WCH9: The Effect of Constant Egg Incubation Temperatures across Life Stages in the Brown Anole

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.

Artificial Light at Night Increases Growth and Reproductive Output in Anolis Lizards

New literature alert!

In Proceedings of the Royal Society B
Thawley and Kolbe

Abstract

Since the invention of electric lighting, artificial light at night (ALAN) has become a defining, and evolutionary novel, feature of human-altered environments especially in cities. ALAN imposes negative impacts on many organisms, including disrupting endocrine function, metabolism, and reproduction. However, we do not know how generalized these impacts are across taxa that exploit urban environments. We exposed brown anole lizards, an abundant and invasive urban exploiter, to relevant levels of ALAN in the laboratory and assessed effects on growth and reproduction at the start of the breeding season. Male and female anoles exposed to ALAN increased growth and did not suffer increased levels of corticosterone. ALAN exposure induced earlier egg-laying, likely by mimicking a longer photoperiod, and increased reproductive output without reducing offspring quality. These increases in growth and reproduction should increase fitness. Anoles, and potentially other taxa, may be resistant to some negative effects of ALAN and able to take advantage of the novel niche space ALAN creates. ALAN and both its negative and positive impacts may play a crucial role in determining which species invade and exploit urban environments.

 

Thawley, C. J., & Kolbe, J. J. (2020). Artificial light at night increases growth and reproductive output in Anolis lizards. Proceedings of the Royal Society B, 287(1919), 20191682.

Green Anole on Cape Cod?

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.”

 

“Scientist Profiles”: Featuring Anole Researchers on AnoleAnnals.org

Greetings, fellow anole aficionados! One of the new features of Anole Annals is the “Meet The Scientists” page. Thus far, we have populated this page with profiles of each anole researcher featured in our series of short films, The Lizard’s Tale.
 
The purpose of this section of the website, however, is not just to showcase the scientists who appear in the videos – after all, they’ve had their 15 minutes of fame! Our goal for this page is actually to highlight the large, diverse community of researchers around the world who study Anolis lizards… Particularly (although not exclusively) those who contribute to this website!
 
Therefore, we’d like to extend an invitation to all anole researchers, particularly those who contribute to Anole Annals, to share their own profile to be featured on the “Meet The Scientists” page.
 
We use a structured biographic format to ensure consistency among all researchers’ profiles. For your profile, please answer the following questions:
 
1)    Where do you work, and what do you do?
 
2)    What aspects of anole biology do you study, and what have you learned?
 
3)    How and why did you start studying anoles?
 
4)    What do you love most about studying anoles?
 
5)    What is your favorite anole species and why?
 
6)    Where can people learn more about you and follow you online?
(this is where you can provide a URL for a lab website, a personal website, a Twitter and/or Instagram account, etc.)
 
7)    What is your position and affiliation? (E.g. “Assistant Professor of Biology, University of Virginia”)
 
And you’ll need to submit one photograph of yourself… Just one, so choose wisely! You can check out the existing researcher profiles here if you want some inspiration.
 
Please submit your profiles via email to: neil[dot]losin[at]gmail[dot]com. You can attach the text of your profile as a Word document, and include a photo of you (in the field, in the lab, or just a glamorous head shot) as a separate .jpg (please don’t just paste the photo into the Word doc). Images should be a minimum of 350×350 pixels, and you should choose an image that can be cropped to a square format for display on the “Meet The Scientists” page.
 
Please send me all materials by Sunday, Feb. 16. Once I receive everyone’s profiles, I’ll get them up onto the site. Thank you in advance for your help with this!

Anole Gene Editing Workshop

CRISPR-based gene editing has been successfully performed in a wide variety of vertebrate species, including fish, amphibians, birds, and mammals. Therefore, it may come as little surprise that we recently added anoles to the list of CRISPR-edited animals. However, to perform gene editing you must get CRISPR reagents (Cas9 protein and a gene specific guide RNA) into the appropriate embryonic stage or cell type of the organism. The most common approach is to inject the Cas9/guide RNA complex (Cas9 RNP) into freshly fertilized eggs using a very fine, hollow glass needle. Injection of fertilized eggs can be relatively straightforward in species with external fertilization, especially if the species can be induced to spawn in captivity. In species with internal fertilization, accessing early stage embryos is more challenging. In mammals, many decades of work have led to very effective methods to isolate, inject, and transfer embryos from a donor female to a host female. By comparison, methods to isolate and manipulate squamate embryos at the single cell stage have not been established. In addition, the females of many squamate species store sperm, making it difficult to determine the precise time point at which oocytes are fertilized. Given these challenges, we decided to try injecting Cas9 RNPs into unfertilized Anolis sagrei oocytes rather than fertilized eggs. In October of 2018, graduate student Ashley Rasys generated the world’s first gene edited reptile, an albino Anolis sagrei, using this approach.

With over 20,000 protein coding genes in the Anolis sagrei genome, there is much interesting anole biology to explore with gene editing! To teach other researchers how to perform gene editing in anoles, we are holding a gene editing workshop at the University of Georgia from June 14, 2020 – June 20, 2020. This workshop is funded through the NSF EDGE program and will walk participants through each step of the gene editing procedure. Students will learn the anesthesia, surgical, and oocyte microinjection methods we have developed in anoles. The course will also cover CRISPR guide design, Cas9 RNP preparation, essential equipment, and screening methods for the creation and detection of gene edited lizards. Since participants will be working directly with lizards, space will be limited to 10 students.

Workshop Application Process

We are now accepting applications for our 2020 gene editing workshop, which will be held at the University of Georgia in Athens, GA from June 14 – June 20. Applicants should prepare a single PDF that contains the following: 1) A one-page description of your research interests that describes why you want to attend the workshop, 2) your CV, and 3) a letter of support from your research advisor (required for graduate student and postdoc applicants). These materials should be emailed to LizardGeneEdit (at) gmail (dot) com. Priority will be given to applicants that have facilities for housing lizards or plans to establish such a facility, so please indicate whether your lab has infrastructure for breeding lizards in your one-page research statement. The application deadline is February 28, 2020. Applicants will be notified whether they have been accepted to participate in the workshop by March 20, 2020. There will be a $100 registration fee for workshop participants, but lodging and meals will be subsidized.

North American Anolis carolinensis is not a distinct species

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.

Egg Incubation Temperature Does Not Influence Adult Heat Tolerance in the Lizard Anolis sagrei

New literature alert!

In Biological Letters
Gunderson, Fargevieille, and Warner

Abstract

Extreme heat events are becoming more common as a result of anthropogenic global change. Developmental plasticity in physiological thermal limits could help mitigate the consequences of thermal extremes, but data on the effects of early temperature exposure on thermal limits later in life are rare, especially for vertebrate ectotherms. We conducted an experiment that to our knowledge is the first to isolate the effect of egg (i.e. embryonic) thermal conditions on adult heat tolerance in a reptile. Eggs of the lizard Anolis sagrei were incubated under one of three fluctuating thermal regimes that mimicked natural nest environments and differed in mean and maximum temperatures. After emergence, all hatchlings were raised under common garden conditions until reproductive maturity, at which point heat tolerance was measured. Egg mortality was highest in the warmest treatment, and hatchlings from the warmest treatment tended to have greater mortality than those from the cooler treatments. Despite evidence that incubation temperatures were stressful, we found no evidence that incubation treatment influenced adult heat tolerance. Our results are consistent with a low capacity for organisms to increase their physiological heat tolerance via plasticity, and emphasize the importance of behavioural and evolutionary processes as mechanisms of resilience to extreme heat.

Help Identify More Cuban Anoles

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

 

1

2

3

4

In the Eye of the Beholder: How Do Anoles Respond to Human Clothing Color?

Water anole (Anolis aquaticus), Costa Rica

Have you ever wondered how your clothing color affects how many anoles you see and catch on a given day? When we go out herping, a lot of us favor some sort of clothing color scheme – whether it’s conscious or subconscious, scientific or superstitious. Since anoles have such excellent color vision, and since they’re so sensitive to the signaling colors of the individuals they interact with, this makes sense, right?

(a) Water anole dewlap; (b) Orange and blue shirts; and (c) green shirts worn in this study.

Bree Putman (Cal. State San Bernardino), Andrea Fondren (an undergraduate researcher), and I teamed up to determine if there was any truth behind the superstition. In an effort to understand how anthropogenic colors affect the behavior of lizards, we designed a study to test whether researcher shirt color would influence the sighting and capture rates of water anoles (Anolis aquaticus). Male water anoles have large orange dewlaps, which is their most conspicuous sexual signal. Using a modified version of the species confidence hypothesis, we predicted that water anoles would be least fearful of anthropogenic colors that most closely resembled the color of their own sexual signals (orange). To test this, a group of us spent the summer surveying water anole populations wearing three different shirt colors: orange, blue, and green. We carefully allocated shirt colors to research teams and study sites, planning it out in advance so that all shirt colors were worn by all researchers and used at all sites evenly in the study.

Our results, published in Biotropica, may make you rethink your own field shirt color choices. Both our sighting rates and our capture rates of water anoles were significantly higher when wearing orange shirts – matching the color of water anole dewlaps. Lizards likely have sensory biases for colors used in their species-specific displays, and we found that this translates into clear differences in behavioral responses to anthropogenic colors. On some level, I think many of us expected that the green shirts, which camouflaged us beautifully in the forest, would have been the most successful. Not so. Looking more broadly, Bree Putman’s previous study on western fence lizards (Sceloporus occidentalis) helps to solidify the idea that lizards are biased toward anthropogenic colors that they themselves “wear.” Western fence lizards have blue sexual signals and – corresponding nicely to our water anole results – these lizards are less fearful of researchers wearing blue clothing. Taken together, these findings remind us that placing a human perspective on animal perception can sometimes lead to flat out wrong conclusions.

As ecotourism is on the rise, it’s worthwhile to consider how the anthropogenic stimuli that ecotourists bring into a species’ native habitat can affect species behavior and survival. Reducing a species’ exposure to more frightening stimuli (such as unfamiliar colors) can reduce stress and disturbance. Something for the ecotourism community to consider as researchers continue to explore ways to minimize disturbance of natural populations, while promoting sustainable use of natural areas.

As for me, it looks like I’ll be buying quite a few orange shirts this year.

 

SICB 2020: Do Large Brown Anoles Get the Most Mating Opportunities?

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.

 

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