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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.

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).

 

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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.

 

SICB 2020: Oviposition Site Choice in the Brown Anole, Anolis sagrei

Abigail Dennis at SICB 2020

Embryonic environment is arguably one of the most influential factors on offspring development and later-life phenotypes. For oviparous species, this critical stage can experience potential fluctuations in moisture, temperature, and oxygen-availability. However, maternal choice in nest-site has the opportunity to buffer embryos from environments that might negatively affect survival or disadvantageous phenotypes. Undergraduate student Abigail Dennis of Trinity University in San Antonio, TX in Dr. Michelle Johnson’s lab, sought to investigate maternal nest-site choice when mothers are given nesting conditions that have been shown to be unfavorable to offspring development. 

To address this, Abigail housed female brown anoles (Anolis sagrei) in groups of 2-3 with 1 males per cage. Within each cage, females were given an option to nest in a heated box or an unheated box (ambient box). The heated nesting box was placed over a thermostat-controlled mat and both boxes were checked for eggs every 2-3 days. When an egg was found, depth and water proportion in surrounding soil were recorded. She predicted that females would avoid the deeper, warmer nesting conditions in the heated box and that nesting depths would be more variable in the ambient box. Thermal readings from the surface and base of the soil were recorded for each box. These temperatures were averaged in the ambient boxes and coupled with depth and temperature models for the heated boxes. Thermal conditions varied from 25.5 to 38 °C, although most nesting sites were found between 26.5 and 31 °C. 

At SICB’s poster session on Monday, Abigail reported that there was no difference between the number of nest sites (N=36) found in heated or ambient boxes. However, females tended to avoid nesting in sites greater than 33 °C and there was a trend suggesting nests in the ambient box were deeper than those in the heated box. Soil moisture readings also did not differ between nest boxes. Abigail speculated that if global change increases surface soil temperatures, females may avoid higher temperatures that would negatively influence offspring development by altering their nest depth. Abigail is writing this work as part of her Senior Thesis and is interested in pursuing graduate work in the evolution of development. 

SICB 2020: Sex-biased Parasitism and the Expression of a Sexual Signal in a Tropical Forest Lizard

Panamanian slender anole (Anolis apletophallus) (Photo Credit: Dr. Christian Cox)

Male sexual signals, and their often-associated distinct phenotypic and behavioral displays, have been hypothesized to have evolved from multiple sources. Two of which include the Good Genes hypothesis, which suggests sexual signals serve as an honest signal to potential mates, and the Immunocompetence Handicap hypothesis, which indicates trade-offs to elaborate signals. However, Dr. Christian Cox, an assistant professor in the Biology Department at Florida International University, thinks of these as a continuum rather than opposing hypotheses. The vibrant dewlaps of Anolis species serve as an excellent model system to address questions related to this continuum. Dr. Cox’s lab has documented sex-biased parasitism, which he discussed on Monday at SICB 2020. 

Dr. Christian Cox

The Panamanian slender anole (Anolis apletophallus) is known to host ectoparasitic trombiculid mites (also known as chigger mites). In this species, males and females are roughly the same size and, as with most anole species, males carry a large, colorful dewlap beneath their chins. Dr. Cox asked whether males and females of this species differ in the attachment site and intensity of mite infestations and whether any other factors (e.g., energetics) might influence infection. Using a combination of field and laboratory studies, Dr. Cox and his colleagues quantified the number of mites, prevalence, and intensity of infestation on individuals. He also measured mass, snout-vent length, and dewlap size. Following this, he collected fat bodies, livers, and gonads to investigate differences in energetics. 

Dr. Cox found that males were more likely to have mites on their dewlap whereas females had more mites in the inguinal and axillary regions. Additionally, large males had more mites than smaller ones and there was a significant correlation between the size of the dewlap and the number of mites. Dr. Cox also found that there was a negative relationship between fat body mass and the total number of mites. In other words, males with a heavier fat body had less mites. In females, there was a positive relationship between gonad size and the number of mites. These findings suggest sex-dependent factors influence ectoparasite load and are indicative of trade-offs to male sexual signals. 

SICB 2020: Arginine Vasotocin Stimulates Chemical Communication and Social Behavior in Anolis carolinensis

Dr. Stephanie Campos presenting her research at SICB 2020

A captive green anole (Anolis carolinensis) (Photo Credit: Dr. Stephanie Campos)

Exocrine signals (e.g., pheromones) and endocrine signals, like those associated with chemosensory organs, stimulate communication among and within species. Dr. Stephanie Campos is especially interested in the endocrine signals of reptiles and highlighted some of her work at SICB 2020. 

Dr. Campos is a postdoctoral research fellow at Georgia State University Center for Behavioral Neuroscience and Neuroscience Institute in Dr. Walt Wilczynski’s lab. She investigated the role of arginine vasotocin (AVT), a modulator of social interaction similar to the mammalian homologue vasopressin, on reptilian chemosensory systems. Previous work in green anoles (Anolis carolinensis) showed that AVT reduces aggressive visual display rates and stimulates females to display more than untreated counterparts.

In this study, Dr. Campos and colleagues tested the role of chemosensory systems in A. carolinensis by injecting resident males with AVT and introducing a male or female intruder. Ten minutes after injecting with AVT or a control solution, an untreated male or female was placed in the tank for thirty minutes. Chemical behavior, including tongue flick, jaw/chin rub, lip smack, lick, fecal/urine deposit, or cloacal rub were recorded.

They found that male intruders displayed more of these chemical behaviors toward individuals treated with AVT than controls. Additionally, males treated with AVT displayed quicker (e.g., tongue-flicking) to female intruders than controls. Dr. Campos speculates that AVT might boost production of odor cues or interior mediated endocrine-mechanisms. This might serve as an indication for multimodal communication in reptiles.

You can learn more about Dr. Campos’ research by following her on Twitter

SICB 2020: Acute Interactions between Green and Brown Anoles

Jordan Bush giving her talk on the interaction between green and brown anoles at SICB 2020

Green and brown anole interacting within Jordan’s enclosures.

As brown anoles (Anolis sagrei) become more and more abundant, many people (trained and citizen scientists alike) are intrigued with exactly how the native green anole (A. carolinensis) will respond. Newspaper articles still report on these interspecific interactions, and some recent research has shown the brown anoles can be quite mean to the native green. Thankfully, it seems that the green anole may simply be moving higher into the canopy and aren’t being merely driven to extinction by the invading brown. However, we do not yet understand the nuances of how green anoles respond when brown anoles first arrive to a new location, and that’s where Jordan Bush, a sixth year PhD student in Dan Simberloff’s lab at the University of Tennessee, Knoxville, comes in.

To understand how green anoles immediately react to the novel presence of brown anoles, Jordan built 5 x 5 x 5m enclosures in which she placed 6 female and 6 male green anoles. These animals then set up territories and became acclimated to their new living space. Jordan quantified baseline behavior and territory sizes (in 3D!!!) for each individual in an enclosure.

Example 3D territories

After 10 days, Jordan introduced brown anoles in these enclosures, either two females and two males or four females and four males to investigate the effects of density, and quantified behavioral and territorial changes in the green anole. Being the careful researcher that she is, she also introduced the same number of green anoles to other enclosures so that she could show that any changes in behavior were not simply due to more animals being present. After 10 days of interacting with the brown anole, Jordan found no change in activity level, home range volume, or perch height, suggesting that, at least within an acute time frame, the green anole can handle its own against the brown anole.

Page 30 of 145

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