Author: Martha Muñoz Page 4 of 8

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.

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.

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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When AA contributors attend scientific conferences, we try our best to post about as many talks and posters as we can visit, but inevitably we simply can’t visit them all. I will be attending the annual meeting for the Society for Integrative and Comparative Biology this upcoming January. This will be the third consecutive year in which I blog about SICB and I want to try a different approach this time. Rather than choosing the talks and posters myself, I want to get your input on what types of research most interest you. If you like to read about new research presented at conferences, then please take the survey provided below. Choose up to three different subject matters and I’ll decide my schedule based on the results. You can access a list of anole-related presentations here. Most presentations can fit into more than one category, but I just want a general idea of what most interests the readers. Now go vote!

With all my preparations for Thanksgiving underway, I had almost forgotten that the highlight of the holiday season is upon us. I am referring, of course, to the annual meeting for the Society for Integrative and Comparative Biology (SICB). Unlike most scientific conferences, which tend to host their meetings during the summer, SICB bucks the trend and meets during the first week of January. To me what is most exciting about SICB is the diversity of work that is presented there. SICB draws biomechanists, ecologists, physiologists, and geneticists, among many others, under the same roof. Thus, for those of us who are interested in anoles, SICB is a one-stop shop for learning about what’s new and exciting in Anolis lizards. In recent years, anoles have had a very strong presence at SICB. At the 2012 meeting in Charleston, South Carolina, there were 26 anole-related talks and posters. Last year’s meeting in San Francisco saw a bit of a lull, as there were only 18 talks and posters focusing on anoles. The program for the 2014 meeting has just been released, and a few quick searches using the terms “Anolis” and “anole” turn up 22 talks and posters. I hope this means that the Anolis presence at SICB is back on the rise. I will be posting about as many talks and posters as I can visit, so stay tuned. The talks and posters are given in alphabetical order by author below.

Anolis talks and posters at SICB 2014.

Anolis sagrei mating. Image from Bob Cox’s lab website 

Although the Evolution meetings are coming to a close, we get to go out on a high note. Christian Cox gave one of the last talks of the day discussing the hormonal basis for gender differences in sexual size dimorphism in anoles. Sexual size dimorphism (SSD), or the tendency for the sexes to differ in the size of different traits, has been widely documented in nature. Usually the male exhibits comparatively larger features, such as bigger body size or larger ornaments. Anoles are an intriguing case of SSD, as the traits that can exhibit dimorphism can vary widely among species. Some species, such as Anolis carolinensis, exhibit SSD in multiple traits, including body size, head shape, and dewlap size. In contrast, other species exhibit minimal SSD. As an example, A. distichus from the Caribbean island of Hispaniola tends to show no SSD in body size or head shape, but has strong SSD in dewlap size.

Christian Cox and his collaborators posit that one mechanism underlying SSD may be a pleiotropic regulator that can couple and decouple dimorphism in different phenotypes and their candidate for this study was testosterone. They conducted experiments manipulating levels of testosterone in adult males and females of Anolis sagrei and assessed how body size, head shape, and dewlap traits changed. Anolis sagrei is a particularly good system for assessing the role of SSD in anoles. Male A. sagrei can be up to 50% larger and three times more massive than females.

To conduct the study, they took three year-old male and female lizards and gave them either testosterone or blank subdermal implants. They maintained lizards under laboratory conditions for two months and then gathered information on morphological dimensions and dewlap characteristics. Under testosterone treatment, males and females grew similarly, whereas males grew faster than females in the control group. This merits restating – they were able to make females grow like males just by applying testosterone! Clearly testosterone has strong effects on male-specific growth patterns.

To determine if testosterone affects metabolism, they measured metabolic rate using stop-flow respirometry. They found that testosterone treatment increased metabolic rate for males and females. Correspondingly, they found that visceral fat bodies were lower in testosterone treated animals, suggesting that increased growth is caused by shunting energy towards growth and away from storage metabolism. They further determined that testosterone treatment increased the size of the humerus and femur, but had no significant effect on jaw length and head width. Because this species exhibits little SSD with respect to head dimensions, perhaps this finding is not surprising, but I would be curious to know whether testosterone influences skull growth in species with SSD in head dimensions, such as A. carolinensis.

Finally, the authors found that testosterone led to increased dewlap size in both males and females. In fact, the dewlaps of testosterone-treated females were comparable in size to those of control males and eroded the sex differences that otherwise existed between them. Testosterone treatment decreased the saturation and brightness in the dewlap, leading the authors to suggest that it accelerates its development, as they posit that this color is representative of the fully developed dewlap in the wild.

Thus, they find strong evidence that testosterone plays a large role in modulating SSD in anoles. In particular, it abolishes differences in growth in various traits except for skull shape. And it can create male-like females as well as forge super-males. It would be interesting to see if, in addition to acquiring a male-like morphology, the females would tend to act like males, as well. Their next step is to conduct testosterone manipulation experiments in A. distichus, a species that has low SSD in body size and head shape, but strong SSD in dewlap size, to determine if the effects of testosterone are repeatable in a system exhibiting a pattern of SSD that is different from A. sagrei.

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Extreme sex differences in the development of body size and sexual signals are mediated by hormonal pleiotropy in a dimorphic lizard. Authors: Cox, Christian L.; Hanninen, Amanda F; Cox, Robert M.

Gamble and Zarkower (2012) Current Biology

Tony Gamble, a postdoctoral researcher working with Dave Zarkower at the University of Minnesota, presented his work on uncovering sex-specific markers in geckos and anoles. Recent years have seen a large impetus to understand how sex chromosomes evolve. Sex chromosomes can be involved in sex-specific adaptation, genetic conflict, and other important modes of evolution. This line of research is particularly imperative in reptiles because not only do we have comparatively little information about sex chromosomes in this group, but different types of sex determining mechanisms have evolved multiple times and so there are likely multiple sex-specific mechanisms and multiple evolutionary transitions are at play (see Figure above).

Traditionally sex chromosomes were discovered by karyotyping, which is a method of separating and identifying the chromosomes. This is problematic in reptiles because the sex chromosomes of many species are homomorphic, meaning they are similarly shaped and, oftentimes, quite small. Gamble and Zarkower tried a different approach – RADseq – for identifying sex chromosomes. RADseq uses restriction enzymes to identify sex-specific markers. Their reasoning is that in XY systems (i.e., males are the heterogametic sex), you would expect males and females to exhibit X-specific markers and males to exhibit sex-specific markers unique to the Y (i.e., the non-recombining region). In ZW systems (i.e., females are the heterogametic sex), you would expect the opposite. In theory, this could prove a cheap and fast way to determine the sex chromosomes of different species and develop sex-specific markers.

The challenge for this study was to determine the sex chromosomes for the crested gecko and for the anole. Unlike the crested gecko, Anolis is genome-enabled and we have evidence that they are an XY system, and so they used anoles to pilot their method and confirm that it works before trying it on the crested gecko. However, anoles are not without their challenges. The sex chromosomes are not only homomorphic, but they are also micromorphic, meaning they are quite small. Furthermore, the Anolis genome was built using a female anole, making finding sex-specific markers on the non-recombining region (i.e., the Y chromosome) that much more challenging. Their RADseq approach worked quite well, however, as they were able to recover a male-specific marker in A. carolinensis, which they were able to confirm with PCR amplification. They repeated their results using more A. carolinensis (from a different clade), A. sagrei, and A. lineatopus, and were able to recover the same locus. When they conducted this method in the crested gecko, they found evidence for a ZW system and, correspondingly, recoverd two female-specific markers. Thus, they found that RADseq will work in a variety of taxa, even if they are not genome-enabled, and can successfully be used to uncover sex-specific markers. A neat application of this method is that, using their sex-specific primers, you can sequence an embryo to determine its sex, something that was not previously possible.