Genome-wide analysis of RNA-chromatin interactions in lizards as a mean for functional lncRNA identification.

BACKGROUND: Long non-coding RNAs (lncRNAs) are defined as transcribed molecules longer than 200 nucleotides with little to no protein-coding potential. LncRNAs can regulate gene expression of nearby genes (cis-acting) or genes located on other chromosomes (trans-acting). Several methodologies have been developed to capture lncRNAs associated with chromatin at a genome-wide level. Analysis of RNA-DNA contacts can be combined with epigenetic and RNA-seq data to define potential lncRNAs involved in the regulation of gene expression. RESULTS: We performed Chromatin Associated RNA sequencing (ChAR-seq) in Anolis carolinensis to obtain the genome-wide map of the associations that RNA molecules have with chromatin. We analyzed the frequency of DNA contacts for different classes of RNAs and were able to define cis- and trans-acting lncRNAs. We integrated the ChAR-seq map of RNA-DNA contacts with epigenetic data for the acetylation of lysine 16 on histone H4 (H4K16ac), a mark connected to actively transcribed chromatin in lizards. We successfully identified three trans-acting lncRNAs significantly associated with the H4K16ac signal, which are likely involved in the regulation of gene expression in A. carolinensis. CONCLUSIONS: We show that the ChAR-seq method is a powerful tool to explore the RNA-DNA map of interactions. Moreover, in combination with epigenetic data, ChAR-seq can be applied in non-model species to establish potential roles for predicted lncRNAs that lack functional annotations.

Sprint training interacts with body mass to affect hepatic insulin-like growth factor expression in female green anoles (Anolis carolinensis).

Locomotor performance is a key predictor of fitness in many animal species. As such, locomotion integrates the output of a number of morphological, physiological, and molecular levels of organization, yet relatively little is known regarding the major molecular pathways that bolster locomotor performance. One potentially relevant pathway is the insulin and insulin-like signaling (IIS) network, a significant regulator of physiological processes such as reproduction, growth, and metabolism. Two primary hormones of this network, insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2) are important mediators of these processes and, consequently, of life-history strategies. We sprint-trained green anole (Anolis carolinensis) females to test the responsiveness of IGF1 and IGF2 hepatic gene expression to exercise training. We also tested how sprint training would affect glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 2 (EEF2). The former is a crucial enzyme for glycolytic function in a cell, and the latter is necessary for protein synthesis. Resistance exercise forces animals to increase investment of resources towards skeletal muscle growth. Because IGF1 and IGF2 are important hormones for growth, and GAPDH and EEF2 are crucial for proper cellular function, we hypothesized that these four genes would be affected by sprint training. We found that sprint training affects IGF and EEF2 expression, such that larger sprint-trained lizards express hepatic IGF1, IGF2, and EEF2 to a lesser extent than similarly sized untrained lizards. These results demonstrate that the IIS, and pathways connected to it, can react in a size-dependent manner and are implicated in the exercise response in reptiles.

Expression of insulin-like growth factors depends on both mass and resource availability in female green anoles (Anolis carolinensis).

The insulin and insulin-like signaling (IIS) network is an important mediator of cellular growth and metabolism in animals, and is sensitive to environmental conditions such as temperature and resource availability. The two main hormones of the IIS network, insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2), are present in all vertebrates, yet little is known regarding the responsiveness of IGF2 in particular to external stimuli in non-mammalian animals. We manipulated diet (low or high quantity of food: low and high diet group, respectively) in adult green anole (Anolis carolinensis) females to test the effect of energetic state on hepatic gene expression of IGF1 and IGF2. The absolute expression of IGF2 in female green anoles was 100 times higher than that of IGF1 regardless of diet treatment, and IGF1 and IGF2 expression interacted with post-treatment body mass and treatment, as did the expression of the purported housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 2 (EEF2). The low diet group showed a negative relationship between body mass and gene expression for all genes, whereas the relationships between body mass and gene expression in the high diet group were either absent (in the case of IGF1) or positive (for all other genes). After accounting for total change in mass, the low diet group expressed IGF2, GAPDH and EEF2 at higher levels compared with individuals in the high diet group of a similar change in mass. These results illustrate that expression of IGF1 and IGF2, and of the housekeeping genes is affected by energe-tic status in reptiles.

Selection at behavioural, developmental and metabolic genes is associated with the northward expansion of a successful tropical colonizer.

What makes a species able to colonize novel environments? This question is key to understand the dynamics of adaptive radiations and ecological niche shifts, but the mechanisms that underlie expansion into novel habitats remain poorly understood at a genomic scale. Lizards from the genus Anolis are typically tropical, and the green anole (Anolis carolinensis) constitutes an exception since it expanded into temperate North America from subtropical Florida. Thus, we used the green anole as a model to investigate signatures of selection associated with colonization of a new environment, namely temperate North America. To this end, we analysed 29 whole-genome sequences, covering the entire native range of the species. We used a combination of recent methods to quantify both positive and balancing selection in northern populations, including FST outlier methods, machine learning and ancestral recombination graphs. We naively scanned for genes of interest and assessed the overlap between multiple tests. Strikingly, we identified many genes involved in behaviour, suggesting that the recent successful colonization of northern environments may have been linked to behavioural shifts as well as physiological adaptation. Using a candidate genes strategy, we determined that genes involved in response to cold or behaviour displayed more frequently signals of selection, while controlling for local recombination rate, gene clustering and gene length. In addition, we found signatures of balancing selection at immune genes in all investigated genetic groups, but also at genes involved in neuronal and anatomical development.

Anolis carolinensis as a model to understand the molecular and cellular basis of foveal development.

The fovea is an anatomical specialization of the central retina containing closely packed cone-photoreceptors providing an area of high acuity vision in humans and primates. Despite its key role in the clarity of vision, little is known about the molecular and cellular basis of foveal development, due to the absence of a foveal structure in commonly used laboratory animal models. Of the amniotes the retina in birds of prey and some reptiles do exhibit a typical foveal structure, but they have not been studied in the context of foveal development due to lack of availability of embryonic tissue, lack of captive breeding programs, and limited genomic information. However, the genome for the diurnal bifoveate reptile species Anolis carolinensis (green anole) was recently published and it is possible to collect embryos from this species in captivity. Here, we tested the feasibility of using the anole as a model to study foveal development. Eyes were collected at various stages of development for histological analysis, immunofluorescence, and apoptosis. We show that at embryonic stage (ES) 10 there is peak ganglion cell density at the incipient central foveal region and a single row of cone photoreceptor nuclei. At ES17 the foveal pit begins to form and at this stage there are 3-4 rows of cone nuclei. Post-hatching a further increase in cone density and lengthening of inner and outer segments is observed. A yellowish pigment was seen in the adult central foveal region, but not in the temporal fovea. At ES14 Pax6 was localized across the entire retina, but was more prominent in the ganglion cell layer (GCL) and the part of the inner nuclear layer (INL) containing amacrine cell bodies. However, at ES17 Pax6 expression in the ganglion cells of the central retina was markedly reduced. Bioinformatic analysis revealed that 86% of human candidate foveal hypoplasia genes had an orthologous gene or DNA sequence in the green anole. These findings provide the first insight into foveal morphogenesis in the green anole and suggest that it could be a very useful model for investigating the molecular signals driving foveal development, and thus inform on human foveal development and disease.

Exercise training reveals trade-offs between endurance performance and immune function, but does not influence growth, in juvenile lizards.

Acquired energetic resources allocated to a particular trait cannot then be re-allocated to a different trait. This often results in a trade-off between survival and reproduction for the adults of many species, but such a trade-off may be manifested differently in juveniles not yet capable of reproduction. Whereas adults may allocate resources to current and/or future reproduction, juveniles can only allocate to future reproduction. Thus, juveniles should allocate resources toward traits that increase survival and their chances of future reproductive success. We manipulated allocation of resources to performance, via endurance exercise training, to examine trade-offs among endurance capacity, immune function and growth in juvenile green anole lizards. We trained male and female captive anoles on a treadmill for 8 weeks, with increasing intensity, and compared traits with those of untrained individuals. Our results show that training enhanced endurance capacity equally in both sexes, but immune function was suppressed only in females. Training had no effect on growth, but males had higher growth rates than females. Previous work showed that trained adults have enhanced growth, so juvenile growth is either insensitive to stimulation with exercise, or they are already growing at maximal rates. Our results add to a growing body of literature indicating that locomotor performance is an important part of life-history trade-offs that are sex and age specific.

Synuclein expression in the lizard Anolis carolinensis.

The synuclein (syn) family comprises three proteins: α-, ß- and γ-syns. In humans, they are involved in neurodegenerative diseases such as Parkinson's disease and in tumors. Members of the syn family were sequenced in representative species of all vertebrates and the comparative analysis of amino acid sequences suggests that syns are evolutionarily conserved, but information about their expression in vertebrate lineages is still scarce and completely lacking in reptiles. In this study, the expression of genes coding for α-, ß- and γ-syns was analyzed in the green lizard Anolis carolinensis by semiquantitative RT-PCR and Western blot. Results demonstrate good expression levels of the three syns in the lizard nervous system, similarly to human syns. This, together with the high identity between lizard and human syns, suggests that these proteins fulfill evolutionarily conserved functions. However, differences between lizard and humans in the expression of syn variants (two different variants of γ-syn were detected in A. carolinensis) and differences in some amino acids in key positions for the regulation of protein conformation and affinity for lipid and metal ions also suggest that these proteins may have acquired different functional specializations in the two lineages.

Sexually dimorphic expression of CREB binding protein in the green anole brain.

Green anoles are seasonally breeding lizards in which male sexual behavior is primarily regulated by an annual increase in testosterone. This hormone activates stereotyped behaviors, as well as morphological and biochemical changes in the brain, with greater effect in the breeding season than in the non-breeding season. This study is the first description of CREB binding protein (CBP) in the reptilian brain, and investigates the possibility that changes in CBP, an androgen receptor coactivator, may facilitate differences in responsiveness to testosterone across seasons. A portion of this gene was cloned for the green anole, and in situ hybridization was performed to examine the expression of CBP in the brains of gonadally intact male and female green anoles across breeding states. Additionally, hormonal regulation of CBP was evaluated across sex and season in animals that were gonadectomized and treated with testosterone or a control. Similar to other vertebrates, CBP was expressed at relatively high levels in steroid-sensitive brain regions. In the anole ventromedial amygdala, CBP mRNA levels were nearly twice as high in gonadally intact females compared to males. In contrast, CBP expression did not differ across seasons or hormone manipulation in this brain region. No significant effects were detected in the preoptic area or ventromedial hypothalamus. This pattern suggests that CBP might influence female-biased functions controlled by the ventromedial amygdala, but is not consistent with a role in mediating seasonal differences in responsiveness to testosterone in these areas associated with reproductive function.

Context-dependent changes in motor control and kinematics during locomotion: modulation and decoupling.

Successful locomotion through complex, heterogeneous environments requires the muscles that power locomotion to function effectively under a wide variety of conditions. Although considerable data exist on how animals modulate both kinematics and motor pattern when confronted with orientation (i.e. incline) demands, little is known about the modulation of muscle function in response to changes in structural demands like substrate diameter, compliance and texture. Here, we used high-speed videography and electromyography to examine how substrate incline and perch diameter affected the kinematics and muscle function of both the forelimb and hindlimb in the green anole (Anolis carolinensis). Surprisingly, we found a decoupling of the modulation of kinematics and motor activity, with kinematics being more affected by perch diameter than by incline, and muscle function being more affected by incline than by perch diameter. Also, muscle activity was most stereotyped on the broad, vertical condition, suggesting that, despite being classified as a trunk-crown ecomorph, this species may prefer trunks. These data emphasize the complex interactions between the processes that underlie animal movement and the importance of examining muscle function when considering both the evolution of locomotion and the impacts of ecology on function.

Arginine vasotocin, steroid hormones and social behavior in the green anole lizard (Anolis carolinensis).

Arginine vasotocin (AVT) is a potent regulator of social behavior in many species, but little is known about its role in reptilian behavior. Here we examine the effect of exogenous AVT on aggressive responding and courtship behavior in the green anole lizard (Anolis carolinensis). Aggressive behavior was stimulated in two ways: (1) mirror presentation (no relative status formed) and (2) size-matched pairs (where a social status is achieved). To elicit courtship behavior, a novel female was introduced into the home cage of a male. Regardless of the behavior condition, male anoles were injected i.p. with either reptile Ringer solution (vehicle) or AVT prior to testing. Animals treated with AVT performed fewer aggressive display bouts during mirror presentation but AVT treatment did not affect the overall number of aggressive display bouts within size-matched pairs. Male courtship behavior was not affected by AVT; however, untreated females displayed more frequently when paired with an AVT-treated male than a vehicle-injected control, suggesting that AVT-treated males were more attractive to females. Regardless of behavior condition, AVT injections led to increases in circulating corticosterone. Overall, we found that AVT tended to reduce aggressive behavior as has been reported for other territorial species. AVT did not perceptibly alter male courtship but did increase the display behavior of untreated females paired with treated males. Our study supports a role for AVT in the regulation of reptile social behavior.

Comparative proteomic analysis of tail regeneration in the green anole lizard, Anolis carolinensis.

As amniote vertebrates, lizards are the most closely related organisms to humans capable of appendage regeneration. Lizards can autotomize, or release their tails as a means of predator evasion, and subsequently regenerate a functional replacement. Green anoles (Anolis carolinensis) can regenerate their tails through a process that involves differential expression of hundreds of genes, which has previously been analyzed by transcriptomic and microRNA analysis. To investigate protein expression in regenerating tissue, we performed whole proteomic analysis of regenerating tail tip and base. This is the first proteomic data set available for any anole lizard. We identified a total of 2,646 proteins - 976 proteins only in the regenerating tail base, 796 only in the tail tip, and 874 in both tip and base. For over 90% of these proteins in these tissues, we were able to assign a clear orthology to gene models in either the Ensembl or NCBI databases. For 13 proteins in the tail base, 9 proteins in the tail tip, and 10 proteins in both regions, the gene model in Ensembl and NCBI matched an uncharacterized protein, confirming that these predictions are present in the proteome. Ontology and pathways analysis of proteins expressed in the regenerating tail base identified categories including actin filament-based process, ncRNA metabolism, regulation of phosphatase activity, small GTPase mediated signal transduction, and cellular component organization or biogenesis. Analysis of proteins expressed in the tail tip identified categories including regulation of organelle organization, regulation of protein localization, ubiquitin-dependent protein catabolism, small GTPase mediated signal transduction, morphogenesis of epithelium, and regulation of biological quality. These proteomic findings confirm pathways and gene families activated in tail regeneration in the green anole as well as identify uncharacterized proteins whose role in regrowth remains to be revealed. This study demonstrates the insights that are possible from the integration of proteomic and transcriptomic data in tail regrowth in the green anole, with potentially broader application to studies in other regenerative models.

The value of comparative approaches to our understanding of puberty as illustrated by investigations in birds and reptiles.

This article is part of a Special Issue "Puberty and Adolescence". Studies of birds and reptiles have provided many basic insights into the neuroendocrine control of reproductive processes. This research has elucidated mechanisms regulating both early development, including sexual differentiation, and adult neuroendocrine function and behavior. However, phenomena associated with the transition into sexual maturation (puberty) have not been a focus of investigators working on species in these taxonomic classes. Research is complicated in birds and reptiles by a variety of factors, including what can be extended times to maturation, the need to reach particular body size regardless of age, and environmental conditions that can support or inhibit endocrine responses. However, careful selection of model systems, particularly those with available genetic tools, will lead to important comparative studies that can elucidate both generalizability and diversity of mechanisms regulating the onset of reproductive maturity.

Seasonal and sexual dimorphisms in expression of androgen receptor and its coactivators in brain and peripheral copulatory tissues of the green anole.

Green anoles are seasonally breeding lizards, with an annual rise in testosterone (T) being the primary activator of male sexual behaviors. Responsiveness to T is decreased in the non-breeding season (NBS) compared to breeding season (BS) on a variety of levels, including displays of reproductive behavior and the morphology and biochemistry of associated tissues. To evaluate the possibility that seasonal changes in responsiveness to T are regulated by androgen receptors (AR) and/or two of its coactivators, CREB binding protein (CBP) and steroid receptor coactivator-1 (SRC-1), we tested whether they differ in expression across season in brains of both sexes and in peripheral copulatory tissues of males (hemipenis and retractor penis magnus muscle). AR mRNA was increased in the brains of males compared to females and in copulatory muscle in the BS compared to NBS. In the hemipenis, transcriptional activity appeared generally diminished in the NBS. T-treatment increased AR mRNA in the copulatory muscle and AR protein in the hemipenis, the latter to a greater extent in the BS than the NBS. T also decreased SRC-1 protein in hemipenis. Interpretations are complicated, in part because levels of mRNA and protein expression were not correlated and multiple sizes of the AR and CBP proteins were detected, with some tissue specificity. However, the results are consistent with the idea that differences in receptor and coactivator expression at central and peripheral levels may play roles in regulating sex and seasonal differences in the motivation or physical ability to engage in sexual behavior.

The maternal energetic environment affects both egg and offspring phenotypes in green anole lizards (Anolis carolinensis).

Animals exist in dynamic environments that may affect both their own fitness and that of their offspring. Maternal effects might allow mothers to prepare their offspring for the environment in which they will be born via several mechanisms, not all of which are well understood. Resource scarcity and forced resource allocation are two scenarios that could affect maternal investment by altering the amount and type of resources available for investment in offspring, albeit in potentially different ways. We tested the hypothesis that maternal dietary restriction and sprint training have different consequences for the offspring phenotype in an oviparous lizard (Anolis carolinensis). To do this, we collected and reared eggs from adult diet-manipulated females (low-diet [LD] or high-diet [HD]) and sprint-trained females (sprint trained [ST] or untrained [UT]) and measured both egg characteristics and hatchling morphology. ST and LD mothers laid both the fewest and heaviest eggs, and ST, UT, and LD eggs also had significantly longer incubation periods than the HD group. Hatchlings from the diet experiment (LD and HD offspring) were the heaviest overall. Furthermore, both body mass of the mother at oviposition and change in maternal body mass over the course of the experiment had significant and sometimes different effects on egg and offspring phenotypes, highlighting the importance of maternal energetic state to the allocation of maternal resources.

Artificial light at night alters diurnal and nocturnal behavior and physiology in green anole lizards.

Artificial light at night (ALAN) disrupts biological rhythms across widely diverse organisms. To determine how energy is allocated by animals in different light environments, we investigated the impacts of ALAN on behavior and physiology of diurnal green anole lizards (Anolis carolinensis). Two groups of 24 adult lizards (half males, half females) were maintained in a controlled lab setting for six weeks. One group was exposed to a simulated natural summer light-dark cycle; the other was exposed to ALAN that simulated urban, nocturnal light exposure. After an acclimation period, we conducted four behavioral trials. One trial examined behavioral time allocation over two 24 h periods, and three others were conducted during mid-day and mid-night: open field tests, to examine exploratory behavior; foraging trials, to examine prey consumption; and social interaction trials, to examine same-sex interactions. We then measured each lizard's snout-vent length and mass of its body, abdominal fat pads, liver, and, for males, testes. Lizards exposed to ALAN were more likely to be awake at night, using nocturnal light to explore, forage, and display to conspecifics. However, during the day, ALAN lizards were less likely to be awake, slower to move, and females displayed less frequently. ALAN lizards had heavier fat pads and testes, but ALAN did not impact body mass, liver mass, or snout-vent length. In sum, ALAN appears to cause a broad shift towards increased nocturnal activity and may alter metabolic and reproductive processes. Future work should examine the fitness consequences of these behavioral and physiological changes.

Transfer of Anolis locomotor behavior across environments and species.

Arboreal animals must learn to modulate their movements to overcome the challenges posed by the complexity of their heterogeneous environment, reduce performance failure, and survive. Anolis lizards are remarkable in the apparent ease with which they conquer this heterogeneity, demonstrating an impressive ability to modulate their locomotor behavior to maintain stable locomotion on widely disparate surfaces. Significant progress has been made towards understanding the impact of substrate structure on the behavioral plasticity of arboreal species, but it is unclear whether the same strategies employed to shift between substrates in one context can be employed to shift between those same substrates in a new context. Is the kinematic shift between broad and narrow perches achieved in a similar way on different inclines? Do all species within an ecomorph make similar adjustments? Here, we analyze the limb movements of two trunk-crown Anolis ecomorphs, A. carolinensis and A. evermanni, running on 6 different surfaces (3 inclinations × 2 perch diameters), from the perspective of Transfer Learning. Transfer learning is that field of machine learning which aims at exploiting the knowledge gained from one task to improve generalization about another, related task. In our setting, we use transfer learning to show that the strategies employed to improve locomotor stability on narrow perches are transferred across environments with different inclines. Further, behaviors used on vertical inclines are shared, and thus transfer well, across perch diameters whereas the relationship between horizontal and intermediate inclines change on different perch diameters, leading to lower transfer learning of shallow inclines across perch diameters. Interestingly, the best incline for transfer of behavior differs between limbs: forelimb models learn best from the vertical incline and hind limb models learn best from horizontal and intermediate inclines. Finally, our results suggest both that subtle differences exist in how A. carolinensis and A. evermanni adjust their behaviors in typical trunk-crown environments and that they may have converged on similar strategies for modulating forelimb behavior on vertical surfaces and hind limb behavior on shallow surfaces. The transfer of behavior is analogous to phenotypic plasticity, which likely plays a key role in the rapid adaptive evolution characteristic of Anolis lizards. This work is an example of how modern statistical methodology can provide an interesting perspective on new biological questions, such as on the role and nuances of behavioral plasticity and the key behaviors that help shape the versatility and rapid evolution of Anolis lizards.

Size but not relatedness drives the spatial distribution of males within an urban population of Anolis carolinensis lizards.

The way that individuals are spatially organized in their environment is a fundamental population characteristic affecting social structure, mating system, and reproductive ecology. However, for many small or cryptic species, the factors driving the spatial distribution of individuals within a population are poorly understood and difficult to quantify. We combined microsatellite data, remote sensing, and mark-recapture techniques to test the relative importance of body size and relatedness in determining the spatial distribution of male Anolis carolinensis individuals within a focal population over a five-year period. We found that males maintain smaller home ranges than females. We found no relationship between male body size and home range size, nor any substantial impact of relatedness on the geographic proximity. Instead, the main driver of male spatial distribution in this population was differences in body size. We also found no evidence for offspring inheritance of their parent's territories. Males were never sampled within their father's territory providing strong support for male-biased dispersal. This study introduces a novel approach by combining standard mark release capture data with measures of pairwise relatedness, body size, and GPS locations to better understand the factors that drive the spatial distribution of individuals within a population.

The Effect of Long Term Captivity on Stress Levels in Anolis carolinensis Lizards.

The effect of long term captivity is a factor that is important for all research utilizing wild caught animals. Despite the fact that it can be considered to be one of the most fundamental potential sources of stress in captivity, it has received a low amount of interest in recent research on lizards. Given the wide variety in ecology and life history among lizards species, it would make sense to investigate the effect of long term captivity on wild caught lizards on a broader scale. In this study we investigated the effect of long term captivity (four months) on the physiology and behavior of male and female Anolis carolinensis lizards. Our results showed no negative effects of four months of captivity on physiological and behavioral measurements in male A carolinensis lizards. Similar results for females were found for all measurements except body mass and tail width. Here our results indicated a potential negative effect of four months of captivity on body mass and tail width in females.

Neural and endocrine responses to social stress differ during actual and virtual aggressive interactions or physiological sign stimuli.

Neural and endocrine responses provide quantitative measures that can be used for discriminating behavioral output analyses. Experimental design differences often make it difficult to compare results with respect to the mechanisms producing behavioral actions. We hypothesize that comparisons of distinctive behavioral paradigms or modification of social signals can aid in teasing apart the subtle differences in animal responses to social stress. Eyespots are a unique sympathetically activated sign stimulus of the lizard Anolis carolinensis that influence aggression and social dominance. Eyespot formation along with measurements of central and plasma monoamines enable comparison of paired male aggressive interactions with those provoked by a mirror image. The results suggest that experiments employing artificial application of sign stimuli in dyadic interactions amplify behavioral, neural and endocrine responses, and foreshorten behavioral interactions compared to those that develop among pairs naturally. While the use of mirrors to induce aggressive behavior produces simulated interactions that appear normal, some behavioral, neural, and endocrine responses are amplified in these experiments as well. In contrast, mirror image interactions also limit the level of certain behavioral and neuroendocrine responses. As true social communication does not occur during interaction with mirror images, rank relationships can never be established. Multiple experimental approaches, such as combining naturalistic social interactions with virtual exchanges and/or manipulation of sign stimuli, can often provide added depth to understanding the motivation, context, and mechanisms that produce specific behaviors. The addition of endocrine and neural measurements helps identify the contributions of specific behavioral elements to the social processes proceeding.

The (dis)advantages of dominance in a multiple male group of Anolis carolinensis lizards.

Male Anolis carolinensis lizards will fight and form social dominance hierarchies when placed in habitats with limited resources. Dominance may procure benefits such as priority access to food, shelter or partners, but may also come with costs, such as a higher risk of injuries due to aggressive interaction, a higher risk of predation or a higher energetic cost, all of which may lead to an increase in stress. While most research looks at dominance by using dyadic interactions, in our study we investigated the effect of dominance in a multiple male group of A. carolinensis lizards. Our results showed that dominant males in a multiple male group had priority access to prey and potential sexual partners but may run a higher risk of predation. We could not confirm that dominant males in a multiple male group had a higher risk of injuries from aggressive interactions or a higher energetic cost by being dominant. Overall our results seem to indicate that dominant male A. carolinensis lizards in a multiple male group obtain clear benefits and that they outweigh the disadvantages.