SICB 2014: Suboptimal Locomotion in Anolis carolinensis

Cusick_FL_carolinensis_2 in grassA majority of biomechanical studies focus on eliciting maximal performance from animals in laboratory conditions, an approach that can make it difficult to apply results from the lab towards understanding performance in nature. Jerry Husak addressed this issue in his talk entitled “Maximal locomotor performance and sprint sensitivity in green anole lizards (Anolis carolinensis).”

By measuring sprint performance on a variety of perches (two different dowel widths, as well as a broad perch with pegs functioning as obstacles) and comparing these performances to sprint performance on a broad, flat surface, Husak showed that green anoles are substantially worse at running on narrow perches and through obstacles than at running on broad, flat surfaces. This confirms that animals moving through their natural habitats are almost certainly sprinting sub-maximally–in nature, green anoles are found most often on perches even narrower than those tested.

Crucially, the morphological correlates of performance varied by perch, suggesting that fine-scale studies of selection on limb and muscle morphology in the wild will require knowledge of how often and in what circumstances lizards navigate different microhabitats and move on different substrates. Coupled with behavioural observations in the wild, this study can pave the way for a more nuanced understanding of body shape evolution in our favourite lizards.

SICB 2014: Dynamic Energy Budget of Green Anoles

Quinn

A figure from Quinn’s poster, showing alternative possible energy budgets in green anoles (click for a better view).

Animals allocate energy that they acquire to a variety of bodily functions and activities. Some of the more important allocations are those toward metabolism and growth, though the relative allocations to these is unclear. McKenzie Quinn, an undergraduate student working with Michele Johnson at Trinity University, presented her work in the third poster session on the dynamic energy budget of green anole lizards. She quantified food intake, excretion, growth, and resting metabolic rate (RMR, the energy required for basic maintenance) of individual lizards over 40 days to create a predictive model to describe how they allocate energy. If metabolism receives a large allocation, then RMR and/or body mass are expected to be significant predictors of energy use. On the other hand, if growth is more important, then aspects of body length (snout-vent length, SVL) are expected to be better predictors.

Interestingly, she found that RMR and body mass were not included in the best model of energy use. Instead, their model building (with AIC criteria, if you’re interested) showed that a decreasing nonlinear function of SVL was the best model. This suggests that metabolic functions are a small, non-significant part of these lizards’ dynamic energy budget. This work was conducted on adult males, so it will of course be interesting to see how this approach might apply to younger individuals or females. However, this is useful information to know for those who wonder how anoles allocate energy in their daily lives.

SICB 2014: Regulation of Anole Limb Development

Readers of AA are very familiar with the dramatic differences in limb length among the anole ecomorphs, but we don’t yet know which genomic regions are involved in the evolution of anole limb length.  Carlos Infante, currently a postdoc in Doug Menke’s lab at the University of Georgia, presented a talk on his work to identify enhancers (short regions of DNA where proteins bind to enhance the transcription of a gene) that are associated with anole limb development.

Carlos first described a series of previous studies that did not find differences in the proteins expressed in the limbs of different anole species, suggesting that the differences in limb length are likely controlled by differences in gene regulation. However, examining a series of enhancer regions that were identified from previous work in mice also did not reveal differences in sequence variation that were correlated with limb length.

So, Carlos and his collaborators are using tools from the field of functional genomics to address this issue, using ChIP-Seq (a method that analyzes interactions between DNA and proteins) to identify active enhancers and promotors in embryonic Anolis carolinensis tissue using antibodies against Pitx1 (a transcription factor involved in hindlimb development) and H3K27ac (an acetylated histone mark).  By comparing results from these two datasets, they could identify enhancers that are expressed in forelimbs, hindlimbs, trunk tissues, or tubercles. Their plan for future work involves using the list they’ve generated of enhancers expressed in both forelimbs and hindlimbs to identify the regulatory regions that control the development of limb morphologies among Anolis species.

SICB 2014: Growth Hormone and Body Size in Anoles

GHIGF

A figure from Eric Mueller’s poster showing the conserved pathway of how growth hormone may affect body size.

Anyone familiar with Anolis lizards is aware of the dramatic variation in body size. Think dwarf twig anole and crown giant. Although the ecological and evolutionary processes that can lead to such variation in body size have been studied, it is still unknown what physiological mechanism explains the variation we see today. Eric Mueller, a graduate student at Southern Illinois University – Edwardsville, presented a poster asking just that question. Specifically, do differences in circulating levels of plasma growth hormone regulate evolutionary changes in body size among anole species of differing size and morphology?

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Anolis carolinensis (L) and A. equestris (R) have dramatic differences in body size but not in growth hormone levels. (photo 1, 2) Species not to scale.

Growth hormone (GH) is secreted by the pituitary gland and has many functions in the body, including promoting muscle and bone growth and increasing protein synthesis (among many, many other things!). It seems a logical candidate mechanism to investigate when it comes to explaining variation in body size. Mueller looked at GH levels in three anoles of varying size:  A. equestris, A. carolinensis, and A. sagrei. GH was higher in A. equestris and A. carolinensis than A. sagrei, supporting his hypothesis. However, there was no difference in GH levels between A. equestris and A. carolinensis despite dramatic differences in adult body size. Looking within species, GH levels were positively correlated with SVL only in A. equestris, and not the other two species.

Although differences in circulating GH may explain some size differences among anole species, as in other studies of anole hormones, things don’t seem to be simple. Mueller hypothesized that other aspects of the GH pathway may be more important. For example, GH receptors, Insulin-like Growth Factor (IGF) levels, and IGF-binding proteins should be examined for a full picture. The GH-IGF axis also interacts with other hormone pathways, such as testosterone, making this a very complex issue. Since endocrine systems are so multi-faceted, and multiple components have the possibility to evolve independently, there is lots of potential for future research that seeks to explain species differences in body size.

 

SICB 2014: Micro-landscape Variation in Reproduction

It was a real pleasure to see Dr. Ray Huey give a presentation that was inspired by research he and his collaborators began in the 1970s on seasonality of reproduction and behavioral thermoregulation in Puerto Rican Anolis cristatellus. Almost 40 years after the publication of that work, Huey and many of the same colleagues (and some new ones) returned to the same areas in Puerto Rico to examine how very fine-scale variation in thermal environment (a few meters!) might lead to variation in reproduction. The investigators (Otero, Huey, and Gorman) studied how reproduction differed between open areas (where lizards carefully thermoregulate) and forested areas (where lizards are thermoconformers) and found striking differences between them. Females in open habitats reproduced most of the year, whereas females in the neighboring forest decreased reproductive in a much more seasonal manner. Differences were largest from October – December, with females in forested habitats essentially shutting down reproduction during those months. This finding was true at two different sites.

These striking differences in reproductive phenology are similar in magnitude to differences seen along elevational gradients, but the difference here is the scale. The females that Huey compared were literally only a few meters away from each other. One important take-home message from these data is that reproduction can vary at the microgeographic scale just as it can at larger geographic scales. While the latter type of study is now common, the former isn’t. Future work should consider how small-scale variation in microhabitat use might influence reproduction so that we can better understand how general this phenomenon is.

One final point that Huey made was how collaborations can not only be an integral part of research, but also a source of personal reward as those collaborations continue over time and result in great friendships. He encouraged young investigators to keep this in mind as they progress through their academic careers.

Editor’s note: this research project has been the subject of previous posts [1,2].

SICB 2014: Arginine Vasotocin and Social Behavior

800px-Vasotocin 800px-Anolis_carolinensis_mating Arginine vasotocin, the reptilian homolog to vasopressin is a potent modulator of social behavior. (photos from Wikipedia)

For many of us, hearing about arginine vasotocin (AVT), or its mammalian homolog vasopressin, conjures up memories from a physiology class about water balance and the antidiuretic effects of the vasopressin system. However, AVT is also a potent neuromodulator and neurotransmitter in the brain that has strong effects on social behavior, such as parental care and pair bonding. Although most of this behavioral research has been done in mammals and birds, it appears that AVT might have similar behavioral functions in anoles! Leslie Dunham, a graduate student at Georgia State University, experimentally assessed the effects of AVT on male and female green anole behavior. By comparing lizards injected with either AVT or a control solution, Dunham was able to examine the effects of AVT on aggression and courtship in males. The behavior of individuals in the two experimental groups was then assessed in the following contexts: aggression toward a mirror, aggression toward another male, and courtship toward a female. She found:

  1. Decreased aggression toward a mirror.
  2. No difference in aggression toward a size-matched male.
  3. No difference in courtship behavior directed at a female.

AVT decreases aggression in other vertebrates, but the lack of an effect on courtship in anoles suggests that AVT may modulate behavior differently in male anoles compared to fish and birds.

But wait, there’s more! Not to be outdone by other types of vertebrates, females in the courtship trials showed some very interesting behavior. Although Dunham focused on the effects of AVT on male behavior, she also measured female behavior during the trials. She found that females displayed more toward males treated with AVT compared to control males. This was despite any detectable behavioral difference between the male treatment groups. Why is this? Although the answer is still unknown, Dunham proposed some plausible and testable hypotheses for what might be happening. First, there may be subtle changes in behavior brought about by AVT that weren’t detected. Second, there may be a role for chemical communication between the sexes during courtship that wasn’t measured. Anoles, and iguanian lizards in general, aren’t known for their reliance on chemical communication, so this possibility is sure to spark some interesting future research.

SICB 2014: Why Curly-Tailed Lizards Curl Their Tails

Continuing with my theme of posting about non-anoles that anolologists find interesting, here’s a summary of a fascinating poster about tail-curling in two species of LeiocephalusL. carinatus (the famous consumer of A. sagrei in the Bahamas) and L. barahonensis. Tail-curling is known to function as a predator-deterrent signal in L. carinatus, but its potential as a social signal has remained unexplored. Bonnie Kircher, a student in Michele Johnson’s lab at Trinity University, set about rectifying this gap.

Having never seen a curly-tail myself, I was surprised to learn that these lizards exhibit a variety of tail-curling behaviours.

A figure from Bonnie Kircher's poster describing how tail-curling was scored in this study.

A figure from Bonnie Kircher’s poster describing how tail-curling was scored in this study.

By scoring the intensity of tail-curling during social encounters as well as during non-social periods, Kircher showed that tail-curling was not used as a social signal in either Leiocephalus species. In an elegant control, she demonstrated that head-bobbing was more frequent in social than in non-social contexts, thus verifying that social contexts were indeed accurately identified.

Kircher also simulated predation by approaching the lizards and observed their use of tail-curling while fleeing. A comparison of the frequency of tail-curling between disturbed and undisturbed lizards confirmed that L. carinatus uses tail-curling as a signal during encounters with potential predators. The same pattern was not observed in L. barahonensis–we therefore don’t yet know why this species curls its tail. Kircher speculates that the behaviour might have been directed at other, undetected, predators, or perhaps plays a role in lending stability to the lizard during locomotion.

This variation in the utilization of the same signal between two closely-related species points to the lability of signal use, and with almost 30 species in the genus, this system is a prime candidate for future work on signal evolution.

SICB 2014: Hemiclitoris Development in Anolis distichus

Anoles display a staggering amount of phenotypic diversity, even in their genital morphology. Traditionally research has focused on characterizing the diversity and function of male genitals, or hemipenes, but females also possess paired genitals, or hemiclitorises, and yet almost nothing is known about them. In fact, female genital morphology is poorly understood across all reptiles. To date, we know that in some species hemiclitorises appear as miniaturized versions of hemipenes, whereas in other species they are unique structures. Further, the timing of sexual differentiation of genital structures appear to differ among lizard clades. Clearly, we need a broader understanding of the form, function, and evolution of female genitalia in reptiles.

In a fascinating poster, Casey Gilman, a graduate student at the University of Massachusetts, Amherst, presented her work on the development and morphology of hemiclitorises in the bark anole, Anolis distichus. Here’s the abstract:

Genitalia are extraordinarily diverse and show remarkably rapid evolution, relative to other morphological traits, across a wide range of animal taxa. Male and female genitalia in many animal groups begin as the same embryonic structures and later go through hormone-mediated differentiation. Surprisingly, little is known about the genetic mechanics of these processes. Even less is known about external genitalia differentiation in reptiles. Unlike other amniote groups, lizards and snakes possess a set of paired reproductive intromittent organs, called hemipenes. In a number of lizard species, females retain miniaturized versions of the male genitalia, called hemiclitorises. In these species, hemiclitorises can be used for taxonomic purposes, as they retain many morphological characteristics of the male genitalia, which are often species-specific. In lizards, the external genitalia of both sexes grow at the same rate until approximately halfway through embryonic development. Following this period, the hemipenes of the males continue to grow while the hemiclitorises of the females regress until they are about half the length of their male counterparts. We investigated the development of male and female external genitalia in Anolis distichus to determine the timing and patterning of growth and regression of these structures using histology, immunohistochemistry and whole mount in situ hybridization.

SICB 2014: Novel Insights into the Evolution of Cold Tolerance

 

Green anole eating a dronefly. Photo from Wikipedia.

 

The tremendous diversity in Anolis lizards is one of the major draws for researchers to work on this system. There are nearly 400 species of anoles and their distribution spans much of the New World. Most of Anolis’ distribution spans environments with very low seaonsality. One exception is Anolis carolinensis, whose range spans much of continental North America, and encompasses highly seasonal environments. Further, unlike most reptiles, A. carolinensis does not hibernate during the winter. Rather, lizards remain active during the cold North American winter months.

Today Shane Campbell-Staton, a graduate student at Harvard University, presented some of his thesis work examining how A. carolinensis adapts to the thermal environment, and how local adaptation influences patterns of gene flow. The work he presented was conducted in collaboration with Scott Edwards and Jonathan Losos at Harvard University and Zachary Cheviron and Anna Bare from the University of Illinois-Urbana Champaign.

Shane first asked whether differences in the thermal environment limit gene flow among populations of A. carolinensis. To answer this question, he examined variation in over 2000 loci for 131 individuals of A. carolinensis and its ancestor, A. porcatus, from Cuba. He leveraged the Anolis genome with double digest RADseq to discover these SNPs and used multiple matrix regression to assess the correlation between genetic distance among populations and geographic and climatic distance. He discovered a significant signal of isolation by temperature, but not isolation by geographic distance or isolation by precipation. This means that populations are likely structured by thermal habitat, and that differences in temperature among localities limit gene flow in A. carolinensis.

Next Shane asked whether there was a signal of local adaptation in physiological tolerance to the thermal environment. He measured heat tolerance (CTmax) and cold tolerance (CTmin) in nearly 200 individuals of Anolis carolinensis. He found a significant positive correlation between temperature seasonality and thermal tolerance (i.e., the difference between CTmin and CTmax), but that most (though not all) of this pattern was driven by variation in cold tolerance across habitats.

Finally, Shane wanted to understand the mechanism that limits cold tolerance for terrestrial ectotherms. Specifically, he wanted to test whether oxygen limitation plays a role in determining how cold tolerant lizards are. The oxygen limitation hypothesis suggests that the ability to transport and utilize oxygen is limited at cold temperatures, and that lizards lose their mobility at low temperatures because they can no longer effectively transport oxygen to their muscles. Under this scenario, lizards that are more tolerant cold should be more efficient at transporting oxygen at cold temperatures than less cold tolerant individuals. To test this hypothesis, he examined CTmin in lizards from different thermal extremes of the species range and found that lizards from more cold-tolerant populations (i.e.,: higher latitude) utilized less oxygen at colder temperatures. His results support the oxygen-limitation hypothesis, and suggest that lizards can achieve a greater tolerance to cold, at least in part, by becoming more efficient at transporting oxygen, thereby reducing their demand for oxygen at lower temperatures.

SICB 2014: Neural Correlates of Communication Modalities in Lizards

 

The six species examined by Robinson and colleagues.

The six species examined by Robinson and colleagues.

Reptiles differ vastly in how they communicate. Some species are predominantly visually-oriented, whereas other species rely almost exclusively on chemical signals for communication. Despite such marked differences in communication modalities, there is surprisingly little known about how communication modalities translate into differences in neuranatomy among species. Chris Robinson, an undergraduate working with Dr. Michele Johnson at Trinity University, presented a study examining the relationship between sense perception and neural density in six species of lizards.

Chris predicted that visually oriented lizards should have larger and more densely packed neurons in two visual centers – the lateral geniculate nucleus (LGN) and the optic tectum (OT) – whereas lizards that employ chemical modalities should have a similar pattern in the nucleus sphericus of the amygdala (NS). He included three iguanid species in this study, the green anole Anolis carolinensis, the curly tail Leiocephalus carinatus, the Texas spiny lizard Sceloporus olivaceous, as well as the whiptail Aspidoscelis gularis, the skink Scincella lateralis, and the Mediterranean house gecko Hemidactylus turcicus. To determine which sensory modalities best characterized each species he performed focal behavioral observations. During these observations, he quantified the number of chemosensory behaviors (rubbing the cloaca on a substrate, licking the air or substrate) and visual behaviors (head bobbing, dewlapping, and tail curling). Chris amassed over 120 hours of behavioral observations, and 10-33 hours per species, which is no small feat.

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