Anole Annals

Anolis sagrei eggs in the field. Photo by Jenna Pruett.

I would like to start by apologizing for the title of this post. I couldn’t help myself. Let’s move on.

How can organisms respond to climate change? There are basically three mechanisms: move, evolve, or acclimate via phenotypic plasticity. Plasticity is potentially very powerful because it drives changes in traits without the generational time lag and population cost of natural selection. Individuals simply adjust on the fly to prevailing conditions. To find out if plasticity can help in a changing world, the following questions have to be addressed: 1) Are relevant traits plastic? and 2) if so, how plastic are they (i.e., how much can they change)?

The thermal tolerance of most organisms is plastic to some degree, and this includes anoles. For example, if you move adult A. carolinensis housed at low temperatures to warmer conditions, their heat tolerance will increase (Corn 1971). Most of the work on thermal tolerance plasticity comes from studies of “reversible” plasticity, in which the plastic trait shift can be erased. In the A. carolinensis example, moving the individuals from the warm conditions back to the original cooler conditions would be associated with a decrease in heat tolerance. Reversible plasticity in thermal tolerance is fairly weak in lizards: on average across taxa, a 1°C increase is body temperature is associated with only about a 0.1°C increase in heat tolerance (Gunderson and Stillman 2015).

Plastic shifts can also be irreversible if they are induced at the right time in the organism’s life cycle, termed “developmental plasticity.” For example, Drosophila usually have greater heat tolerance as adults when they develop under warm versus cool temperatures (MacLean et al. 2019). Overall however, relatively little is known about the presence and strength of developmental plasticity in thermal tolerance. This is especially the case in lizards. Few studies exist and, importantly for this audience, none have focused on anoles (reviewed in Refsnider et al. 2019).

Heat tolerance of adult A. sagrei that developed as embryos under different temperature regimes.

In a new paper with Dan Warner and Amelie Fargevieille, we tested for developmental plasticity in the heat tolerance of the Cuban brown anole, Anolis sagrei (Gunderson, Fargevieille & Warner, 2020). Eggs laid by females maintained in the lab were incubated under one of three different fluctuating thermal regimes (cool, warm, and hot) that mimicked temperature dynamics measured in nests in the field. Minimum temperatures of each treatment were similar, but they differed in the maximum temperatures experienced during the day. After hatching, all lizards were raised under common garden conditions until sexual maturity, at which point we measured heat tolerance. With this design, we isolated the effect of embryonic conditions on the thermal physiology of reproductive adults. As far as we know, this is the first study to use this design in a reptile system.

We found no evidence for developmental plasticity: embryonic temperature did not influence adult heat tolerance. One conclusion that might be drawn from our work is that developmental plasticity will be of little to help to anoles as the climate warms, meaning behavioral and evolutionary processes could be particularly important in dealing with changing temperatures. Additionally, developmental plasticity may play a minor role in driving observed differences in the thermal physiology of anoles from different thermal environments, making evolutionary divergence a more likely explanation.

But these inferences must be taken with a huge grain of salt. Plasticity itself evolves, and therefore what we find in one or even a few species may not be broadly representative. We will have to wait for more data to emerge to get a clearer picture of the ecological and evolutionary implications of developmental plasticity in reptile thermal traits.

Cellular processes, including metabolism and longevity, are regulated by the insulin and insulin-like signaling network. This cascade of internal systems is regulated by two similar but unique hormones, IGF1 and IGF2. Expression of these hormones not only differs among species, but also varies throughout the lifespan of individuals. For example, in humans IGF1 and IGF2 expression remains on into adulthood. However, in rodents IGF2 expression is switched off shortly after birth; thus, IGF2 post-natal effects have largely been ignored because of the lack of expression in mice. 

Ph.D. Candidate, Abby Beatty, of Dr. Tonia Schwartz’s lab at Auburn University, sought to address this imbalance by using the Cuban brown anole (Anolis sagrei) which, like humans, expresses IGF2 into adulthood. To do this, Abby first quantified gene expression of IGF1 and IGF2 hormones by using quantitative PCR on embryonic, juvenile, and adult A. sagrei liver cDNA. She compared this expression across a variety of taxa including, but not limited to, mice, zebra finches, and eastern fence lizards. Secondly, Abby mined adult liver transcriptomes for all amniotes in NCBI and quantified the expression of IGF1 and IGF2. 

She found that, in contrast to the mice used for many biomedical models, IGF2 is expressed in adult birds and reptiles and in many mammals. Abby speculates that our understanding of IGF expression is biased, with laboratory mice serving as a default for many human-mediated models, and warrants the use of other species to study the function of IGF2. 

Measuring Anolis thermal tolerances has been a hallmark of many studies since the heydays of thermal physiological studies in the mid-to-late 1900’s. However, studies examining the factors affecting thermal tolerances of embryos are still relatively sparse. In the symposium “Beyond CTmax and CTmin: Advances in Studying the Thermal Limits of Reptiles and Amphibians” at the ninth World Congress of Herpetology, Joshua Hall – PhD candidate in the Warner lab at Auburn University – explored critical thermal maximum temperatures in Anolis sagrei during development. He sought to determine (1) How we should measure embryonic CTmax? (2) What is the ecological relevance of embryonic CTmax? And (3) Are there differences between acute and chronic CTmax?

Previous work from Pruett and Warner, determined that constant incubation temperatures resulted in a chronic CTmax of 35°C for A. sagrei. Meanwhile, Joshua tested three methodologies of creating an acute CTmax during incubation including: heat shock, thermal ramp, and thermal fluctuations. All three methodologies showed an acute CTmax of ~45/46°C; there was consistency across methodologies as well as an extremely large difference found between chronic and acute CTmax. Additionally, Josh examined what data were available via the Reptile Developmental Data Base to examine chronic CTmax in nine other squamate species (ranging from 28-36°C). Of those nine species from previously collected data, four had measures of acute CTmax, and in all four cases the acute CTmax was higher than the chronic CTmax. Lastly, Josh recommends that researchers use the terminology acute and chronic when describing CTmax and that more work should be done to better determine the relationships between chronic and acute CTmax in an ecological context. Super cool work, looking into the future of thermal physiological work!

Ectotherms are well known for having an inordinate fraction of their biology linked to thermal conditions. Many of their demographic vital rates and life-history traits are influenced by temperature-dependent physiological processes. This connection between temperature and physiology is particularly apparent during embryonic development, especially in oviparous species lacking parental care after eggs are laid. Jenna Pruett, a PhD candidate in the Warner lab at the University of Auburn, investigated the effect of constant egg incubation temperatures across life stages in the brown anole. Many studies of this nature lack enough temperature treatments to fully characterize the thermal reaction norm and frequently do not follow the offspring past hatching. Jenna sought to fill these knowledge gaps by answering the questions:  1) How does constant incubation temperature affect embryonic development? 2) Do these effects vary across a small geographic scale? and 3) Do effects carry over into later life stages?

To do this Jenna incubated ~350 brown anole (Anolis sagrei) eggs from different locations across eight different constant incubation temperatures. When examining hatching success, temperature seemed to be the only driver of success. Meanwhile, hatchling mass had a significant interaction between temperature and location potentially indicating that lizards at specific locations respond differently to different thermal regimes during development. Overall, she found that geographic variation doesn’t impact hatching success but changes how phenotypes respond to temperature.

The second part of the experiment involved a large release and recapture experiment on experimental spoil islands off the coast of Florida. Hatchlings were released early and late in the summer and then were recaptured the following fall and spring to determine survival to recapture. Jenna found that survival to recapture was influenced by incubation temperature, release date, and an interaction between the two, showing that timing is everything and that in this case the optimal temperature for the greatest survival varied across life stages.

Green anole in Cape Cod in January

Veronica Worthington writes from Cape Cod: “This September I found an anole  in my unheated, open greenhouse. I snapped a picture of him and he scurried off. Cold weather sets in, below freezing off and on, and I figure the anole  must not have made it but to my surprise a few days ago, January 14th,  I see him again and he’s perfectly fine. I have no idea how he could’ve gotten here, I have not brought any plants in to the greenhouse in a few years and I have no neighbors that could’ve had a pet lizard. Have you heard anything about anoles migrating north?”

First sighting, September 2019

Veronica then added in a subsequent email: “I find it so curious that this little guy ended up in my backyard. And that he has been able to survive all this time. No matter who I tell they say he must have arrived as a hitchhiker on a plant But I have not brought any plants into the greenhouse in a few years and it is always unheated in winter and the doors and sides are open all summer. I don’t know how far they travel naturally catchy but I can’t imagine that this little guy would’ve traveled very far on his own. I don’t have any neighbors close to me  that keep reptiles. The first picture is of him two days ago and the second picture is of him five months ago. Both times that I have seen him he is exactly where I saw him the last time, on a bag of wool. I raise sheep and that’s where the wool came from.”

The green anole, A. carolinensis, is the only native anole in North America. Over the years, the question of whether it is distinct from the Cuban A. porcatus has been debated–morphological differences are pretty minor, other than the Cubans generally being a bit larger.

Now, in an open access paper published last year in Ecology and Evolution, Johanna Wegener and colleagues have driven the final nail in the coffin of the idea that North American carolinensis is a distinct species.

For some time, we have known that carolinensis is nested phylogenetically in the western clade of porcatus, rending porcatus paraphyletic. This phylogeny indicates that North American populations are the result of a colonization event from western Cuba, perhaps 6-12 million years ago (see references in Wegener et al. paper).

From Wegener et al. (2019). Florida populations are usually referred to as “ carolinensis,” Cuban populations as “porcatus“

The novel contribution of the Wegener et al. paper is to look for evidence of hybridization between recently introduced “porcatus” from Cuba and native “carolinensis.” And she found it in spades! The abstract, pasted at the bottom of this post, provides some more details and, of course, you can read the paper itself.

So, Florida populations of the green anole are derived from Cuban populations, and the two readily interbreed when given a chance. Given these facts, there is no justification for treating North American populations as a distinct species. The morphological differences that do exist–quite minor–are the result of geographic variation. Paraphyly plus no reproductive isolation = one species!

But now here’s where it gets interesting. By the rules of zoological nomenclature, the older name has precedence, and so this single species takes the name Anolis carolinensis. That’s right: A. carolinensis is the correct name for Cuban green anoles! I’m sure that won’t go over so well in some quarters.

But it gets more interesting! Cuban Anolis porcatus as currently recognized is not a monophyletic entity, as shown in the attached figure, based on Glor et al. (2005). As the figure shows, eastern populations of porcatus are more closely related to A. allisoni (remember, North American populations are nested in the western clade). Given that the species-level distinctness of allisoni has not been question, most systematists would recognize the two clades of porcatus as different species. Thus, the eastern clade retains the name porcatus.

Bottom line: both A. carolinensis and A. porcatus occur in Cuba!

Abstract

In allopatric species, reproductive isolation evolves through the accumulation of genetic incompatibilities. The degree of divergence required for complete reproductive isolation is highly variable across taxa, which makes the outcome of secondary contact between allopatric species unpredictable. Since before the Pliocene, two species of Anolis lizards, Anolis carolinensis and Anolis porcatus, have been allopatric, yet thisvperiod of independent evolution has not led to substantial species‐specific morphologicalvdifferentiation, and therefore, they might not be reproductively isolated. Invthis study, we determined the genetic consequences of localized, secondary contactvbetween the native green anole, A. carolinensis, and the introduced Cuban green anole, A. porcatus, in South Miami. Using 18 microsatellite markers, we found that the South Miami population formed a genetic cluster distinct from both parental species. Mitochondrial DNA revealed maternal A. porcatus ancestry for 35% of the individuals sampled from this population, indicating a high degree of cytonuclear discordance. Thus, hybridization with A. porcatus, not just population structure within A. carolinensis, may be responsible for the genetic distinctiveness of this population. Using treebased maximum‐likelihood analysis, we found support for a more recent, secondary introduction of A. porcatus to Florida. Evidence that ~33% of the nuclear DNA resulted from a secondary introduction supports the hybrid origin of the green anole population in South Miami. We used multiple lines of evidence and multiple genetic markers to reconstruct otherwise cryptic patterns of species introduction and hybridization. Genetic evidence for a lack of reproductive isolation, as well as morphological similarities between the two species, supports revising the taxonomy of A. carolinensis to include A. porcatus from western Cuba. Future studies should target the current geographic extent of introgression originating from the past injection of genetic material from Cuban green anoles and determine the consequences for the evolutionary trajectory of green anole populations in southern Florida.