Updates on the Development of Anolis as a “Model Clade” of Integrative Analyses of Anatomical Evolution

Staging series page 1

The first plate from the Sanger et al. (2008) Anolis staging series.

Long time readers of this blog will likely remember the many posts I’ve made trumpeting the utility of anoles for integrative analysis of anatomical diversity, studies that gain perspective from disparate biological fields. The community has come a long way since we published the first staging series of anole embryology only nine years ago. To some this may be old news, but I still find this pace exciting and personally motivating. Decades of ecological and evolutionary studies have created a strong foundation upon which to build new insights about the molecular and developmental underpinnings of anatomical diversity. My lab’s questions boil down to trying to shed light on the developmental origins of adaptive anatomical variation. Otherwise stated, where did the requisite phenotypic variation arise from during the adaptive radiation of anoles. The inherently comparative nature of these studies led me to use anoles as a “model clade,” a group of species that provides the capacity to obtain evolutionary insights the way that “model species” have provided pure developmental biologists and geneticists the power to deduce insights in their areas.

One of the highest hurdles to the progression of Anolis as a model system has been long-term access to living embryos. Although comparative biology is a powerful approach for evolutionary studies, one of the hallmark lessons of modern Evo-devo is the need to experimentally validate the candidate molecular changes associated with anatomical evolution. If I hypothesize that Gene X underlies some phenotypic difference between two species, I must 1) show that it is expressed at the time when the difference arises and 2) somehow tweak the expression of Gene X at that time and in that tissue to show that the changes parallel those observed in nature. To do this you must have access to an embryo in culture, unencumbered from its opaque shell.

Over the past several years several people have been working on ways to gain access to lizard embryos. The first report of a culturing method was by Tschopp et al., who used lentivirus to trace cell migration into the genitalia and limbs. I have not personally been able to consistently replicate those conditions, especially for later embryos. Bonnie Kircher and I, however, recently published two relatively “simple” culturing protocols as part of a new book, Avian and Reptilian Developmental Biology. One of the challenges of earlier culturing attempts was bacterial and fungal growth. As a first step to combatting these invaders, we developed a protocol to sterilize the eggs, soaking the eggs in a weak bleach solution (yes, a literal bleach solution). From there we were off and running.

The first method we describe, following from advice from Raul Diaz, has worked on eggs a few days old to those that are nearly half way through their incubation period. Using a fine pair of scissors, we separate the outer opaque lays of the shell from the inner membranes that surround the embryo and yolk. This bag-of-embryo is then transferred to a small culture dish with a nutrient rich media and drugs to further combat bacterial and fungal contamination. This culturing system has worked well for up to ten days, roughly from the time the limbs are developing digits to the time that the limbs have visible scales on them. (Check out the video!) In principle, this method will allow better access to the embryo for viral injection or the application of small molecule inhibitors that disrupt particular signaling pathways.

Be warned, the second method is a little more Frankensteinian. Because the membranes cover the embryo, visualizing development remains difficult. To circumvent this problem, we developed a protocol where we explant a piece of anole tissue, such as the developing

A developing A. sagrei foot explanting onto a chicken embryo

A developing A. sagrei foot explanting onto a chicken embryo

limb, to a chicken embryo. Both anole and chicken seem to fare well at 33 degrees Celsius, below the standard incubation conditions of the chicken and above that of our anoles. Development appears to proceed normally in the explanted tissue, just as it does would in an embryo within its own shell. These experiments still have a relatively low success rate, but when the explant takes, it works well. To better visualize the tissue for imaging we also stained the tissue with a vital fluorescent dye before the transfer, giving the tissue a wonderful Halloween feel.

The work is far from over. These culturing protocols are just the first step and will not work for all applications. More technically challenging steps especially await those that want to manipulate the anole genome or target distinct patterns of gene expression. This is only the start of what’s to come. For more details about these protocols you can download the chapter here.

Knight Anoles Introduced to Another Island: Abaco, Bahamas

Photo by Joel Sartore

The knight anole is really getting around these days: Turks & Caicos, Grand Bahama, Grand Cayman and many other islands. Now they’ve  made it to Abaco, Bahamas, where one individual was captured and possibly two others seen (see article in IRCF Reptiles and Amphibians)Abaco Scientist has an insightful discussion of introduced reptiles and amphibians on Abaco.

Female Green Anole with Sand on Her Head–Been Egg-Laying?

Photo-chronicler of Floridian natural history Karen Cusick has done it again. We’ve been captivated by her backyard photos before, but here’s photo of a female green anole with sand on its snout. Been digging holes to bury her eggs, maybe? And while Karen observed the little lady lizard, it suddenly darted into the bushed and emerged with a meal!

 

Festive Anole Invades British Columbia!

The plant in question

Well, at least one A. sagrei did. Gavin Hanke’s, Curator of Vertebrate Zoology at the Royal BC Museum in Victoria, BC, reported on the arrival of one stowing away in a tropical plant. Anoles do seem to have a knack of getting around in plants, fruit and other contrivances.

Anolis Lizards Have Their Own Homing Device

Carolina Anole

Lizards are active creatures, often running around in new territories to explore and find food. Sometimes they encounter challenges that keep them from running too far. When they wander away from home, how do they get back? It’s a question that’s led researchers to study this topic.

After watching the daily routines of Anolis lizards by using tracking devices placed on their backs, researcher Manuel Leal learned that they return to the same home again and again. This established the next question which was to find out how the lizards knew how to get back. Birds have a similar ability to find their way home. Although the exact method has not been discovered, it’s possible that lizards have similar abilities and functioning as birds in finding their own again.

They Claim Their Homes

Anolis lizards, especially males, claim trees as home territories, fighting to keep any newcomers off their bit of land. They’ve proven that they remember exactly where they stake out their claim, and like all animals, they like structure in their environment, including the location of where they spend their days and nights. Some studies prove that after disorienting the lizards and placing them a far distance from their home, they can still find their way back within 24 hours.

Then They Listen

U.S.  Geological Society geologist John Hagstrum proposed that in order to get back home, pigeons use sounds wave; extensive studies on pigeons show that they use low-frequency sound waves to create an acoustic map of where they are. This way, they can identify predators and safe spaces to land. Some have wondered whether  Anolis lizards might have similar capabilities that are advantageous for homing.

It’s hard to argue that lizards use any other method to get where they came from. Tests have proven shown results that indicate that lizards don’t have the ability to use any magnetic senses or distinguish polarizing light. Still, they manage to baffle scientists who wonder at their complex societies and developed capabilities.

If you want to learn a little more on the topic of how Anolis lizards find their way home, you can see a short film done by Days Edge Productions that follows Leal as he conducts his study.

An Update on Taking Toepad Pictures

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I’ve taken more than four hundred toepad pictures using the new macro photography technique I introduced  in an earlier post and I’ve learned a few tricks that I want to share in this update.

First and foremost, I highly recommend this approach. For those of you looking to capture a lot of toepad data, particularly in the field, this kit is way faster and more portable than using a flatbed scanner and the images I’m getting are at least as sharp.

forefoot

A few tips:

  • Petri dishes work great as a clear platform to place the lizard feet on. I found that the 60 mm diameter dishes were much easier to balance atop the lens (~40 mm in diameter) than the larger dishes I’d originally shown.
  • I cut and taped a scale bar to one edge of the petri dish so I wouldn’t have to worry about juggling a lizard and a tape measure.
  • Make sure you have several petri dishes – they scratch fast – and keep some ethanol and a kimwipe close at hand.

IMG_6314

  • The app that lets you remotely trigger your iPhone is absolutely maddening. Do not download it. I’m not even going to relink the name. Instead, I suggest a much more stable alternative: connect your phone to your computer with the USB cable, open QuickTime Player, select File > New Movie Recording and click the down arrow next to the record button. This will give you the option to select your attached iPhone as a recording device. This live-view is far more stable and less frustrating. *Windows and android users I’m afraid I haven’t had an opportunity to sort out a solution for those platforms. If you know of something that works, please include in the comments!

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Unfortunately, through the live view all you can see is whether the lizard is in position. You cannot remotely trigger the shutter this way. That means you’ll need a second pair of hands to help. I found it worked best when my partner was in charge of putting the ID tag in the frame after I’d placed the lizard foot and then pushing the volume button on the side of the phone to trigger the camera shutter.

  • Lighting is really important. I suggested a headlamp in the previous post providing an oblique light source through the diffuser around the lens. I tried using a microscope fiber optic light source but I was really unhappy with the “warmth” of the light. I found that the white-LEDs in my headlamp produced a much more realistic looking image (see above). Also, make sure you don’t have any light sources above/behind the subject. Backlighting confuses the camera’s auto-contrasting and results in dark and sometimes unfocused images.

red dewlap

Florida Greens and the Suprascapular Spot

Miami-Dade county, Florida; 18 March 2017

Miami-Dade county, Florida; 18 March 2017

After scampering about much of North America the past few decades, I once again live  in my hometown of Ormond Beach, Florida — on the northern edge of Volusia county. When I was a kid, back in the late 70s and early 80s, I spent much of my time tangling with and studying our local anoles. The Carolina greens (A. carolinensis) were dominant back then, covering our walls, windows, trees, and (sometimes by forced measure) our ear lobes. Every now and then I’d find a Cuban brown (A. sagrei) — usually around the shopping centers and strip malls. Nowadays, of course, that coin has flipped. The Carolina greens have moved back up into the higher foliage and the Cuban browns dominate our shrubs, walls, and windows.

I remember actually finding a Cuban brown anole on our property in 1984 or so. I was in 4th grade, drunk on Star Wars and lizards. I managed to catch the little non-native lizard and put it in my anole terrarium (a homemade wood-and-open-screen enclosure my dad and I built). I was in the habit of catching anoles (and the occasional snake), keeping and watching them for a day or two, and then releasing them back into the yard. Needless to say, the Carolina green already in the enclosure wasn’t too thrilled with his new roommate. Though guilt eventually kicked in the following day, I admit I was somewhat delighted by the defensive/discomfort color play of that poor Carolina green. Usually, they’d be cool, smooth emerald green with very little patterning… but distressed or riled up Carolina greens certainly know how to put on a good color and pattern show.

Soon enough, I released the Carolina green back into the yard and kept the Cuban brown for another day or two. This little moment of tension, however, leads me to the point of this post: the distress patterns of our local Carolina green anoles. More specifically, I’m interested in the presence of a supraspacular dark spot that shows up with some individuals. It’s a dark spot with light trim that sometimes appears just above and behind the front shoulder line — as seen in this particularly ornate individual photographed in Miami-Dade county on 18 March 2017:

This Miami-Dade individual really stuck out to me. It’s patterning was distinct. It was quite large. It had that supraspacular spot. Most notably, it was still wielding quite a bit of green. Could this be A. porcatus? Like many naturalist-lizard enthusiasts, I tend to catch myself up in the eternal cycle of porcatus-or-not? when I’m in south Florida. Heh. Nowadays,  my assumptions generally fall on the side of A. carolinensis unless I’m with somebody more in-the-know who can tell me differently with confidence; this hasn’t happened yet. Honestly, I have a hard time seeing a clear difference between the two. I’m glad I’m not alone.

Though distinct, this fabulously mottled Green wasn’t the only Green I’ve photographed with that supraspacular spot. Here’s an impressive male tangling with a Cuban brown anole in the Lower Keys of Monroe county, Florida, on 08 June 2007:

Further north, in my home territory, I’ve only noticed and photographed two individuals with that spot, albeit with less figure-ground contrast between the spot and the trim.

Orange county, Florida (05 September 2011):

Anolis carolinensis, 05 September 2011

Alachua county, Florida (05 December 2011):

Anolis carolinensis, 05 December 2011

Both were in WTF-dark-mode (as I call it).

Of note, I spent a few years in Valdosta, Georgia, intensely watching anoles.

Condition Dependence of Shared Traits Differs between Sympatric Anolis Lizards

A male slender anole (Anolis limifrons)

A male slender anole (Anolis limifrons)

A walk through a tropical rainforest can reveal astonishing forms and colors of organisms – from vibrant poison frogs and coral snakes to the vegetative camouflage of stick insects and other cryptic creatures. Perhaps some of the most dramatic displays of variation can occur between the sexes, where males and females can differ so greatly in appearance that they resemble different species. Research in many systems has demonstrated that much of this variation is driven by sexual selection, the force responsible for the evolution of traits that are important for acquiring mates. Individuals may invest as much energy as possible into such sexually selected traits because doing so will give them a competitive advantage for mate acquisition. These traits are therefore considered condition dependent, as their expression is dependent upon the energetic condition of the individual that possesses them. While condition dependence has been the subject of many studies, it is not well known how it may vary between closely related species that share the same traits. If closely related species vary in condition dependence of their shared traits, then this implies that condition dependence could be important for the evolutionary diversity of sexually selected traits.

The rainforest at the La Selva Biological Station in Costa Rica

The lowland rainforest at the La Selva Biological Station in Costa Rica

Together with students from Grinnell College and Reed College, and as part of an OTS (Organization for Tropical Studies) course that I took as an undergraduate at the University of Virginia, we took to the lowland jungles of Costa Rica to answer this question. We studied two anole species from Costa Rica, the slender anole (Anolis limifrons) and the ground anole (Anolis humilis). Specifically, we tested whether several traits that they had in common exhibited condition dependence, including dewlap size, aspects of jaw morphology, and sprinting speed. To test for condition dependence, we first calculated two conventional indices of body condition, the residual index and the scaled mass index, which both take into account an organism’s mass, given its length. We then obtained residuals from the relationship between our variables of interest (dewlap size, jaw width, jaw length, and sprint speed) and snout-vent length (a measure of body length), which allowed us to control for the fact that trait sizes often scale with the overall size of an animal. Finally, we used bivariate linear regressions to test the effect of our indices of body condition on our residual traits of interest, with a significant positive relationship suggesting condition dependence. We found that dewlap size (a trait important for sexual signaling) and jaw width (a trait important for bite force and male combat) exhibited condition dependence in ground anoles, but not in slender anoles. In contrast, neither sprint speed nor jaw length were condition-dependent in either species. Importantly, the presence of condition dependence in one species, but not the other, implies that the condition dependence of shared traits is evolutionarily labile. Additionally, by detecting condition dependence in the dewlap of ground anoles, which have a larger dewlap given their body length when compared to slender anoles, our findings may indicate that the strength of sexual selection differs between these two species. Lastly, our research suggests that variation in condition dependence of the dewlap among species could contribute to the extraordinary diversity of dewlaps in the Anolis genus.

If you would like to read the full paper, published in the Journal of Experimental Zoology Part A, go to:http://onlinelibrary.wiley.com/doi/10.1002/jez.2076/epdf

Anole Ecomorph Watches 50%–Today Only!

Note: the watch on the bottom right is not one of ours!

Note: the watch on the bottom right is not one of ours!

It’s that time again. For one day only, Zazzle.com is offering 50% off the Ecomorph line of watches. Sale Code:

COOLZAZSTYLE

And we’re open to suggestions for new species to feature on a lovely wrist fob. Suggest away!

Signals and Speciation: Do Dewlap Color Differences Predict Genetic Differences?

Dewlap and genetic differences between co-occurring Anolis distichus and A. brevirostris

Dewlap and genetic differences between Anolis distichus and A. brevirostris at sites where they co-occur on Hispaniola.

Here at Anole Annals, we’re all familiar with the replicated evolution of different anole ecomorph types in the Greater Antilles. However, divergence into these different ecomorph classes is not enough to explain how the group became so speciose on these islands. Additional factors must therefore have promoted speciation throughout the history of the group.

One potential factor is the flashy anole dewlap. Dewlap diversification across anoles has led to the remarkable array of dewlap color, pattern and size we see today. If dewlap differences did indeed drive speciation in anoles, or are involved with the maintenance of species boundaries, we might expect that as differences in dewlap color and pattern increases between species, genetic differentiation will also increase through fewer hybridization events.

In our study that just came out in the Journal of Herpetology, Rich Glor, Anthony Geneva, Sabina Noll and I set out to test this using two widespread species from the Anolis distichus species complex, A. distichus and A. brevirostris. These two species co-occur in many locations on Hispaniola and, while they often differ in dewlap color where they do co-occur (yellow with an orange patch vs. all pale yellow), in other areas, they co-occur with similarly pale dewlaps. Using mitochondrial DNA, microsatellite and AFLP data, we investigated patterns of genetic differentiation at four sites: two where the species differ in dewlap color, one where the species share the same dewlap color, and another where pale dewlapped A. brevirostris co-occurs with two A. distichus subspecies (one with a similarly pale dewlap and the other with an orange dewlap).

In general, we found that A. distichus and A. brevirostris looked like “good species,” with strong genetic differentiation and little evidence of hybridization, even at a site where they share the same dewlap color. This suggests that dewlap color differences are not associated with genetic differentiation in a manner one might expect if dewlaps were involved in the speciation process or in maintaining species boundaries. However, at the site where A. brevirostris co-occurs with two A. distichus subspecies with both similar and dissimilar dewlap colors, we found some evidence of hybridization and the species were not as highly genetically differentiated. This discrepancy suggests that site-specific factors could be influencing the dewlap’s role in speciation or maintaining species boundaries. For example, as Leo Fleishman’s and Manuel Leal’s work has shown (e.g. 1, 23), the dewlap’s effectiveness as a signal is dependent on the light environment. Further understanding about the environmental differences among our study sites, how species utilize the available light microhabitats within each site, and how the dewlap looks to anoles at each site could provide more insight into our findings.

On the other hand, perhaps we need to be looking beyond the dewlap and focusing instead on whole signaling displays. Anole behavioral displays can also be strikingly different among species (e.g. 1) and may instead be the key to understanding species diversification in Greater Antillean anoles.

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