Anole Annals

Anole Annals contributor Martha Muñoz of Harvard University won the second annual Raymond B. Huey Award for her presentation discussing the role of behavior in the evolution of Anolis cybotes. The Huey Award, sponsored by the Division of Ecology and Evolution of the Society of Integrative and Comparative Biology, is given for the Best Student Presentation in the division.

Behavior is thought to play two contrasting roles during evolutionary diversification. First, behavior can expose individuals to novel environments, thereby driving physiological and morphological change. Second, behavior can be used to compensate for environmental differences, thereby impeding organismal change. In her talk, Martha described how she tested these two contradictory hypotheses in a clade of trunk-ground anoles that span a wide environmental range.

The Anolis cybotes species complex occurs in Hispaniola from sea level up to 2,500 meters in elevation. By comparing two populations of a lowland generalist, A. c. cybotes, to two independently derived high-altitude specialists, A. c. armouri and A. c. shrevei, Martha was able to detect signatures of adaptation to high elevation. First, Martha asked whether physiological evolution had occurred. She found that body temperatures in the field were not significantly different at high and low elevations, despite the fact that lizards experience air temperatures 15 degrees cooler, on average, at high elevation. In addition, there were no significant differences in preferred body temperature (measured in the lab) among the four populations, and in each case the preferred body temperature matched the field body temperature. These results clearly support a lack of change in the thermal physiology of these lizards despite occupying very different thermal environments.

Martha then tested whether behavioral inhibition was the cause of the observed stasis in thermal physiology. By recording the perch sites of lizards in the field, Martha found that low-elevation lizards perch primarily on trees while high-elevation lizards have shifted to perching primarily on rocks. To quantify how this perch shift affects a lizard’s thermal environment, Martha deployed a series of copper lizard models at each site. The copper models closely mimic the thermal properties of a live lizard, so the temperatures recorded by the models are essentially those experienced by a non-thermoregulating lizard (i.e., the operative temperature). By placing the copper models on both rocks and trees at each site, she was able to assess the thermal properties of each perch type. Martha found that at low elevation, models on both trees and rocks achieved temperatures in the lizards’ preferred temperature range, and sometimes models on rocks got dangerously hot. At high elevation, however, only models placed on rocks achieved temperatures in the preferred range, while models on trees remained too cool. These results support the hypothesis that behavioral inhibition (perch switching) is preventing evolution in thermal physiology.

In a final twist, Martha asked whether evolutionary stasis is also observed in morphology. Morphology is known to correlate with microhabitat in Anolis lizards and is rapidly evolvable, and so stasis would be a surprising result. Martha found the high-elevation populations have significantly flatter and wider heads, a common feature of rock-dwelling lizards, compared to low-elevation populations. She found no differences in limb length or lamellae number. Martha hypothesized that for head morphology, perch switching was a form of behavioral drive that promoted evolutionary change.

Martha concluded by emphasizing that niches are multidimensional, and, therefore, evolution can occur along multiple niche axes simultaneously. By examining adaptation to both the thermal niche (body temperature) and structural niche (morphology) in this study, she revealed that behavioral drive and behavioral inhibition—previously thought to be incompatible—can in fact occur simultaneously in the same organism.

Congratulations, Martha, on your award-winning talk!

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.

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?

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.

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].

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.