Anole Annals

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.

Figure from Nicholson et al. (2007) showing variation in dewlap color among various species of anoles.

As Nick Crawford, recent Ph.D. of Boston University, points out, the genomics era allows scientists unprecedented access to understanding the genetic basis of adaptation and, by extension, the genetics of speciation. For his doctoral thesis, Nick focused on understanding the genetics of colorful adaptation in Anolis lizards, which is genome-enabled. Adaptive radiations provide lots of variation among closely related organisms, making anoles a great system for studying the genetics of adaptation.

One feature of anoles that really stands out is how colorful they are. Just a casual glance at some of the color variation in dewlaps among species reveals that color is likely an important component of species diversification in anoles. Nick focused on Anolis marmoratus, a colorful anole from the Caribbean island of Guadeloupe. Anolis marmoratus is an excellent choice for studying the genetics underlying color. This species exhibits strong geographic variation in coloration and, as I discussed in my talk a few days ago, lacks a strong signal of genetic structure. In this case, searching for the genes underlying local adaptation can be conducted without the confounding effects of population structure.

One of Nick’s slides showing the ranges of A. m. marmoratus (orange color) and A. m. speciosus (blue) on the islands of Basse Terre (left) and Grande Terre (right) in Guadeloupe.

Nick focused on A. m. marmoratus, which has red marbling on its head, and A. m. speciosus, which has a blue head and, oftentimes, a blue body and tail. These two species are clinally distributed along the eastern side of Basse Terre and A. m. speciosus ranges into the nearby island of Grande Terre (see Figure 1). Rather than use RAD tags, Nick sequenced the genomes for 20 individuals (10 each per subspecies). For every 5 kb along the genome, Nick measured divergence using various metrics of structure and assessed sequence divergence.

Overall, Nick found that about 2% of the genome falls within divergent regions for these two subspecies. Importantly, he found divergence in two genes involved in carotenoid pigmentation and one gene involved in melanosome transport. Divergence in the two carotenoid genes could very well underlie the color divergence in A. m. marmoratus, which has distinct red marbling on its head. These genes fall in regions containing several fixed single nucleotide polymorphisms (SNPs) in a row. Nick suggests that these are likely single haplotypes that are being selected in different environments. Finally, he found no evidence of coding sequence changes, and so he posits that the modifications are probably cis-regulatory in nature. For many years we have been waiting to find out how divergence in coloration occurs in anoles. After seeing Nick’s work, it appears we are closer than ever before to understanding local color adaptation at a genomic level, so stayed tuned to his work for more to come.

Yesterday, Matt McElroy presented a phylogenetic analysis of the Puerto Rican radiation of anoles. The work was focused around the “stages of radiation” hypothesis that states that divergence occurs along different niche axes at different points in time. In the case of anoles, it has long been argued that the last stage in radiation is divergence of ecomorphologically similar species into different climatic niches.

McElroy constructed a phylogeny for 180 individuals of eight species, encompassing the geographic distribution of these species (most of which occur island-wide). Ten genes were sequenced, nine nuclear and one mitochondrial. The resulting phylogeny was well-resolved and in agreement with previous phylogenetic hypotheses, indicating that ecomorphs evolved relatively early in the radiation and that closely related sister taxa pairs are usually members of the same ecomorph, but differ in climate–the one exception–which always has struck me as odd, but apparently is correct–is the sister taxon relationship between the deep rainforest trunk ground species A. gundlachi and the xeric grass-bush species, A. poncensis.

The time of divergence was estimated for each of the four sister taxa pairs, indicating that there were three phases of radiation. The deepest split, pegged at 15 mya, was between the two trunk-crown species, A. evermanni and A. stratulus. At 10 mya, two pairs split simultaneously, the aforementioned one above and the two trunk-ground species, A. cooki and A. cristatellus. Both of these pairs include one species that occurs in the xeric southwestern portion of Puerto Rico, perhaps not a coincidence? Finally, 5 mya, the two grass-bush species, A. pulchellus and A. krugi diverged. These latter two species have recently been shown to hybridize, and McElroy’s data confirms that this is the only one of the four pairs in which hybridization occurs, perhaps due to their recency of divergence?

This is a fabulous example of detailed phylogenetic work spanning both interspecific comparisons and including the extensive degree of phylogeographic divergence that occurs within many anole species. More work of this sort is needed on anoles on the other three islands of the Greater Antilles. The monophyletic Jamaican radiation would be a good starting point.

The study of geographic variation has long been a foundation of evolutionary biology. As Martha Muñoz explained, in recent years attention has focused on whether geographic variation is the first stage in the process of speciation resulting from divergent selective pressures. Making the case for such ecological speciation requires demonstrating the occurrence of divergent selective pressures correlated with environmental differences and that the resulting  phenotypic differences lead to a reduction in genetic exchange among populations in different populations.

Martha reported on a study of the highly variable Guadeloupean anole, Anolis marmoratus. Detailed population level sampling confirmed that differences in body color correlate with environmental differences, with bluer populations in wetter habitats. These results strongly suggest the role of divergent natural selection as the cause, particularly because the differences were replicated across two different transects. Moreover, the fact that the color differences occur only in males suggests a role for divergent sexual selection.

Coalescent analyses of molecular genetic differences suggested that these differences occurred in the presence of gene flow, i.e., a parapatric model of divergence, rather than in allopatry. However, estimates of ongoing gene flow find no evidence of reduced gene flow across environmental  borders. In other words, even though selection is driving phenotypic differences, these differences are not leading to a reduction in genetic exchange–selection does not appear to be leading to speciation, contrary to the ecological speciation hypothesis.

These results are in agreement with studies on several other Lesser Antillean anoles. Martha pointed out that in situ cladogenetic speciation only occurs in anoles on the large islands in the Caribbean, suggesting that the lack of opportunity for allopatry on small islands precludes speciation from occurring, even in the presence of strong divergent selection.

These results were recently published in Molecular Ecology and are further discussed in a previous post.