Dewlap size in male water anoles associates with consistent inter-individual variation in boldness

Dewlap size in male water anoles associates with consistent inter-individual variation in boldness Male sexually selected signals can indicate competitive ability by honestly signaling fitness- relevant traits such as condition or performance. However, behavior can also influence contest out- comes; in particular, boldness often predicts dominance rank and mating success. Here, we sought to determine whether male ornament size is associated with consistent individual differences in boldness in water anoles Anolis aquaticus. We measured the relative size of the dewlap, a flap of skin under the chin that is a sexually selected ornament in Anolis lizards, and tested for associa- tions with responses to a novel and potentially risky environment: time to emerge from a refuge into an arena and number of head scans post-emergence. We found that individuals consistently differed in both time to emerge and head scanning (i.e., individual responses were repeatable), and that dewlap size was negatively related to number of head scans. This suggests that ornament size could indicate male boldness if scanning represents antipredator vigilance. We found that males that had larger relative dewlaps were also in better body condition, but boldness (i.e., head scan- ning) was not related to condition. Lastly, we found consistent differences in behavior between tri- als, showing that anoles were becoming habituated or sensitized to the testing arena. Overall, our study shows that in addition to indicating condition and performance, dewlap size could also hon- estly indicate male boldness in Anolis lizards. Key words: Anolis aquaticus, behavioral syndrome, mating success, personality, signal, sexual selection, temperament Many animals possess sexually selected signals that act as reliable because more conspicuous signals (or more time devoted to signal- indicators of fitness-relevant traits (e.g., male quality). Males tend to ing) are more likely to attract predators and/or be energetically convey information in the form of coloration, ornamentation, and/ demanding (Engqvist et al. 2015). Male sexual signals often display or behaviors that conspecific males use to assess potential competi- that, in spite of these costs, the individual has survived and possesses tors and that females use to assess potential mates (Andersson and superior fighting or competitive ability (Zahavi 1977). Often, the Iwasa 1996; Berglund et al. 1996). Countless studies show a link be- magnitude of male signals positively associates with physical traits tween variation in signals and in mating success and/or survival that affect contest outcomes such as body size, body condition, or (e.g., Møller 1988; Pryke et al. 2001; Papeschi and Dessı `-Fulgheri performance (e.g., bite force) (David et al. 1998; Vanhooydonck 2003). However, signals should also inherently involve costs et al. 2005a; Byers et al. 2010). V C The Author(s) (2018). Published by Oxford University Press. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy041/5025953 by guest on 13 July 2018 2 Current Zoology, 2018, Vol. 0, No. 0 Behavioral traits could also influence the outcomes of male–male resistance to being handled (Re ´ ale et al. 2007). Thus, multiple competitions. For instance, boldness often consistently differs behaviors that serve different functions or are dictated by different among individuals, forming a personality trait in which behavioral processes (and that are not necessarily correlated—see Watanabe types (e.g., bold versus shy type) are relatively inflexible in their et al. 2012; Beckmann and Biro 2013) can be combined into the sin- responses (Sih et al. 2004). Boldness broadly encompasses various gle personality trait of boldness. For our study, boldness might be animal behaviors that indicate an individual’s response to a risky en- associated with dewlap size because these traits may share underly- vironment or situation (Re ´ ale et al. 2007). Bold individuals, those ing physiological components—testosterone has been linked to dew- lap size in anoles (Husak et al. 2007; Cox et al. 2009) and to that are more risk-prone, are often more dominant and successful at boldness in other species (Pellis and McKenna 1992; Raynaud and acquiring mates compared to less bold individuals (Reaney and Schradin 2014). Behavioral traits related to boldness are also often Backwell 2007; Colle ´ ter and Brown 2011; Ballew et al. 2017; heritable (Brown et al. 2007; Carrete et al. 2016; Ballew et al. Scherer et al. 2017). Because of their enhanced ability to acquire 2017), so can be subject to evolution following natural selection in resources, males that are bolder tend to also have higher growth subsequent generations. We measured boldness in water anoles as rates and be larger in body size (Brown and Braithwaite 2004; Adriaenssens and Johnsson 2010; Shine et al. 2016), which might two distinct behaviors that may represent different processes: (1) la- also be preferred by potential female mates (Endler and Houde tency to enter a novel arena from within a refuge, a common ap- 1995; Rosenthal and Evans 1998). Indeed, females of some species proach used to measure boldness in various taxa (see: Seda et al. use male boldness over male sexual coloration as their primary mate 2012; Michelangeli et al. 2016a; Shine et al. 2016; Lapiedra et al. choice metric, such that females guppies prefer bolder males even if 2017), and (2) number of head scans during the first minute within their sexual signal is weak (Godin and Dugatkin 1996). Female liz- the novel arena. Latency to emerge from refuge might represent a la- ards also prefer bold males in high risk environments (Teyssier et al. tent response to handling as an observer has to place the lizard into 2014). the arena, while head scans are typically assumed to represent anti- Because of the importance of animal personality in mating com- predator vigilance (i.e., scanning for danger), increasing with risk (Baldellou and Henzi 1992; Mathot et al. 2009; Monclu ´ s et al. petition, some sexual traits may convey information about behavior- 2015). We predicted that bold individuals would have low latencies al types. If male sexual traits indicate competitive ability, and if to emerge and exhibit fewer head scans. We also predicted that these bolder males are more competitive, then sexual traits may positively measures of boldness would positively associate with relative dew- associate with boldness. Furthermore, bold behavior may be related lap size in male water anoles. to the direct development of sexual traits if bold individuals are bet- ter at acquiring resources. Some research has examined the link be- tween boldness and male coloration. In guppies, there is a positive Materials and methods association between orange coloration, courtship, and boldness in males (Re zucha and Reichard 2016). In lizards, boldness and aggres- Study site and species siveness associate with distinct color morphs that also represent dif- We performed our study on water anoles Anolis aquaticus at Las ferent reproductive strategies (Healey and Olsson 2008; Yewers Cruces Biological Station in San Vito, Costa Rica. Anolis aquaticus et al. 2016; Pellitteri-Rosa et al. 2017). Fewer studies have examined is a medium-sized lizard (adult size: 52–77 mm, snout–vent length; the link between boldness and other male sexual traits, like orna- Ma ´ rquez and Ma ´ rquez 2009) with a range throughout southwestern ment size. One study found no link between boldness and tail Costa Rica and northwestern Panama (Savage 2005). The water length, a sexually-selected trait, in barn swallows, Hirundo rustica anole’s habitat is defined by small streams on lowland and pre- (Saino et al. 2014), while another found an association between montane slopes, and its ecology is strongly linked to water: predator cephalic crest development and boldness in the river blenny fish avoidance and foraging almost entirely occurs streamside. Male Salaria fluviatilis (Fabre et al. 2014). water anoles have large orange–red dewlaps, which are used as sex- Here, we sought to determine whether variation in a male sexually ual signals. Little is known about water anole social structure, selected signal, the dewlap, associates with boldness in an Anolis liz- though populations appear to be dense in patches of preferred habi- ard. Anolis lizards (commonly called anoles) have been models of tat, and streamside refugia and display sites are actively contested adaptive radiation and ecomorphological specialization, and the rela- among males (L.S., personal observation). tionship of various morphological traits to fitness is well known The study took place over a 5-week period starting in late June (Losos 2009). The dewlap is an extensible fold of skin below the chin 2016. We captured anoles either by hand or by noosing from a sec- more commonly found in males than females. Research on variation tion of the Rio Java within the biological reserve. We only captured in dewlap presence, size, and coloration within and among species has adult and near-adult (> 50 mm snout–vent length, SVL) male anoles been crucial in understanding signal function, evolution, and associa- for our study. Although certain trapping methods might target a tions with speciation (Nicholson et al. 2007; Ingram et al. 2016). non-random portion of the population, a previous study examining Dewlap size can be an honest indicator of male bite force, jumping different capture techniques of lizards found no effect of trapping ability, and body size or condition (Vanhooydonck et al. 2005a, method on selecting certain behavioral types (Michelangeli et al. 2005b, Irschick et al. 2006; Henningsen and Irschick 2012; Curlis 2016b). After capture, we transported anoles to the field station to et al. 2017) and hence functions as an honest signal of a male’s com- measure morphological traits and perform behavioral trials. Each petitive ability. No work to date has yet determined whether the dew- anole was kept in captivity for no more than 72 h and their behav- lap can also honestly signal male boldness, another trait that has the iors were tested over the course of 2 days. We housed them individu- potential to influence male–male contests and female choice. ally in enclosures (123 193 13.5 cm) within a temperature- For our study, we looked at whether relative dewlap size in male controlled room (20–23 C) that had a 12: 12 light/dark cycle typical water anoles Anolis aquaticus associates with boldness. Boldness for this species. The walls of each enclosure were covered so that the can include behaviors such as risk-taking, response to predators anoles were not able to see each other or the testing arena. After (e.g., antipredator behaviors), response to threats (e.g., a trap), and transporting them back to the station, we allowed the anoles to Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy041/5025953 by guest on 13 July 2018 Putman et al.  Dewlap size and boldness in anoles 3 acclimate to their environment for at least 12 h before we began the Braithwaite 2004; Adriaenssens and Johnsson 2010). Prior to analy- behavioral experiment (see ‘Behavioral experiment’ section). All ses, we looked for correlations among relative dewlap size, body anoles were returned to their place of capture at the Rio Java once condition, and SVL, and found that relative dewlap size was signifi- cantly positively correlated with body condition (Pearson’s they completed the behavioral experiment. r ¼ 0.332, P ¼ 0.019), consistent with results from past studies on anoles (Henningsen and Irschick 2012; Curlis et al. 2017). Morphological measurements However, because the strength of the correlation was relatively low Once anoles were brought back to the station, we recorded SVL and (Figure S1, Supplementary Material), we elected to include all mor- mass. When we were finished doing these measurements for each phological traits into our models as predictor variables and assessed anole, we used forceps to extend the dewlap to its greatest extent collinearity between these variables by calculating the variance infla- and placed the anole on a white sheet of paper next to a ruler. We tion factor (VIF), which measures how much of the variance of an then took a photo of the dewlap extended and later calculated the estimated coefficient increases if predictors are correlated. VIFs> 10 area of the dewlap using the computer software Image J. We took generally indicate high multicollinearity, and values< 3 are ideal photos using an Olympus Tough TG4 (Olympus America Inc., (i.e., multicollinearity is not an issue). Thus, for all analyses, we Center Valley, Pennsylvania, USA). looked at the effects of relative dewlap size, body condition, and SVL on boldness (TTE and no. of scans). Because TTE and no. of Behavioral experiment scans were not correlated (Pearson’s r ¼ 0.217, P ¼ 0.162), they may We measured boldness by examining how anoles responded to being be part of separate personality axes, and so we analyzed them separ- placed into a refuge within a novel arena (i.e., a risky environment). ately. In Supplementary Material, we provide results from models in These trials were conducted in the same light- and temperature- which we tested for the effects of dewlap size and body condition controlled room (20–23 C) in which the anoles were housed. Each separately to demonstrate that the inclusion of these two correlated behavioral test was performed in the morning and twice over 2 days variables into a single model did not alter our overall results. with 24 h in between the two tests, and the order in which anoles We ran linear mixed models (LMMs; lme4 package in R—Bates were tested was randomized each day. One person (K.R.A.) orches- et al. 2015) with relative dewlap size, body condition, and SVL as trated the behavioral trials. For a trial, an anole was taken from its fixed effects, and anole identity and trial number as random effects, to housing enclosure and placed in a 11.53 73 5cm (L3 W3 H) explain variation in head scanning and TTE. Even though TTE con- cardboard refuge (a new one was used each trial), which was then tained time-to-event data, we were able to meet LMM assumptions placed into the novel testing arena that was roughly through a log transformation of the data. Number of scans was 33.53 203 21 cm (L3 W3 H). The testing arena was made of clear square-root transformed to meet model assumptions. We also per- plastic, but the sides were covered with paper on the outside so that formed a Cox-proportional hazards regression analysis (survival pack- anoles could not see beyond the arena walls. The floor of the arena age in R—Therneau 2015) with paired data (i.e., individuals were was covered with white paper that was changed between each trial. tested twice) to examine the TTE data in order to verify that the Trials were video recorded using an iPhone 5 C (Apple Inc., LMM on TTE produced qualitatively similar results to a model that Cupertino, CA, USA) affixed directly above the arena, and behav- accounts for right-censored data. We included relative dewlap size, iors were quantified from video recordings after the study. The ob- body condition, and SVL as independent variables and anole identity server started video recording prior to placing the refuge (which and trial number as clustered variables in the survival analysis. housed the anole) into the arena. Once the refuge was in position, Repeatability (also known as the intraclass correlation coefficient) the observer left the room for the 30 min duration of the trial. The provides insights into the components contributing to variability in testing arena was cleaned thoroughly with dishwashing soap be- the data, and at the level of individual, it provides support that tween trials to prevent any olfactory cues left behind by the previous among-individual variation is caused by intrinsic factors (Re ´ ale et al. anoles that may interfere with future tests. 2007; Nakagawa and Schielzeth 2010). To measure the repeatability At the end of our study, video recordings of the trials were of behaviors, we used the rptR package in R, which calculates repeat- assigned a random number and one person (B.J.P.), blind to anole ability as the group-level variance over the sum of group-level and re- identity, trial number, and date of trial, quantified the anoles’ be- sidual variance (Stoffel et al. 2017). The statistical significance of havioral responses. Two behavioral responses were measured: (1) repeatability is tested through likelihood ratio tests (LRT) by compar- how long it took the anole to fully emerge into the arena (time to ing a model including the grouping factor of interest to one excluding emerge, TTE), and (2) number of head scanning events during the it. We calculated the adjusted repeatability, which controls for the first minute after emerging. We defined a head scan as a lateral addition of fixed effects (in our case, relative dewlap size, body condi- movement of the head from an initial motionless position. We tion, and SVL) identified from linear mixed models (LMM) fitted in assumed that each of these measures negatively associated with the rptR package and based on 1000 bootstrapping runs (which esti- boldness (i.e., bold individuals should have lower latencies to re- mates uncertainty around repeatability). We used the same LMM spond and exhibit fewer head scans). If an anole never emerged structures as above to estimate repeatability of TTE and head scan- from refuge during the trial, we assigned it the maximum value of ning at the level of anole identity to determine whether behaviors 30 min. were repeatable across time within individuals, the definition of per- sonality (Re ´ ale et al. 2007). We also examined repeatability at the Statistical analyses level of trial number, which might arise due to habituation or sensi- To get relative values of morphological measurements (to account tization to the testing arena. Significant repeatability at the level of for allometric scaling), we calculated the residuals from linear trial would signify that anoles consistently behaved differently be- regressions of log SVL on log of square-root dewlap size and log tween trials; for instance, behavioral responses might decline with SVL on log of cube-root body mass (to obtain body condition, time, indicating some level of phenotypic plasticity (e.g., habituation), which also has the potential to associate with boldness; Brown and but individuals can still maintain their relative differences in rank Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy041/5025953 by guest on 13 July 2018 4 Current Zoology, 2018, Vol. 0, No. 0 order (e.g., “bold” individuals still have relatively lower values than indicating that they could be less wary of novel or dangerous situa- “shy” individuals even if there is some overlap). All tests were done in tions. Head scanning was repeatable within individuals, indicating R (v. 3.2.1) and alpha was set to 0.05. that it could be representative of the personality trait of boldness, or response to a risky environment/situation. Previous work in various Results In total, we tested 26 adult male water anoles, 24 of which were tested twice in the behavioral experiment, and 15 of which emerged from the refuge during both trials (i.e., we could examine repeatabil- ity of head scanning post-emergence for these individuals). We looked at whether male dewlap size, body condition, and/or SVL associated with two behavioral responses within a novel envir- onment, head scanning and time to emerge from refuge (TTE). We found that as relative dewlap size increased, the number of head scans performed by anoles decreased (P ¼ 0.044, Table 1, Figure 1). There was no effect of body condition (P ¼ 0.678, Figure 1) or SVL (P ¼ 0.866) on head scanning (Table 1), which was also the case for models in which each of these predictor variables were tested separ- ately (Table S1, S2; Supplementary Material). Another linear mixed model found that none of the predictor variables influenced TTE, al- though SVL had a marginally non-significant effect (P ¼ 0.078, Table 1). Results were qualitatively similar from the survival ana- lysis: TTE was not affected by relative dewlap size (hazard ratio ¼ 0.426, Z¼0.206, P ¼ 0.837) or body condition (hazard ratio ¼ 0.089, Z ¼ -0.205, P ¼ 0.838), and SVL had a marginally non-significant effect (hazard ratio ¼ 0.962, Z¼1.658, Figure 1. Relationship of relative dewlap size, body condition, and number of P ¼ 0.097). All predictor variables in the LMMs had VIFs< 2, indi- head scans in a novel environment for adult male water anoles. Body condi- cating that multicollinearity between variables had little influence tion was positively correlated with dewlap size, but not with head scanning. on the variance of the model coefficients. Points on plot are slightly jittered to reduce overlap. We found that both TTE and number of head scans were repeat- able within individual anoles (TTE: R ¼ 0.4646 0.151 SE, LRT ¼ 6.20, df ¼ 1, P ¼ 0.006, Figure 2A; no. of scans: R ¼ 0.5636 0.174, LRT ¼ 6.95, df ¼ 1, P ¼ 0.004, Figure 2B), indi- cating that these behaviors represent personality with significant consistent differences among individuals. We also found no. of scans was repeatable at the level of trial, albeit low (R ¼ 0.1466 0.159, LRT ¼ 3.84, df ¼ 1, P ¼ 0.025), such that anoles scanned more dur- ing the second trial compared to the first (Figure 2). Repeatability of TTE at the level of trial was low and not statistically significant (R ¼ 0.0626 0.091 SE, LRT ¼ 1.60, df ¼ 1, P ¼ 0.103). Discussion In our study, male water anoles with larger relative dewlaps scanned a novel environment less than males with smaller dewlaps, Table 1. Results of the linear mixed models predicting head scan- ning behavior and time to emerge from refuge (TTE) in male water anoles Response Predictor variable b6 SE t P variable Head scans (Intercept) 1.5116 1.358 1.113 0.266 Relative dewlap size 27.1406 3.546 22.014 0.044 Body condition 5.1026 12.295 0.415 0.678 SVL 0.0036 0.020 0.169 0.886 TTE (Intercept) 20.0146 1.309 20.011 0.991 Relative dewlap size 1.1916 3.398 0.351 0.726 Body condition 4.5076 11.321 0.398 0.691 Figure 2. Spaghetti plots showing individual anole differences in (A) time to SVL 0.0356 0.020 1.756 0.079 emerge (TTE) from a refuge into the novel environment between trial 1 and trial 2, and (B) number of head scans in the novel environment between trial Predictor variables that met statistical significance are in bold. 1 and trial 2. Each line represents an individual anole. Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy041/5025953 by guest on 13 July 2018 Putman et al.  Dewlap size and boldness in anoles 5 lizard species have found male coloration to reliably indicate person- reach statistical significance). We found that anoles consistently ality differences (Healey and Olsson 2008; Yewers et al. 2016; scanned more during the second trial compared to the first, which, if Pellitteri-Rosa et al. 2017). This is the first study, to our knowledge, scanning represents vigilance, would suggest a more risk-averse re- to show that a non-color sexual trait, dewlap size, could be an hon- sponse, and suggests that anoles were becoming sensitized, and not est indicator of male personality in Anolis lizards. habituated, to the testing arena. Either way, we show that anoles Dewlap size in various species of anole is known to reliably indi- exhibited phenotypically plastic responses to change over time, but cate male size, condition, and performance (Vanhooydonck et al. still maintained consistent individual differences in behavior, appar- 2005a, 2005b; Irschick et al. 2006; Henningsen and Irschick 2012; ent in the significant repeatability in TTE and head scanning at the Curlis et al. 2017; Petelo and Swierk 2017). Here, we show that level of individual. even with a relatively small number of individuals tested, dewlap Finally, relative dewlap size was positively correlated with body size also indicated consistent differences in scanning a novel and po- condition, but not with body size (SVL) in our study. In other spe- tentially risky environment. Head scans are a measure of antipreda- cies of anole, dewlap size is also condition-dependent (Henningsen tor vigilance behavior in other animals (Baldellou and Henzi 1992; and Irschick 2012; Curlis et al. 2017). A separate study also found Mathot et al. 2009; Monclu ´ s et al. 2015). Vigilance helps animals that larger male water anoles have disproportionately larger dew- assess risk and detect potential predators, but reduces time spent on laps and weaponry compared to smaller males (Petelo and Swierk foraging or other fitness-related activities (Lima and Bednekoff 2017). Alternatively, these traits could all share a proximate mech- 1999; Putman and Clark 2015; Clermont et al. 2016). Our results anism, such as a common neuroendocrine mechanism (Kralj-Fi ser demonstrate that males with larger dewlaps perform less surveil- et al. 2010). However, even though males in better condition had lance behavior when entering a new environment. Although scan- larger relative dewlaps, and those with larger dewlaps exhibited ning could also be used to detect things other than predators or fewer head scans, body condition was not related to head scanning danger, e.g., mates, it is unlikely that the context in which they were or time to emerge in a novel environment. The correlation between tested (in a novel arena and without mate cues), anoles were per- body condition and dewlap size was relatively weak and this likely forming mate-searching in our study. If anoles with larger dewlaps is why we failed to detect condition-dependent boldness. Whether spend less time being vigilant, this could increase their available these traits associate with greater reproductive success remains to be time to defend territory and/or forage. However, this type of bold- tested. For instance, females might prefer males with larger dewlaps ness might also increase their risk of predation (Smith and Blumstein merely due to its relationship with body condition and not necessar- 2010). Future work could assess these trade-offs in free-ranging ily boldness. Bold behavior could be the mechanism that produces individuals. larger dewlaps (if bold individuals spend more time foraging and We did not find a relationship between dewlap size and latency gaining energy to put toward ornament size). Further studies (with to emerge from a refuge into the novel environment, and this, in larger sample sizes) on the interplay between these three traits and part, could be due to our relatively low sample size of individuals on female preferences will advance our knowledge on the relative tested. Latency to emerge was repeatable within individual anoles, importance of sexual selection on boldness in anoles. suggesting it can be part of a personality trait, but it appears to be distinct from head scanning since these behaviors were not corre- lated or similarly affected by dewlap size. Time to emerge from a Acknowledgments refuge is a common measure of boldness in studies on various spe- For their support and mentorship, we thank Daniel Kinsey, Randall Werk, cies, including lizards (Seda et al. 2012; Michelangeli et al. 2016a; Brian Grebliunas, Dan Blumstein, Greg Pauly, Juan Moreira, Scott Walter, Shine et al. 2016; Lapiedra et al. 2017). This suggests that head and Rodo Quiros Flores. For help with field and lab work, we thank the ladies scanning might not be a measure of boldness, but some other per- of team “Lizard Wizards”: Sattie Whitefoot, Amber Morgan, Jennet Chang, sonality trait, but it seems more likely that head scanning might rep- and Kimberly Guo. For help with statistical analyses, we thank Bob Wong. resent a different type of boldness. Even though these behaviors, to Use of animals was reviewed and approved by Yale University (IACUC ap- us, may represent animals responding to risk (i.e., boldness), they proval #2016-20103), and research permits were obtained from the Ministry may result from different perceptions of risk by the animals and of the Environment and Energy, Republic of Costa Rica (128-16-ACLAP). have different functions. For instance, latency to emerge from refuge could represent a response to a recent predator encounter (i.e., human handling the lizard) while scanning could represent predator Funding detection (i.e., vigilance). This is supported by a study done on her- This study was completed with funding from the National Science mit crabs, which found responses after a predator attack loaded on Foundation and the Organization for Tropical Studies, which supported the a separate axis than behaviors related to predator detection, even authors during the Native American and Pacific Islander Research Experience though all behaviors measured fell under the umbrella of boldness (NAPIRE) at Las Cruces. (Watanabe et al. 2012). Regardless, male water anoles with larger sexual signals scan their environment less, and this could be a risky strategy if scanning allows individuals to detect danger. Supplementary Material We also found that head scanning was repeatable at the level of Supplementary material can be found at https://academic.oup.com/cz. trial, indicating that anoles consistently behaved differently in the se- cond trial compared to the first. 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Dewlap size in male water anoles associates with consistent inter-individual variation in boldness

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Abstract

Male sexually selected signals can indicate competitive ability by honestly signaling fitness- relevant traits such as condition or performance. However, behavior can also influence contest out- comes; in particular, boldness often predicts dominance rank and mating success. Here, we sought to determine whether male ornament size is associated with consistent individual differences in boldness in water anoles Anolis aquaticus. We measured the relative size of the dewlap, a flap of skin under the chin that is a sexually selected ornament in Anolis lizards, and tested for associa- tions with responses to a novel and potentially risky environment: time to emerge from a refuge into an arena and number of head scans post-emergence. We found that individuals consistently differed in both time to emerge and head scanning (i.e., individual responses were repeatable), and that dewlap size was negatively related to number of head scans. This suggests that ornament size could indicate male boldness if scanning represents antipredator vigilance. We found that males that had larger relative dewlaps were also in better body condition, but boldness (i.e., head scan- ning) was not related to condition. Lastly, we found consistent differences in behavior between tri- als, showing that anoles were becoming habituated or sensitized to the testing arena. Overall, our study shows that in addition to indicating condition and performance, dewlap size could also hon- estly indicate male boldness in Anolis lizards. Key words: Anolis aquaticus, behavioral syndrome, mating success, personality, signal, sexual selection, temperament Many animals possess sexually selected signals that act as reliable because more conspicuous signals (or more time devoted to signal- indicators of fitness-relevant traits (e.g., male quality). Males tend to ing) are more likely to attract predators and/or be energetically convey information in the form of coloration, ornamentation, and/ demanding (Engqvist et al. 2015). Male sexual signals often display or behaviors that conspecific males use to assess potential competi- that, in spite of these costs, the individual has survived and possesses tors and that females use to assess potential mates (Andersson and superior fighting or competitive ability (Zahavi 1977). Often, the Iwasa 1996; Berglund et al. 1996). Countless studies show a link be- magnitude of male signals positively associates with physical traits tween variation in signals and in mating success and/or survival that affect contest outcomes such as body size, body condition, or (e.g., Møller 1988; Pryke et al. 2001; Papeschi and Dessı `-Fulgheri performance (e.g., bite force) (David et al. 1998; Vanhooydonck 2003). However, signals should also inherently involve costs et al. 2005a; Byers et al. 2010). V C The Author(s) (2018). Published by Oxford University Press. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy041/5025953 by guest on 13 July 2018 2 Current Zoology, 2018, Vol. 0, No. 0 Behavioral traits could also influence the outcomes of male–male resistance to being handled (Re ´ ale et al. 2007). Thus, multiple competitions. For instance, boldness often consistently differs behaviors that serve different functions or are dictated by different among individuals, forming a personality trait in which behavioral processes (and that are not necessarily correlated—see Watanabe types (e.g., bold versus shy type) are relatively inflexible in their et al. 2012; Beckmann and Biro 2013) can be combined into the sin- responses (Sih et al. 2004). Boldness broadly encompasses various gle personality trait of boldness. For our study, boldness might be animal behaviors that indicate an individual’s response to a risky en- associated with dewlap size because these traits may share underly- vironment or situation (Re ´ ale et al. 2007). Bold individuals, those ing physiological components—testosterone has been linked to dew- lap size in anoles (Husak et al. 2007; Cox et al. 2009) and to that are more risk-prone, are often more dominant and successful at boldness in other species (Pellis and McKenna 1992; Raynaud and acquiring mates compared to less bold individuals (Reaney and Schradin 2014). Behavioral traits related to boldness are also often Backwell 2007; Colle ´ ter and Brown 2011; Ballew et al. 2017; heritable (Brown et al. 2007; Carrete et al. 2016; Ballew et al. Scherer et al. 2017). Because of their enhanced ability to acquire 2017), so can be subject to evolution following natural selection in resources, males that are bolder tend to also have higher growth subsequent generations. We measured boldness in water anoles as rates and be larger in body size (Brown and Braithwaite 2004; Adriaenssens and Johnsson 2010; Shine et al. 2016), which might two distinct behaviors that may represent different processes: (1) la- also be preferred by potential female mates (Endler and Houde tency to enter a novel arena from within a refuge, a common ap- 1995; Rosenthal and Evans 1998). Indeed, females of some species proach used to measure boldness in various taxa (see: Seda et al. use male boldness over male sexual coloration as their primary mate 2012; Michelangeli et al. 2016a; Shine et al. 2016; Lapiedra et al. choice metric, such that females guppies prefer bolder males even if 2017), and (2) number of head scans during the first minute within their sexual signal is weak (Godin and Dugatkin 1996). Female liz- the novel arena. Latency to emerge from refuge might represent a la- ards also prefer bold males in high risk environments (Teyssier et al. tent response to handling as an observer has to place the lizard into 2014). the arena, while head scans are typically assumed to represent anti- Because of the importance of animal personality in mating com- predator vigilance (i.e., scanning for danger), increasing with risk (Baldellou and Henzi 1992; Mathot et al. 2009; Monclu ´ s et al. petition, some sexual traits may convey information about behavior- 2015). We predicted that bold individuals would have low latencies al types. If male sexual traits indicate competitive ability, and if to emerge and exhibit fewer head scans. We also predicted that these bolder males are more competitive, then sexual traits may positively measures of boldness would positively associate with relative dew- associate with boldness. Furthermore, bold behavior may be related lap size in male water anoles. to the direct development of sexual traits if bold individuals are bet- ter at acquiring resources. Some research has examined the link be- tween boldness and male coloration. In guppies, there is a positive Materials and methods association between orange coloration, courtship, and boldness in males (Re zucha and Reichard 2016). In lizards, boldness and aggres- Study site and species siveness associate with distinct color morphs that also represent dif- We performed our study on water anoles Anolis aquaticus at Las ferent reproductive strategies (Healey and Olsson 2008; Yewers Cruces Biological Station in San Vito, Costa Rica. Anolis aquaticus et al. 2016; Pellitteri-Rosa et al. 2017). Fewer studies have examined is a medium-sized lizard (adult size: 52–77 mm, snout–vent length; the link between boldness and other male sexual traits, like orna- Ma ´ rquez and Ma ´ rquez 2009) with a range throughout southwestern ment size. One study found no link between boldness and tail Costa Rica and northwestern Panama (Savage 2005). The water length, a sexually-selected trait, in barn swallows, Hirundo rustica anole’s habitat is defined by small streams on lowland and pre- (Saino et al. 2014), while another found an association between montane slopes, and its ecology is strongly linked to water: predator cephalic crest development and boldness in the river blenny fish avoidance and foraging almost entirely occurs streamside. Male Salaria fluviatilis (Fabre et al. 2014). water anoles have large orange–red dewlaps, which are used as sex- Here, we sought to determine whether variation in a male sexually ual signals. Little is known about water anole social structure, selected signal, the dewlap, associates with boldness in an Anolis liz- though populations appear to be dense in patches of preferred habi- ard. Anolis lizards (commonly called anoles) have been models of tat, and streamside refugia and display sites are actively contested adaptive radiation and ecomorphological specialization, and the rela- among males (L.S., personal observation). tionship of various morphological traits to fitness is well known The study took place over a 5-week period starting in late June (Losos 2009). The dewlap is an extensible fold of skin below the chin 2016. We captured anoles either by hand or by noosing from a sec- more commonly found in males than females. Research on variation tion of the Rio Java within the biological reserve. We only captured in dewlap presence, size, and coloration within and among species has adult and near-adult (> 50 mm snout–vent length, SVL) male anoles been crucial in understanding signal function, evolution, and associa- for our study. Although certain trapping methods might target a tions with speciation (Nicholson et al. 2007; Ingram et al. 2016). non-random portion of the population, a previous study examining Dewlap size can be an honest indicator of male bite force, jumping different capture techniques of lizards found no effect of trapping ability, and body size or condition (Vanhooydonck et al. 2005a, method on selecting certain behavioral types (Michelangeli et al. 2005b, Irschick et al. 2006; Henningsen and Irschick 2012; Curlis 2016b). After capture, we transported anoles to the field station to et al. 2017) and hence functions as an honest signal of a male’s com- measure morphological traits and perform behavioral trials. Each petitive ability. No work to date has yet determined whether the dew- anole was kept in captivity for no more than 72 h and their behav- lap can also honestly signal male boldness, another trait that has the iors were tested over the course of 2 days. We housed them individu- potential to influence male–male contests and female choice. ally in enclosures (123 193 13.5 cm) within a temperature- For our study, we looked at whether relative dewlap size in male controlled room (20–23 C) that had a 12: 12 light/dark cycle typical water anoles Anolis aquaticus associates with boldness. Boldness for this species. The walls of each enclosure were covered so that the can include behaviors such as risk-taking, response to predators anoles were not able to see each other or the testing arena. After (e.g., antipredator behaviors), response to threats (e.g., a trap), and transporting them back to the station, we allowed the anoles to Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy041/5025953 by guest on 13 July 2018 Putman et al.  Dewlap size and boldness in anoles 3 acclimate to their environment for at least 12 h before we began the Braithwaite 2004; Adriaenssens and Johnsson 2010). Prior to analy- behavioral experiment (see ‘Behavioral experiment’ section). All ses, we looked for correlations among relative dewlap size, body anoles were returned to their place of capture at the Rio Java once condition, and SVL, and found that relative dewlap size was signifi- cantly positively correlated with body condition (Pearson’s they completed the behavioral experiment. r ¼ 0.332, P ¼ 0.019), consistent with results from past studies on anoles (Henningsen and Irschick 2012; Curlis et al. 2017). Morphological measurements However, because the strength of the correlation was relatively low Once anoles were brought back to the station, we recorded SVL and (Figure S1, Supplementary Material), we elected to include all mor- mass. When we were finished doing these measurements for each phological traits into our models as predictor variables and assessed anole, we used forceps to extend the dewlap to its greatest extent collinearity between these variables by calculating the variance infla- and placed the anole on a white sheet of paper next to a ruler. We tion factor (VIF), which measures how much of the variance of an then took a photo of the dewlap extended and later calculated the estimated coefficient increases if predictors are correlated. VIFs> 10 area of the dewlap using the computer software Image J. We took generally indicate high multicollinearity, and values< 3 are ideal photos using an Olympus Tough TG4 (Olympus America Inc., (i.e., multicollinearity is not an issue). Thus, for all analyses, we Center Valley, Pennsylvania, USA). looked at the effects of relative dewlap size, body condition, and SVL on boldness (TTE and no. of scans). Because TTE and no. of Behavioral experiment scans were not correlated (Pearson’s r ¼ 0.217, P ¼ 0.162), they may We measured boldness by examining how anoles responded to being be part of separate personality axes, and so we analyzed them separ- placed into a refuge within a novel arena (i.e., a risky environment). ately. In Supplementary Material, we provide results from models in These trials were conducted in the same light- and temperature- which we tested for the effects of dewlap size and body condition controlled room (20–23 C) in which the anoles were housed. Each separately to demonstrate that the inclusion of these two correlated behavioral test was performed in the morning and twice over 2 days variables into a single model did not alter our overall results. with 24 h in between the two tests, and the order in which anoles We ran linear mixed models (LMMs; lme4 package in R—Bates were tested was randomized each day. One person (K.R.A.) orches- et al. 2015) with relative dewlap size, body condition, and SVL as trated the behavioral trials. For a trial, an anole was taken from its fixed effects, and anole identity and trial number as random effects, to housing enclosure and placed in a 11.53 73 5cm (L3 W3 H) explain variation in head scanning and TTE. Even though TTE con- cardboard refuge (a new one was used each trial), which was then tained time-to-event data, we were able to meet LMM assumptions placed into the novel testing arena that was roughly through a log transformation of the data. Number of scans was 33.53 203 21 cm (L3 W3 H). The testing arena was made of clear square-root transformed to meet model assumptions. We also per- plastic, but the sides were covered with paper on the outside so that formed a Cox-proportional hazards regression analysis (survival pack- anoles could not see beyond the arena walls. The floor of the arena age in R—Therneau 2015) with paired data (i.e., individuals were was covered with white paper that was changed between each trial. tested twice) to examine the TTE data in order to verify that the Trials were video recorded using an iPhone 5 C (Apple Inc., LMM on TTE produced qualitatively similar results to a model that Cupertino, CA, USA) affixed directly above the arena, and behav- accounts for right-censored data. We included relative dewlap size, iors were quantified from video recordings after the study. The ob- body condition, and SVL as independent variables and anole identity server started video recording prior to placing the refuge (which and trial number as clustered variables in the survival analysis. housed the anole) into the arena. Once the refuge was in position, Repeatability (also known as the intraclass correlation coefficient) the observer left the room for the 30 min duration of the trial. The provides insights into the components contributing to variability in testing arena was cleaned thoroughly with dishwashing soap be- the data, and at the level of individual, it provides support that tween trials to prevent any olfactory cues left behind by the previous among-individual variation is caused by intrinsic factors (Re ´ ale et al. anoles that may interfere with future tests. 2007; Nakagawa and Schielzeth 2010). To measure the repeatability At the end of our study, video recordings of the trials were of behaviors, we used the rptR package in R, which calculates repeat- assigned a random number and one person (B.J.P.), blind to anole ability as the group-level variance over the sum of group-level and re- identity, trial number, and date of trial, quantified the anoles’ be- sidual variance (Stoffel et al. 2017). The statistical significance of havioral responses. Two behavioral responses were measured: (1) repeatability is tested through likelihood ratio tests (LRT) by compar- how long it took the anole to fully emerge into the arena (time to ing a model including the grouping factor of interest to one excluding emerge, TTE), and (2) number of head scanning events during the it. We calculated the adjusted repeatability, which controls for the first minute after emerging. We defined a head scan as a lateral addition of fixed effects (in our case, relative dewlap size, body condi- movement of the head from an initial motionless position. We tion, and SVL) identified from linear mixed models (LMM) fitted in assumed that each of these measures negatively associated with the rptR package and based on 1000 bootstrapping runs (which esti- boldness (i.e., bold individuals should have lower latencies to re- mates uncertainty around repeatability). We used the same LMM spond and exhibit fewer head scans). If an anole never emerged structures as above to estimate repeatability of TTE and head scan- from refuge during the trial, we assigned it the maximum value of ning at the level of anole identity to determine whether behaviors 30 min. were repeatable across time within individuals, the definition of per- sonality (Re ´ ale et al. 2007). We also examined repeatability at the Statistical analyses level of trial number, which might arise due to habituation or sensi- To get relative values of morphological measurements (to account tization to the testing arena. Significant repeatability at the level of for allometric scaling), we calculated the residuals from linear trial would signify that anoles consistently behaved differently be- regressions of log SVL on log of square-root dewlap size and log tween trials; for instance, behavioral responses might decline with SVL on log of cube-root body mass (to obtain body condition, time, indicating some level of phenotypic plasticity (e.g., habituation), which also has the potential to associate with boldness; Brown and but individuals can still maintain their relative differences in rank Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy041/5025953 by guest on 13 July 2018 4 Current Zoology, 2018, Vol. 0, No. 0 order (e.g., “bold” individuals still have relatively lower values than indicating that they could be less wary of novel or dangerous situa- “shy” individuals even if there is some overlap). All tests were done in tions. Head scanning was repeatable within individuals, indicating R (v. 3.2.1) and alpha was set to 0.05. that it could be representative of the personality trait of boldness, or response to a risky environment/situation. Previous work in various Results In total, we tested 26 adult male water anoles, 24 of which were tested twice in the behavioral experiment, and 15 of which emerged from the refuge during both trials (i.e., we could examine repeatabil- ity of head scanning post-emergence for these individuals). We looked at whether male dewlap size, body condition, and/or SVL associated with two behavioral responses within a novel envir- onment, head scanning and time to emerge from refuge (TTE). We found that as relative dewlap size increased, the number of head scans performed by anoles decreased (P ¼ 0.044, Table 1, Figure 1). There was no effect of body condition (P ¼ 0.678, Figure 1) or SVL (P ¼ 0.866) on head scanning (Table 1), which was also the case for models in which each of these predictor variables were tested separ- ately (Table S1, S2; Supplementary Material). Another linear mixed model found that none of the predictor variables influenced TTE, al- though SVL had a marginally non-significant effect (P ¼ 0.078, Table 1). Results were qualitatively similar from the survival ana- lysis: TTE was not affected by relative dewlap size (hazard ratio ¼ 0.426, Z¼0.206, P ¼ 0.837) or body condition (hazard ratio ¼ 0.089, Z ¼ -0.205, P ¼ 0.838), and SVL had a marginally non-significant effect (hazard ratio ¼ 0.962, Z¼1.658, Figure 1. Relationship of relative dewlap size, body condition, and number of P ¼ 0.097). All predictor variables in the LMMs had VIFs< 2, indi- head scans in a novel environment for adult male water anoles. Body condi- cating that multicollinearity between variables had little influence tion was positively correlated with dewlap size, but not with head scanning. on the variance of the model coefficients. Points on plot are slightly jittered to reduce overlap. We found that both TTE and number of head scans were repeat- able within individual anoles (TTE: R ¼ 0.4646 0.151 SE, LRT ¼ 6.20, df ¼ 1, P ¼ 0.006, Figure 2A; no. of scans: R ¼ 0.5636 0.174, LRT ¼ 6.95, df ¼ 1, P ¼ 0.004, Figure 2B), indi- cating that these behaviors represent personality with significant consistent differences among individuals. We also found no. of scans was repeatable at the level of trial, albeit low (R ¼ 0.1466 0.159, LRT ¼ 3.84, df ¼ 1, P ¼ 0.025), such that anoles scanned more dur- ing the second trial compared to the first (Figure 2). Repeatability of TTE at the level of trial was low and not statistically significant (R ¼ 0.0626 0.091 SE, LRT ¼ 1.60, df ¼ 1, P ¼ 0.103). Discussion In our study, male water anoles with larger relative dewlaps scanned a novel environment less than males with smaller dewlaps, Table 1. Results of the linear mixed models predicting head scan- ning behavior and time to emerge from refuge (TTE) in male water anoles Response Predictor variable b6 SE t P variable Head scans (Intercept) 1.5116 1.358 1.113 0.266 Relative dewlap size 27.1406 3.546 22.014 0.044 Body condition 5.1026 12.295 0.415 0.678 SVL 0.0036 0.020 0.169 0.886 TTE (Intercept) 20.0146 1.309 20.011 0.991 Relative dewlap size 1.1916 3.398 0.351 0.726 Body condition 4.5076 11.321 0.398 0.691 Figure 2. Spaghetti plots showing individual anole differences in (A) time to SVL 0.0356 0.020 1.756 0.079 emerge (TTE) from a refuge into the novel environment between trial 1 and trial 2, and (B) number of head scans in the novel environment between trial Predictor variables that met statistical significance are in bold. 1 and trial 2. Each line represents an individual anole. Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy041/5025953 by guest on 13 July 2018 Putman et al.  Dewlap size and boldness in anoles 5 lizard species have found male coloration to reliably indicate person- reach statistical significance). We found that anoles consistently ality differences (Healey and Olsson 2008; Yewers et al. 2016; scanned more during the second trial compared to the first, which, if Pellitteri-Rosa et al. 2017). This is the first study, to our knowledge, scanning represents vigilance, would suggest a more risk-averse re- to show that a non-color sexual trait, dewlap size, could be an hon- sponse, and suggests that anoles were becoming sensitized, and not est indicator of male personality in Anolis lizards. habituated, to the testing arena. Either way, we show that anoles Dewlap size in various species of anole is known to reliably indi- exhibited phenotypically plastic responses to change over time, but cate male size, condition, and performance (Vanhooydonck et al. still maintained consistent individual differences in behavior, appar- 2005a, 2005b; Irschick et al. 2006; Henningsen and Irschick 2012; ent in the significant repeatability in TTE and head scanning at the Curlis et al. 2017; Petelo and Swierk 2017). Here, we show that level of individual. even with a relatively small number of individuals tested, dewlap Finally, relative dewlap size was positively correlated with body size also indicated consistent differences in scanning a novel and po- condition, but not with body size (SVL) in our study. In other spe- tentially risky environment. Head scans are a measure of antipreda- cies of anole, dewlap size is also condition-dependent (Henningsen tor vigilance behavior in other animals (Baldellou and Henzi 1992; and Irschick 2012; Curlis et al. 2017). A separate study also found Mathot et al. 2009; Monclu ´ s et al. 2015). Vigilance helps animals that larger male water anoles have disproportionately larger dew- assess risk and detect potential predators, but reduces time spent on laps and weaponry compared to smaller males (Petelo and Swierk foraging or other fitness-related activities (Lima and Bednekoff 2017). Alternatively, these traits could all share a proximate mech- 1999; Putman and Clark 2015; Clermont et al. 2016). Our results anism, such as a common neuroendocrine mechanism (Kralj-Fi ser demonstrate that males with larger dewlaps perform less surveil- et al. 2010). However, even though males in better condition had lance behavior when entering a new environment. Although scan- larger relative dewlaps, and those with larger dewlaps exhibited ning could also be used to detect things other than predators or fewer head scans, body condition was not related to head scanning danger, e.g., mates, it is unlikely that the context in which they were or time to emerge in a novel environment. The correlation between tested (in a novel arena and without mate cues), anoles were per- body condition and dewlap size was relatively weak and this likely forming mate-searching in our study. If anoles with larger dewlaps is why we failed to detect condition-dependent boldness. Whether spend less time being vigilant, this could increase their available these traits associate with greater reproductive success remains to be time to defend territory and/or forage. However, this type of bold- tested. For instance, females might prefer males with larger dewlaps ness might also increase their risk of predation (Smith and Blumstein merely due to its relationship with body condition and not necessar- 2010). Future work could assess these trade-offs in free-ranging ily boldness. Bold behavior could be the mechanism that produces individuals. larger dewlaps (if bold individuals spend more time foraging and We did not find a relationship between dewlap size and latency gaining energy to put toward ornament size). Further studies (with to emerge from a refuge into the novel environment, and this, in larger sample sizes) on the interplay between these three traits and part, could be due to our relatively low sample size of individuals on female preferences will advance our knowledge on the relative tested. Latency to emerge was repeatable within individual anoles, importance of sexual selection on boldness in anoles. suggesting it can be part of a personality trait, but it appears to be distinct from head scanning since these behaviors were not corre- lated or similarly affected by dewlap size. Time to emerge from a Acknowledgments refuge is a common measure of boldness in studies on various spe- For their support and mentorship, we thank Daniel Kinsey, Randall Werk, cies, including lizards (Seda et al. 2012; Michelangeli et al. 2016a; Brian Grebliunas, Dan Blumstein, Greg Pauly, Juan Moreira, Scott Walter, Shine et al. 2016; Lapiedra et al. 2017). This suggests that head and Rodo Quiros Flores. For help with field and lab work, we thank the ladies scanning might not be a measure of boldness, but some other per- of team “Lizard Wizards”: Sattie Whitefoot, Amber Morgan, Jennet Chang, sonality trait, but it seems more likely that head scanning might rep- and Kimberly Guo. For help with statistical analyses, we thank Bob Wong. resent a different type of boldness. Even though these behaviors, to Use of animals was reviewed and approved by Yale University (IACUC ap- us, may represent animals responding to risk (i.e., boldness), they proval #2016-20103), and research permits were obtained from the Ministry may result from different perceptions of risk by the animals and of the Environment and Energy, Republic of Costa Rica (128-16-ACLAP). have different functions. For instance, latency to emerge from refuge could represent a response to a recent predator encounter (i.e., human handling the lizard) while scanning could represent predator Funding detection (i.e., vigilance). This is supported by a study done on her- This study was completed with funding from the National Science mit crabs, which found responses after a predator attack loaded on Foundation and the Organization for Tropical Studies, which supported the a separate axis than behaviors related to predator detection, even authors during the Native American and Pacific Islander Research Experience though all behaviors measured fell under the umbrella of boldness (NAPIRE) at Las Cruces. (Watanabe et al. 2012). Regardless, male water anoles with larger sexual signals scan their environment less, and this could be a risky strategy if scanning allows individuals to detect danger. Supplementary Material We also found that head scanning was repeatable at the level of Supplementary material can be found at https://academic.oup.com/cz. trial, indicating that anoles consistently behaved differently in the se- cond trial compared to the first. 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