Author: Jerry Husak Page 2 of 3

You lookin’ at me? Photo credit to Tim Norriss

It’s often said that winning isn’t everything. This may be true for humans and the games we play, but, unfortunately, for most animals losing a contest can have serious implications for whether they survive or reproduce. The study of animal contests has been thoroughly studied in males, and we know that losing to a rival can mean you get less or no mating success. However, we know far less about the consequences of winning and losing if you are a female. Jess Magaña and Matt Lovern (from Oklahoma State University) asked what happens to females after they win or lose a contest, and they had one of my favorite talk titles ever: “Small and large lizards agree in defeat but react differently to victory.”

They studied brown anole females, which are known to show aggression toward each other. Winners and losers were pre-determined by residency in a cage. Females who got to compete in their home cage were winners, and those who were placed into another lizard’s cage were the losers. They were allowed to interact, and then Magaña monitored their reproduction thereafter. Previous work had shown that losers laid eggs that hatched more quickly, suggesting that offspring were given less yolk and would perhaps be less successful because of it.

Comparisons between winners and losers reveleaed surprisingly little difference in most reproductive traits, such as egg size, time to hatch, and sex ratio. However, when they looked at the effects of body size on reproductive traits, there was a marked difference between winners and losers. In losers, investment in reproduction was unrelated to body size. In winners, though, size was related, and size reflects age in this species. Small (young) winners laid eggs that hatched quickly, but large (old) winners laid eggs that took longer to hatch. They interpreted this as different strategies of investing in potential future reproduction: old winners should invest in current offspring, whereas a young winners should invest in potential future offspring. This interesting finding highlights the fact that there is still much to be learned about the subtlety of how a mother’s environment and experiences can shape her offsprings’ life.

Brown anole with and without crest shown at the whole-animal (left) and histological (right) level. Photo from Ademi and Rand poster.

If you’ve ever been around brown anoles, you know that the males can be pretty aggressive. Part of that aggression involves the enlargement of a crest along the neck and back. The crest is caused by fluid rapidly rushing into the tissue of the crest. How this works has been discussed here before, but Matt Rand’s research group at Carleton College continues to try to unravel what hormonal pathways are responsible for crest formation. Ademi and Rand used an experimental approach to discover what molecular receptors are activated to cause crest formation. Body-wide and local injection of a variety of chemicals and drugs gave some tantalizing clues as to how it works.

Local injections to stimulate cAMP activity caused crest formation locally (top), whereas body-wide injection caused whole-crest formation (bottom). Photo from Ademi and Rand poster.

They found after several inhibitory and stimulatory drug manipulations that crest erection is likely stimulated by epinephrine acting on a Beta-2 like adrenergic receptor that stimulates cyclic AMP (cAMP) activity to cause vasodilation (enlarging of blood vessels) and fluid entrance into the crest. This activity that starts with the B2-adrenergic receptor is essentially the same function as that seen in mammalian circulatory systems, including us. They also stimulated cAMP activity without stimulating the B2-like adrenergic receptor and found similar results. You can see how dramatic the response was below, where they used local injection to cause crest formation only at the site of injection! The use of epinephrine binding to a B2-like adrenergic receptor as the molecule of communication makes the rapid time-course of crest formation make sense. There are still some unknown aspects as to how the vasodilation mechanistically causes the fluid release in the crest, but they are actively studying it.

Are anoles like sherpas? Photo from Reddit

When you’re used to living at low to moderate elevations, it can be challenging to visit high-altitude places. The declining partial pressure of oxygen at high altitude makes it difficult for your body to deliver the same amount of oxygen to tissues. This is why National Football League players often struggle to play in Denver (see playoffs next week!). However, organisms that live at high elevations, including humans, have evolved a number of ways to deal with living in such oxygen-challenged environments. We know less about whether the same aspects of the cardiovascular change in different organisms, even among relatively closely related species. Well, what better group of organisms to address such questions than anoles!

Virtually nobody reading this blog will be unfamiliar with the story of the Greater Antillean ecomorphs, and they are great to use for questions related to elevation and adaptations to deal with it. They live along steep elevational gradients within an island, and such gradients exist across islands. Although, the Caribbean anoles have been the subject of numerous studies that have shown convergent evolution in body size and shape, as well as locomotor performance and endocrine function, we know much less about how they deal with elevational challenges at the cardiovascular level.

Species studied and locations in the Dominican Republic. Photo from Webber et al.’s poster.

Miguel Webber, an undergraduate in the laboratory of Michele Johnson at Trinity University, along with Brittney Ivanov, studied several blood physiology traits in 13 species across five ecomorphs in the Dominican Republic to determine whether elevation has been an important driving force in the evolution of oxygen delivery mechanisms. Although looking at an impressive number of traits that included hematocrit (the proportion of red blood cells), hemoglobin concentration, and red blood cell size, Miguel only found hemoglobin concentration to be positively related with elevation when looking across species.

One of the more interesting findings was that none of the blood physiology variables that Miguel measured were ecomorph specific. However, this makes sense because members of an ecomorph live across wide geographic areas and across elevational gradients. Physiological studies such as Miguel’s are offering interesting insights into how anoles have adapted to their environments and emphasizes that ecomorph membership does not determine everything.

Brown anole photo by Amber Brace.

The immune system can be costly, even for anoles. However, despite a large amount of work on natural populations examining when and why animals use their immune system, as well as what it energetically costs, it remains poorly understood whether a larger (more costly) immune response to pathogens offers more protection. In other words, is a major immune response worth all the cost? This is what Amber Brace, a graduate student in Marty Martin’s lab at the University of South Florida has been trying to test. Amber used experimental malaria infections in introduced brown anoles in Florida to determine whether the high costs of an immune response would result in better protection from the disease. Although malaria naturally occurs in Floridian brown anoles,  Amber first had to develop an experimental protocol to successfully infect lizards. She gave one group a low dose of malaria, and another group a high dose. Interestingly, only the high-dose group became infected. Once this was worked out, she could then test how experimental infection would affect individuals.

Since malaria ultimately results in the bursting of red blood cells, she predicted that a higher malaria burden would be positively related to the change in number of immature red blood cells (from pre- to post-infection), and this is exactly what she found. This shows that individuals with greater malarial infections are compensating for lost red blood cells by producing more. Perhaps most importantly, she found a negative relationship between malaria burden and the change in number of white blood cells. This suggests that individuals greatly increasing one group of immune cells (white blood cells) are able to decrease their malaria burden. Thus, it appears that an enhanced immune response does, in fact, offer added protection, and the high costs of an activated immune system are worth the  investment.

Life cycle of malaria, showing infection of red blood cells (erythrocytes). Photo from malariasite.com

Anolis stratulus, one of the species studied. Photo by Jerry Husak.

Anoles are no strangers to urban environments. In fact, many anole species seem to do just fine in cities. However, they do face a number of different challenges not present in their native environments. One example is the perches on which anoles move. Andrew Battles, a graduate student in Jason Kolbe’s lab at the University of Rhode island, was interested in exploring how the perch use of two anole species differed between natural populations and urban populations, and what that habitat use might do to their running performance. Andrew studied Anolis cristatellus and A. stratulus on Guana Island in the British Virgin Islands to measure perch smoothness/roughness, perch use, and sprinting performance on various perch types.

Lizards were found most often on artificial perches, instead of natural perches, in urban environments. This is interesting, because such artificial substrates tend to be vertically oriented and significantly smoother compared to natural perches like tree branches and trunks. As predicted, lizards ran more slowly on substrates that are smooth and more vertical, and this was most pronounced in the larger male A. cristatellus compared to the smaller female A. cristatellus and both sexes of A. stratulus. Thus, while optimal substrate use might be inclined, rough, natural perches, these anoles are using smoother, more vertical, artificial perches in urban environments. This fits into a theme present at this year’s SICB meeting that animals often move in ways that seem counter-intuitive at first. How such perch decisions might influence fitness remains an open question. Future work will investigate how availability of perches and alternative escape strategies influence perch selection.

Image of an anole with a regenerated tail. The point of breakage (and regeneration) is shown with an arrow. Image from Wired.com

It’s happened to us all: you try so hard not to break the tail when you catch an anole, but inevitably it happens to one. As readers of Anole Annals know, many species of lizards, including anoles, lose their tails as a defense mechanism. While losing a tail, called autotomy, has known detrimental effects on social status in males and reduced locomotor capacity, we know less about other potential costs for a strategy that is intended to keeps lizards alive to reproduce another day. McKenzie Quinn, an undergraduate in Michele Johnson’s lab at Trinity University, wanted to know how losing so much tissue, and then replacing it, might take away available resources from other important processes. She measured changes in egg number, egg size, body size, and fat mass in the liver over the course of three weeks after experimental removal of the tail in green anoles. These females were compared to a control group that did not have their tails removed.

Lizards who had their tails autotomized re-grew their tails over the course of the experiment, whereas control groups that had intact tails had minimal tail growth. Surprisingly, there was no difference between the two groups in any of the traits measured. Females with autotomized tails had just as much growth, just as many eggs of the same size, and just as much fat accumulated in the liver. This suggests that in a laboratory setting females are not taking resources away from growth and reproduction to re-grow a tail. Field studies and additional manipulations of resource availability in the future may help us understand what costs are associated with such an intriguing and seemingly costly defense strategy.

Species studied by Kircher et al. Image credit to Bonnie Kircher.

Readers of Anole Annals are likely familiar with the amazing convergent evolution of habitat use and morphology in Caribbean anoles, but the corresponding divergent and convergent evolution of social behavior has recently captured the interest of anolologists. The species differences in social behavior would seem to be due to differences in how much testosterone, a steroid hormone that regulates behavior in many other vertebrates, but this does not appear to be the case. Bonnie Kircher, formerly of Michele Johnson’s lab at Trinity University and currently at the University of Florida, examined what other aspects of hormone signaling might be responsible for the diversity of social behavior seen in Hispaniolan anoles. Since hormones can only act on tissues that have receptors for them, it is possible that variation in hormone receptors might explain behavioral differences independent of hormone levels circulating in the blood. Since the behavioral differences in anoles involve variation in pushup displays and dewlap extensions, it seems intuitive that there may be differences in receptors for testosterone (androgen receptors) in the muscles responsible for these displays.

Bonnie studied six species of anoles that vary in pushup and dewlap display frequency: A. bahorucoensis, A. brevirostris, A. carolinensis, A. coelestinus, A. cybotes, and A. olssoni. After measuring display frequencies in these six species, the investigators quantified the number of androgen receptors in two muscles that are important for pushup displays (biceps) and dewlap displays (ceratohyoid). As predicted, the results showed that species with higher rates of pushup displays have more androgen receptors in their biceps than species with lower pushup frequencies. Interestingly, this was not the case for the ceratohyoid muscle, which controls dewlap extensions. There was no relationship between androgen receptor density of the ceratohyoid and dewlap display frequency. These results are a tantalizing clue to the still-enigmatic mechanism(s) that underlies anole behavioral diversity.

Brown anole photo by Dan Warner.

Although there is a vast literature on how resource availability affects physiology, behavior, and reproduction (among many other things), we know surprisingly little about the composition of individual diets in nature. To truly know whether you are what you eat, you have to understand what it is you are eating. Dan Warner from the University of Alabama at Birmingham set out to do just that with some very interesting preliminary data on an island population of brown anoles in Florida. He trapped potential prey in two very different habitat types on the island: interior forest and open shoreline. The shoreline had mostly marine-sourced prey items (amphipods), whereas the forest had more terrestrial insects, like roaches. Dr. Warner then wanted to know if these differences in diet would affect body composition of anoles in those habitats.

The methods here are the best part. Dr. Warner used Quantitative Magnetic Resonance (QMR) technology, typically used for rodent lab animals, to determine body composition. He found that there was a very strong match between the QMR estimates of lean and fat mass compared to chemical carcass analysis of the same individuals. And, the QMR measures only take about 5 minutes to do! This non-invasive, non-lethal way to estimate body composition has huge implications for studies that seek to tie those characteristics to components of organismal fitness, namely survival and reproductive success. It doesn’t work to track survival on individuals sacrificed for chemical carcass analysis. He also suggests that this now-validated method will be important to test whether typical measures of body condition (such as mass-length residuals) are actually good estimates. It doesn’t sound good for our typical measures of condition, but he will tell that story soon!

Returning to diet’s effect on body composition, the results showed that lizards in the interior of the island had more fat mass and less lean mass than lizards found on the shoreline. He plans to continue the research by repeating it on replicate islands with similar habitat types, as well as look at long-term consequences of variation in body composition. This new approach will open the door for fascinating research to come, so stay tuned!

Anolis proboscis, showing the male-specific proboscis. Photo by D. Luke Mahler.

A long–time favorite here at Anole Annals, the Ecuadorian Horned Anole (Anolis proboscis) made an appearance at SICB. Diego Quirola and colleagues from Pontificia Universidad Católica del Ecuador described the use of the proboscis during social interactions. They captured male and female anoles and videotaped staged male-male and male-female interactions. From the videos they were able to quantify behavioral patterns of these fascinating lizards. They did some very anole-like behavior, but they definitely have a flair all their own! With such a fascinating, chameleonic appendage one would expect some important functions of the proboscis, and one would not be disappointed. Watching videos that Diego had on display revealed social behavior very reminiscent of chameleons, with males puffing up, curling their tails, and swaying while doing the more typical anole dewlap extensions.

Then there’s the proboscis. This structure, much like the dewlap, is used during both courtship and agonistic interactions. In both contexts, males actually lift the proboscis. Yes, they can move the proboscis up and down, something not seen in chameleons with rostral appendages (no, we don’t know how they do it!). Diego suggests that the proboscis is lifted to either stimulate females or allow the male to bite the nape as other lizards do while copulating. Males also display a behavior called “proboscis flourishing” where the proboscis is prominently displayed while moving the head side to side. During agonistic interactions it may serve as a dominance indicator, though they are still working on those analyses. Proboscis anoles seem to be at the low end of aggression for anoles, but males occasionally fight and lock jaws. During male fights the proboscis likely gets in the way, and it appears to be purposely lifted during these fights. It’s possible that they lift it to keep the rival male from latching onto their snout, or it could be moved so that they can get better bites in. I was very much looking forward to learning more about these anoles, and I was not disappointed. As more work is done on these fascinating anoles we’ll be able to better understand why it has evolved such an interesting, and un-anole-like appendage, as well as the unique behavior that is associated with it.

Why the long face?

When most people think of vertebrate sexual dimorphism (differences between the sexes), they think of elephant seals or red deer. Most of us here, of course, think of the pronounced dimorphism in size and shape in many anole species. Indeed, anoles have served as excellent model systems for the study of sexual dimorphism, particularly the evolutionary forces that give rise to it. Although there has been significant progress since Darwin in our understanding of why sexual dimorphism evolves, we have made less progress in the HOW. That is, what mechanisms during development give rise to what are often extreme differences between the sexes when their genomes are so similar?

When we think of vertebrates where males are larger or shaped differently than females, and have weapons or ornaments, we almost immediately think of testosterone as a mechanism underlying the sex differences. Once sexual maturity happens, the testes start cranking out testosterone, thus causing a change in the male’s phenotypic trajectory. While there is certainly evidence for circulating testosterone to have this effect in some lizards, is this always the case, and does it apply to specific body parts and not just overall size? Aside from the circulating hormone, how are receptors involved in the development of dimorphism? In a new paper by Sanger et al., a novel developmental pathway of sexual dimorphism is described for lizards in the carolinensis clade, which are striking in their elongation of male faces relative to females.

Figure 1a. from Sanger et al. (2014), showing the differences in head shape dimorphism among anole clades. Note the long male face in A. maynardi, a member of the carolinensis clade.

Sanger et al. tested whether sex differences in several different pathways led to the observed head shape dimorphism in A. carolinensis compared to two non-carolinensis species (A. cristatellus and A. sagrei) that exhibit shorter male faces. They show, using a combination of developmental and molecular genetic techniques, that the extreme elongation of male heads in carolinensis lizards is not due to an androgen pathway (i.e., testosterone) or the somatropic axis (i.e., insulin-like growth factor). Instead, they found a significant shift in the estrogen pathway. Specifically, at sexual maturity, males decrease expression of estrogen receptors (erβ), which is the beginning of a signaling cascade, ultimately resulting in up-regulation of genes involved in skeletogenesis in the skull of males.

Figure 4 from Sanger et al. (2014), showing the molecular pathway underlying facial elongation in A. carolinensis.

This identification of a novel mechanism for the development of sexual dimorphism will certainly stimulate further evo-devo research in anoles and beyond. For starters, is the same pathway responsible for male facial elongation in other species in the carolinensis clade, or are more ‘traditional’ mechanisms operating there? This important research highlights that investigators need to consider all aspects of signaling systems, including circulating hormones, their receptors, and signal cascades that result from activation of a particular pathway. Clearly this paper by Sanger et al. is an excellent step in the right direction for understanding how developmental pathways lead to adult difference in anoles, and it will also steer other investigators to consider a diversity of developmental mechanisms in their quest to elucidate how adults end up the way they do.

Sanger TJ, Seav SM, Tokita M, Langerhans RB, Ross LM, Losos JB, Abzhanov A. 2014. The oestrogen pathway underlies the evolution of exaggerated male cranial shapes in Anolis lizards. Proceedings of the Royal Society B 281:20140329.