. Shine. 2006. Invasion and the evolution
of speed in toads. Nature 439:803.
Qualls, F. J., and R. Shine. 2000. Post-hatching environment contributes greatly to
phenotypic variation between two populations of the Australian garden skink,
Lampropholis guichenoti. Biological Journal of the Linnean Society 71:315-341.
Sakai, A. K., F. W. Allendorf, J. S. Holt, D. M. Lodge, J. Molofsky, K. A. With, S.
Baughman et al. 2001. The population biology of invasive species. Annual review
of ecology and systematics 32:305-332.
Sinclair, B. J., C. M. Williams, and J. S. Terblanche. 2012. Variation in thermal
performance among insect populations. Physiological and Biochemical Zoology
85:594-606.
Sinervo, B., F. Mendez-De-La-Cruz, D. B. Miles, B. Heulin, E. Bastiaans, M. Villagrán-
Santa Cruz, R. Lara-Resendiz et al. 2010. Erosion of lizard diversity by climate
change and altered thermal niches. Science 328:894-899.
Stearns, S. C. 1992, The evolution of life histories, v. 249. Oxford, Oxford University
Press
Travis, J. M., and C. Dytham. 2002. Dispersal evolution during invasions. Evolutionary
Ecology Research 4:1119-1129.
Warner, D. A., and R. Shine. 2007. Fitness of juvenile lizards depends on seasonal timing
of hatching, not offspring body size. Oecologia 154:65-73.
110