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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.

A few more photos. 1-3 are from Cienaga de Zapata area and 4 is from a cave near Havana (Parque Escaleras de Jaruco).

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Rachana applying fluorescent powder to a wild brown anole

If you’ve ever tried to note how often lizards mate, you’ve likely found yourself staring at an individual for hours at a time, sometimes with little to no movement at all, let alone observing copulations! Further, if you’re unable to catch the animal after your behavioral observations, you may not be able to draw any conclusions about traits that influence how successful an individual is at mating with another.

Rachana Bhave, a fourth year PhD candidate in Bob Cox’s lab at University of Virginia, studies pre- and post-copulatory sexual selection in brown anoles (Anolis sagrei). One of her interests includes estimating mating rates in the wild and, in particular, testing if traits such as body size directly influence these rates. Given the power required to detect selection statistically, using simple behavioral observations can be inefficient. Further, because selection is a measure of covariance between phenotype and fitness, one needs phenotypic values for each individual within her analyses. Thankfully, Rachana was able to come up with a robust technique to estimate mating rates using an island population of brown anoles in Florida: fluorescent powders!

To understand how size affects mating rate in the brown anole, Rachana and colleagues caught 153 adult male lizards in May and 128 adult male lizards in July, weighed them, and then assigned them to one of four fluorescent powder treatments. Each mass quartile was painted with a unique color of fluorescent powder on their cloaca and released to their initial capture location. After two days, all females on the island were captured and their cloaca were examined under UV light to look for the presence and color of fluorescent powder, which would suggest that she mated with a painted male. Using this technique, Rachana found that within two days, 24% of the captured females had mated in May and 48% had mated in July. These rates were shockingly high for such a short time frame!

A) Powdering an adult male brown anole; B) copulating brown anoles; C) powder visible on the cloaca of a female brown anole, evidence of copulation
Images from Rachana’s poster

Further, she found that both larger males and larger females mated significantly more than smaller males and females across the two sampling periods. Interestingly, 2% of females had multiple colors on their cloacas, which suggests they mated multiple times with males from different size classes in the two-day span. Because multiple matings within the same size class would be undetectable, this is likely an underestimation of multiple matings in the wild.

Next, Rachana plans to quantify male reproductive success using genetic parentage analysis to begin to tease apart how pre- and post-copulatory selection influences selection. We are all looking forward to her results next year! Meanwhile, you can take a look at her poster to find out more on her website.