All posts by Thomas Sanger

About Thomas Sanger

Thom Sanger is an Assistant Professor at Loyola University in Chicago. His lab specializes on understanding the developmental bases of Anolis lizard diversity.

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.

A Call, No A Plea, For Anole Eggs and Hatchlings

One of the long-faced members of the carolinensis clade, Anolis brunneus.

One of the long-faced members of the carolinensis clade, Anolis brunneus.

Several years ago, I wrote a series of papers and blog posts about the diversity of anole head shape and its developmental origins. My colleagues and I touched on disparate topics such as whether the head differences among species are similar to post cranial ecomorphology, whether the patterns of cranial modularity are conserved across anoles, and the developmental bases of sexual dimorphism in skull shape.

Since starting my own lab at Loyola University in Chicago last year, I am revisiting these projects on skull evolution. Like in much of science, I have found that my early forays into this area created more questions than answers. Understanding the diversity of skull shape among anoles and other iguanid lizards will be one of the first focal areas of my new lab. We are currently mining museum collections to understand how the variation in anole skulls compares to iguanid lizards more broadly. The ultimate goal, however, is to return to questions about the developmental origins of this variation. Just how many different ways has development been modified to generate all the variation we observe in adult anatomy? We do not yet know.

This is where my attention turns to you. To thoroughly flesh out the developmental origins of anatomical diversity, I must have robust sampling of species across the iguanid phylogeny. I am asking the community to please think of me and my students if you have extra breeding animals, eggs, or hatchlings of any species of anole or another iguanid lizard. I am happy to help offset the cost of the animals or collaborate in a mutually beneficial manner.

One of the most exciting species that have recently had the fortune to work with is Anolis hendersoni. For its body size, this species has one of the longest faces of all anoles. In this case I was contacted by the owner of Backwater Reptiles who had several A. hendersoni adults that we are hoping to get eggs from over the next year at Loyola. The folks at Backwater have been great to discuss “exotic” anoles with as they occasionally receive species like A. woodi, A. cybotes, and Chamaeleolis, all of which could be great additions to my project. This is just one example of how I am trying to broaden the sampling for this project. I ask you, the broader anole community, to help me increase my sampling further. I sincerely thank anyone that has leads for me in advance.

Teaching Kids How To Dewlap

Anolis lizards have established their place in the annals of college textbooks. There are also a growing number of resources available for elementary and high school teachers to bring the biology of anoles into their classrooms as well. The Howard Hughes Medical Institute (in collaboration with Jonathan Losos) developed several online modules around anoles: one on the diversity of Anolis lizards, another on speciation, and a virtual lab integrating those topics. Michele Johnson also has several classroom exercises on here website, LizardsandFriends.org, some of which have been discussed on AA previously (here and here). I am writing today to share another exercise with our readers that was a recent success with a group of young scientists-to-be.

Dewlapping fifth graders at GEMS 2016

Dewlapping fifth graders at GEMS 2016

I recently introduced Anolis lizards to a group of fifth and sixth grade students at a conference aimed at getting young girls interested in the STEM professions. With around 130 girls learning about topics ranging from gemstones, programming, seeds, and urban wildlife the event was a undeniable success. My session introduced the diversity of topics that our community addresses with Anolis lizards. After explaining to students how they could figure out what lizards are anoles at the local pet stores (dewlaps and toepads), I used anoles to demonstrate how animals can communicate without talking. My exercise amounts to a game of charades where the students have a dewlap, a display-action-pattern, and a key representing four species from Puerto Rico (thanks to Travis Ingram). The display patterns are not as complex as real dewlap displays, but were made to allow the students to easily act them out and distinguish between the patterns and it worked great. The kids thought this was a lot of fun and it gave me the opportunity to pepper the discussion with additional comments about animal communication. I originally designed the exercise for fourth through seventh graders, but a curious three-year-old played along just as well during one session. I would be happy if other people used this exercise for their own outreach activities. It can be downloaded here.

In closing I will add that the students were impressed by the brown anole I brought with me. I imagine I would have left a more lasting impression if I brought a knight anole. Things to remember for next year.

On the Origin and Diversification of the (Hemi)Penis: Anolis Takes Center Stage

Over the last decade the term “model species” has taken on new meaning. Species that were once the building blocks for distinct disciplines have taken on new importance in comparative evolutionary studies that integrate perspectives across biological disciplines. Nowhere is this better illustrated than with Anolis lizards. For decades anoles were a workhorse of ecologists and evolutionary biologists, but have, more recently, been embraced by developmental biologists, genomicists, physiologists, and neurobiologists among others. This disciplinary expansion is perhaps most evident with the rapid increase of penis/hemipenis research that has been published using anoles within the year.

For many herpetologists, including those focused on anoles, the hemipenis is ripe with taxonomic characters, easily allowing for the identification of new species. Julia Klaczko and colleagues recently demonstrated that features of the hemipenis are some of the most rapidly evolving characters among anoles, a group already well known for its rapid anatomical evolution. Independent from these taxon-specific interests, developmental biologists became interested in the anole hemipenis because of its unique anatomy compared to other amniotes. Marissa Gredler and members of the Cohn Lab used anoles as one of their reptilian models of external genital development in what is arguably the broadest embryological survey of reptilian phallus development to date. In parallel, Patrick Tschopp and colleagues probed the cellular and molecular regulation of early phallus development among anoles, snakes, chickens and mice, demonstrating that the hemiphalluses (hemipenes and hemiclitores) and hindlimbs of squamates utilize similar molecular networks at the earliest embryonic stages of morphogenesis. Now, just within the last month, two more papers have used anoles in studies of phallus evolution and development, one using cutting-edge molecular techniques to better understand the relationship between limbs and external genitalia and the other addressing the fundamental question of external genital homology using museum specimens that are more than 100 years old.

Before getting into the findings of this new research, lets lay out some of the dirty details of penis evolution. First and foremost, the penises of amniotes are extremely diverse. Squamates have paired lateral phalluses while other clades have a single midline phallus. Each of the amniote lineages uses hydrostatic pressure to achieve an erection, yet accomplish this using different bodily fluids (lymph or blood). In mammals sperm is transferred to the female through a closed urethral tube, but other groups utilize an open channel. Most birds (97%) and the tuatara, have absent or highly reduced phalluses and reproduce with the famed “cloacal kiss.” These large differences in anatomy should not overshadow the spines, bulges, corkscrews, and dramatic differences in size that give species their distinctive features. But with such striking variation, we are forced to wonder how many times the penis evolved. Perhaps the amniote ancestor possessed an intromittent phallus capable to transferring sperm to the female that later diversified in each lineage independently. Or, perhaps the last common amniote ancestor used cloacal apposition to foster internal fertilization and unique phallus morphologies evolved independently at the origin of each lineage. Because adult anatomy provides few clues to phallus homology, Thom Sanger (me), Marissa Gredler, and Marty Cohn looked towards the embryo for help.

Table 1 from Sanger et al. 2015 summarizing phallus variation in amniotes

Table 1 from Sanger et al. 2015 summarizing phallus variation in amniotes

The tuatara, a species lacking an adult phallus, has presented a problem in attempts to reconstruct the last common ancestor of amniotes because it raises the distinct possibility that reproduction through cloacal apposition was the ancestral condition. Continue reading On the Origin and Diversification of the (Hemi)Penis: Anolis Takes Center Stage

When Does Sexual Dimorphism Arise in Crown-Giant Anoles?

Size dimorphism among Anolis habitat specialists from Butler et al. 2003

In recent years the Anolis community has shown greater interest in understanding the developmental bases of anole diversity. As these data accumulate, we can start to synthetically understand the ecological (ultimate) and developmental (proximate) factors that regulated different aspects of anatomical diversification in anoles. Differences between males and females (i.e., sexual dimorphism) is one area that has received a considerable amount of attention among anole biologists interested in obtaining this integrative understanding of anatomical diversity. Over the last decade a number of papers have been published examining the evolutionary patterns of anatomical differences, ecological correlations, and the developmental/physiological processes underlying dimorphism in anoles.

Variation in body proportion (i.e., shape dimorphism) among Anolis habitat specialists from Butler et al 2003.

Compared to other anoles, crown-giant anoles have relatively low levels of size dimorphism, but vary greatly in body proportion. Males and females tend to vary in relative limb length, head proportions, and in the dimensions of their adhesive toe pads. In a recently published paper Vanhooydonck et al. examine the timing of divergence between male and female A. baracoae. They raised 23 individuals (9 males, 14 females) for 3.5 years, repeatedly measuring three anatomical traits and bite force 11 times over this time period. The authors found that bite force and dewlap size exhibit significant differences in growth between the sexes. Their analysis further suggests that these traits diverge at different times during ontogeny – bite force diverges during juvenile growth while dewlap size does not diverge until sexual maturity – illustrating the independent regulation of dimorphic traits during development. Head length and hindlimb length did not appear to have sexual differences, although it would be interesting to also perform a formal analysis of adult size and shape dimorphism on A. baracoae to see if this species has similar dimorphic trends compared to other crown-giant anoles.

The developmental bases of size dimorphism in A. sagrei from Cox et al. 2009

These results are consistent with other studies that show a mosaic pattern of male and female phenotypic divergence. Using a longitudinal study of male and female A. sagrei, Cox et al. 2009 showed us that body size dimorphism in this species begins early in juvenile life, only three weeks after hatching. Sanger et al. showed that dimorphism in facial length can emerge through two distinct developmental strategies, one early in ontogeny and one at the time of sexual maturity that appears to be clade-specific. Additional research on that compares across traits and among species will further elucidate the number of ways that dimorphism can arise in anoles. Further work that overlays an ecological perspective onto these patterns will also allow a more thorough understanding of whether natural or sexual selection is the primary driver of these differences in timing. As this future work progresses new insights into the evolutionary processes of anatomical diversification are sure to follow.

Green Anole Window Decals

We once discussed what the appropriate term would be for a group of anoles, but what about a family of anoles? Is there such a thing? If not, there is now.

Just in case stick figures aren’t an appropriate representation for your family group, look no further than the green anole window decals made personally by herp. lover Andrew. These aren’t yet available on the web, but Andrew may knock a few out for you if you write him directly. I did and now I have one of the most stylish minivans in Florida!Anole family

SICB 2015: Does Dorsal Crest Erection Arise from an Inflammatory Response?

Morgan Gerace and her SICB 2015 poster on dorsal crest erection in A. sagrei

Morgan Gerace and her SICB 2015 poster on dorsal crest erection in Anolis sagrei

We perviously learned about new research on the mechanisms of dorsal crest erection in the brown anole, Anolis sagrei, being done by the Rand Lab at Carleton College. This is a interesting new research area with relevance to our understanding of anole signaling and anatomy that is being carried out by Rand and a team of impressive undergraduate students. Rand Lab student, Morgan Gerace, presented a second Rand Lab poster on this topic at SICB 2015. Following up on the first poster, in which the authors found no evidence of the involvement of muscles, cartilage, or vascular sinus in crest erection, Morgan tested whether crest erection is due to an inflammatory response. By injecting male anoles with the an adernegenic receptor  antagonist, interfering with the lizard’s fight-or-flight response, Morgan determined that crest erection may be the result of an inflammatory-like response. Conversely, injection of with epinephrine, essentially supercharging the fight-or-flight response, facilitated a crest erection response. This exciting work by this set of motivated undergraduates draws new attention to the physiological control mechanisms of this under-studied display mechanism.

Dorsal crest of Anolis sagrei

Dorsal crest erection in  Anolis sagrei

SICB 2015: Transcriptomic Analysis of Anole Growth Mechanisms

Photograph of a male Anolis sagrei from Christian Cox’s website.

Squamates vary widely in the magnitude and direction of body size dimorphism, which refers to the tendency for the sexes to exhibit different body sizes. Some lineages possess male-baised dimorphism while others have female-baised. The effects of testosterone on mediating sexual size dimorphism in different squamate lineages has long been the study of the Cox lab at the University of Virginia.  Christian Cox (of no relation to his advisor) has now reported some exciting steps forward in the search for the mechanisms regulating body size dimorphism in the brown anole, Anolis sagrei. Cox is in the process of carrying out a  transcriptome-wide analysis of the genes responsible for sexual dimorphism, with particular focus on examining the genes along the insulin growth factor-growth hormone axis (IGH-GH), which is the same pathway that was reported about yesterday.  In his experiment Cox implanted testosterone pellets under the skin of juvenile male and female lizards and then looked for differences in size and gene expression. Increased levels of circulating testosterone prompted increases in body size in both males and females grew to larger sizes, indicating that females have not lost the ability the respond to testosterone. But to better understand the growth axis controlling this difference Cox took a large step forward by also comparing gene expression in the liver of experimental (implant) and control (intact) animals. As the liver is a major regulator of growth via its regulation of the IGH-GH, Cox expected that this tissue would respond to testosterone treatment. This is precisely what Cox found. Specifically, he found a number of genes that are naturally regulated in different ways in males and females and additional genes that responded to the testosterone treatments. To conclude, Cox pointed out that an important next step will be to compare castrated lizards to those intact lizards with the testosterone implant to more clearly elucidate the gene network directly responding to testosterone. But perhaps the most exciting work will come with Cox and his collaborators examining the growth mechanisms of species with male-baised and female-baised patterns of dimorphism to more thoroughly understand how evolution has reshaped these gene regulatory networks during squamate evolution.

 

SICB 2015: How Do Anoles Get Big?

photo from;http://www.saumfinger.de/anolis_equestris.html photo by:Uwe Bartlet ?

One of the largest anoles, Anolis equestris (photo by Uwe Bartlett)

From the diminutive twig anole to the monstrous crown-griant anoles, Anolis lizards vary dramatically in their body size. Much research has focused on the patterns of body size variation among Caribbean species, how changes in body size are correlated with habitat differences among species, and rates of body size evolution upon invasion to new islands, yet an important question remains to be addressed in this body of literature, “how do anoles change body size?” S. Griffis and Dr. D. Jennings of Southern Illinois University at Edwardville are attempting to address this among Cuban anoles by searching for DNA sequence differences in known growth factories. But they are using what might be considered an unlikely model for lizard body size variation: dogs. Several years ago, Elaine Ostrander’s lab at the NIH uncovered that coding differences in the growth factors IGF were responsible for the body size variation in dogs. To a mechanist like myself, it was a surprise that this variation could be traced to coding differences in the genes, not to the levels of circulating growth factors. The authors of this poster are following Ostrander’s lead by looking for coding differences in genes involved with the IGF growth axis. But to keep their options open they are also collecting data on circulating hormone levels. When complete, if there are differences in the IGF growth axis contributing to differences in body size, Griffis and Jennings will find it.

SICB 2015: A Synthesis of Sexual Selection and Life History Perspectives

Anolis sagrei mating. Taken from the Cox lab website.

Anolis sagrei mating. Taken from the Cox lab website.

On the first day of SICB 2015 Robert Cox gave an interesting talk about reproductive investment and sexual selection in lizards. At the center of his talk was the striking notion that males and females are different biologically, yet should still be integrated into cohesive theories of sexual selection. According to Dr. Cox, past theory has generated mutually exclusive ideas about the costs of reproduction for each sex. Whereas theories about females have focused on life history and investment in the egg and offspring, theories about males have focused on mating investment. Cox stressed that this is overly simplified and doesn’t reflect biological reality,  as males and females also share many of the same costs of reproduction as well. Issues like growth, survivorship, energy storage, and parasite load are shared between the sexes. Dr. Cox is now trying to test how sex-specific reproductive mechanisms affect these shared reproductive constraints by surgically removing the gonads of each sex. Preliminary analyses show that parasite load appears to be a shared effect among the sexes regardless of the underlying mechanism (testosterone derived from testes versus estrogen derived from the ovaries). Studies directly comparing the underlying mechanisms of sexual dimorphic anatomy, physiology, and behavior are critical for the further development of sexual selection theory and for improving our understanding of anoles. Studies like Dr. Cox’s are an important step in that direction.

 

SICB 2015: How Do Lizards Move in Nature?

Jerry HusakHow do lizards move in nature? Note the added emphasis on “in nature.” For many years people have studied the mechanics and patterns of of lizard movement and anoles have played an important role in this research. But today Jerry Husak of the University of St. Thomas in St. Paul reminded us that most of this research has focused on characterizing maximum performance ability, despite the fact that  animals rarely achieve this level of activity in nature. For example, most of the time many lizards are merely scurrying about on the ground and not sprinting at their full ability. Hence, although measuring maximal spring speed in the lab is a common theme, this measurement may not actually reflect what animals do in nature. Dr. Husak also stressed to the audience that animal locomotion is context dependent. Specifically, a lizard’s speed depends on whether it is moving in grass or over rocks, and whether it is foraging or fleeing from a predator. During his enlightening discussion, which included a description of him trying to sprint on a frozen Minnesota sidewalk, Dr. Husak described a series of biotic and abiotic factors that should be incorporated into models of terrestrial lizard movement.  Finally, he concluded by challenging our obsession with maximum sprint speed once again by asking whether running at top speed can lead animals to make to costly mistakes. Based on a set of foraging data, he showed that this may be the case.  Dr. Husak’s talk highlighted the importance of understanding the natural habits of lizard behavior and performance. 

SICB 2015: Convergence in Body Shape among Squamates

P. Bergmann

Patterns of Convergence in the Body Shape of Squamate Reptiles

SICB 2015 is off and running and what better way to kick it of than with a lizard talk? Phillip Bergmann of Clark University filled the 8:15 time slot on day one with an intriguing evaluation of broad-scale body shape convergence among squamates. This is a perennial topic on Anole Annals due to the well-studied patterns of convergence among Anolis lizards and, indeed, Dr. Bergmann highlighted anoles early in his talk. He asked whether common functional (ecological) situations lead to body shape convergence at large scales. Rather than search for global patterns of convergence, Dr. Bermann used hypotheses specific to the transformations that occur when lineages transitioned into new habitats. As he pointed out, it is not surprising to find convergence in body shape occurring throughout squamate – after all, convergence is ubiquitous across the tree of life. He concluded his talk highlighting what he feels are some of the most pressing “Big Questions” regarding convergence which included the methods we use to detect convergence, the role of constraints in shaping convergence, and elucidating the mechanisms underlying convergence. Ultimately it was a thought-provoking talk both from the perspective of squamate organismal diversity and the topic of convergence more broadly.

That Fascinating Phallus

I probably would have never said this a few years ago, but penises are absolutely fascinating. The phalluses of terrestrial vertebrates exhibit an incredible diversity of shapes and sizes with some possessing elaborate coils, barbs, bony spines, and multiple lobes. Many of us learn about the rapid evolution of sexual characters in our undergraduate classrooms, but until recently I, for one, did not fully appreciate the striking diversity of this organ until immersing myself in the subject area.

Many biologists study the penis under the umbrellas of different research disciplines, but relatively little work has been performed to explain its anatomical diversity. For example, how many times has a penis/phallus evolved among terrestrial vertebrates? This may seem like a trivial question, but the diversity in form, function, and physiology in the adult phallus actually makes this question difficult to address. Historically there has been much conjecture, but little data to support whether the mammalian penis, squamate hemipenes, and phallus of turtles, crocodilians, and basal birds share a single evolutionary origin or are independently derived. But where comparative anatomy has struggled, comparative developmental biology has recently forged ahead. Within the last several months two independent groups have published a total of seven new research articles that help us resolve the question of phallus homology.

Figure 1 from Gredler et al. 2014 illustrating phallus  diversity among amniotes. The darker lines illustrate the sulcus spermaticus which goes on to become internalized in mammals as the urethra.

Figure 1 from Gredler et al. 2014 illustrating phallus diversity among amniotes. The darker lines illustrate the sulcus spermaticus which goes on to become internalized in mammals as the urethra.

I previously wrote about a series of five papers published from the Cohn lab (University of Florida) describing the embryology and gene expression patterns for the developing phallus. Since then this group has published a sixth paper synthesizing this wealth of information, using it to lay out a number of outstanding questions regarding phallus development and evolution. More recently, the Tabin lab (Harvard University) published a paper comparing the cellular-level origins of the genitalia in the laboratory mouse, green anole, house snake, chick, and python. I have had the distinct pleasure of working with both groups as their “anole guy.” Although these studies vary widely in their experimental and comparative breadth, together they have shed much needed light on the evolution of vertebrate genitalia. Here my goal is to discuss how this new wave of research changes what we now know, what we don’t know, and what we think we know regarding the evolution of external genitalia among vertebrates. Take a look at the original research papers for details of the developmental analyses, which represent many technical steps forward in our use of anoles as a laboratory model system and intellectual advancements in our understanding of genital development.

During the gradual transition of life onto land, vertebrates evolved the amniotic egg to facilitate their departure from moist environments. Continue reading That Fascinating Phallus

Anoles Moving North, Way North

A recent paper by Matt Helmus, Luke Mahler, and Jonathan Losos highlighted the ways in which globalization has influenced the distribution of Caribbean Anolis lizards. At the heart of this research was the relationship between commercial shipping traffic and lizard biogeography. Two more recent observations can now extend these findings well beyond the Caribbean, much, much farther north.

First, Twitter user  recently posted a story about a stow away green anole that he found on pallet in Edmonton, Canada. This was a shipment of oil field supplies that originated in Houston, TX and that took ten days to reach its destination. This male green anole survived the trip all the way to Canada and is now housed in a new terrarium. The tweet originally posted October 16th and since then the anole has shed and appears to have adapted well to its new home.

An anole in the great white north.

The Canadien green anole.

Photo by Randi Duun

In separate case of stowaway lizards, another anole survived a transatlantic journey to Denmark in a shipment of bananas and was discovered incapacitated on the floor of the stockroom where incoming bananas are fumigated. The photo is too small for me to be certain, but this appears to be an Anolis cybotes female. According to the original post by Randi Duun in the “Anoles” Facebook group, the shipment originated in Colombia, Costa Rica, or the Dominican Republic so this would be consistent with an A. cybotes hitchhiker. It would be interesting to know how long a shipment like this takes, but I bet that it is longer than ten days port-to-port. Regardless, just like the globetrotting green anole, this anole is healthy following its journey, housed in a terrarium and enjoying Danish mealworms.

In contrast to the research described by Helmus et al, it is probably safe to assume that despite the perseverance of these anoles, and any others that make their way towards the arctic circle in subsequent shipments, escapees will not be establishing viable introduced populations.

 

Find the Anole: Squamates Versus Archosaurs

Regular readers of Anole Annals may remember the “Find the Anole” series that has been popular over the last few years. It has been a while since we enjoyed such fun times, so I wanted to breathe new life into this classic challenge.

Earlier today I visited Dinosaur World in Plant City, Fl. and enjoyed the contrast between Mesozoic and Cenozoic  reptile diversity. It was very exciting. Below are two images from their grounds for your enjoyment. Can you find and identify the anoles in these photos? A far bigger challenge may be to identify the dinosaurs illustrated by these statues.

Find the anole 1

Find and identify the anole.

On a separate note, if you are ever passing through central Florida with your families, stop by Dinosaur World. The interpreters were quite good with our kids, there are over 200 life-sized (and colorful) dinosaur statues, they clearly state that the earth is 4.5 billion years old, and there are no humans riding dinosaurs. I was pleasantly surprised by all of this in this part of the country. Its worth a few hours of your time!

Find the anole and identify the species.

There Is Grandeur in This View of Reptilian Genitalia

There is considerable variation in phallus morphology among the major groups of amniotes (phallus used herein to be inclusive of both the penis and clitoris). Just for starters, while most clades – including mammals, birds, turtles, and crocodilians – have a single midline phallus, squamates have paired hemiphalluses. Although herpetologists have long appreciated morphological variation in the hemipenis for its systematic value, understanding the nuances of anatomical homology, homoplasy, and novelty at this larger scale has not been as widely addressed. Recently, the Cohn lab of the University of Florida (of which I am now a member) undertook this challenge from a developmental perspective, studying development of external genitalia in representatives of each reptilian clade: the ball python (Python regius), the pond slider (Trachemys scripta), three duck species, the American alligator (Alligator mississippiensis), and who else, but the green anole (Anolis carolinensis). A synthetic review of the complete series will have to wait for another post, but reprints of each paper are available on the lab’s website to hold over the most curious. But because of the growing interest in anole nether regions, I will briefly highlight the recent findings regarding hemiphallus development in the green anole.

Fig. 2 of Gredler et al. illustrating the development of paired genital and cloacal swellings.

Fig. 2 of Gredler et al. illustrating the development of paired genital and cloacal swellings.

The Wade lab has previously shown that both male and female green anoles develop similar hemiphalluses during the early stages of genital morphogenesis, which then later differentiate into sex-specific reproductive structures. Building upon this observation, Gredler et al. described the embryology of the green anole hemiphallus from the earliest stages of morphogenesis through sexual differentiation. Hemiphallus development begins around the time of oviposition when three sets of paired swellings appear between the cloaca and the developing hindlimb bud, reminiscent of what is observed in other amniote clades. These swellings expand and meet at the midline to form the external lips of the cloaca or remain lateral to the cloaca and mature into the hemiphalluses. Following morphogenesis, the male hemipenis continues to elongate as it forms its distinctive lobes and sulcus spermaticus while the female hemiclitores gradually regress into the cloaca. Further details of the developmental anatomy of internal reproductive structures and gene expression patterns of several key molecules associated with genital morphogenesis are described in the paper.

Fig. 4 of Gredler et al. illustrating sexual differentiation of the hemiphalluses

Fig. 4 of Gredler et al. illustrating sexual differentiation of the hemiphalluses. Red arrow highlights the formation of the sulcus spermaticus.

Although there is some variation among squamates in the relative timing of the emergence and fusion of the paired swellings associated with hemiphallus development, these results are largely consistent with classical embryological descriptions of squamate genitalia (summarized by Raynaud and Pieu in Biology of the Reptila volume 15). But the revival of this body of literature in a comparative and molecular context brings new research questions to our collective table. As discussed by Gredler et al., the seemingly modular relationship between the genital swellings, cloaca, and limb buds may be particularly interesting in the context of repeated body elongation and limb loss among squamates. Better understanding of the relationship between cloacal and phallus development may also shed new light on the mechanisms of reproductive isolation, the coevolution of male and female reproductive organs, and evolving patterns of sexual conflict. Furthermore, there remain open many mechanistic questions regarding the molecular patterning of the hemiphalluses and which processes are hormone dependent that can now be more thoroughly addressed using the newly available sex-specific molecular markers. Considering the growing literature on hemipenis variation and expanding access to genomic resources in Anolis, these may be particularly fruitful areas for future investigation.

 

Distribution of Keratins in Lizard Scales

Keratins are the structural proteins of skin, hair, nails, feathers, and scales. There are 54 described  keratins in humans, a subset of which has also been found in the green anole genome. Distinct combinations of keratins in skin appendages are what give these tissues their unique properties such as flexibility, rigidity, or cornification (i.e., the process of forming an epithelial barrier). Lizards have a number of specialized scale types, likely due to the distinct distribution of keratins in those scales. Dating back at least a decade, Lorenzo Alibardi and colleagues have been making great progress describing the keratin gene family in lizards and describing the distribution of these proteins across the body. Alibardi has recently added to this long series with a description of keratin localization in the lamellae of Anolis carolinensis. Because I am not an expert in keratin biology I will let the Abstract give you the details:

ABSTRACT Knowledge of beta-protein (beta-keratin) sequences in Anolis carolinensis facilitates the localization of specific sites in the skin of this lizard. The epidermal distribution of two new beta-proteins (betakeratins), HgGC8 and HgG13, has been analyzed by Western blotting, light and ultrastructural immunocytochemistry. HgGC8 includes 16 kDa members of the glycine-cysteine medium-rich subfamily and is mainly expressed in the beta-layer of adhesive setae but not in the setae. HgGC8 is absent in other epidermal layers of the setae and is weakly expressed in the beta-layer of other scales. HgG13 comprises members of 17-kDa glycine-rich proteins and is absent in the setae, diffusely distributed in the beta layer of digital scales and barely present in the beta-layer of other scales. It appears that the specialized glycine-cysteine medium rich beta-proteins such as HgGC8 in the beta-layer, and of HgGC10 and HgGC3 in both alpha- and beta layers, are key proteins in the formation of the flexible epidermal layers involved in the function of these modified scales in adaptation to contact and adhesion on surfaces.

Fig. 10 from Alibardi 2014

Fig. 10 from Alibardi 2014

 

Fluorescent Lizard Skeletons Used to Precisely Measure Growth

Several weeks ago, Anole Annals highlighted a recent paper that uncovered the molecular bases of craniofacial dimorphism in the carolinensis clade of Anolis lizards (for full disclosure, I am the lead author of that paper). Hidden deep within that research is a relatively new technique for precisely measuring rates of skeletal growth that may be of interest to the community. I briefly introduced this technique several years ago in a post about methods of skeletal preparation, but with the details of this method now available it is worth highlighting once more.

Double labeled facial skeleton of A. carolinensis. Green label (calcein) and red label (alizarin complexone) separated by 30 days.

Because some images shouldn’t be lost in the supplementary materials. Double labeled facial skeleton of A. carolinensis. Green label (calcein) and red label (alizarin complexone) separated by 30 days.

Growth in body size can often be measured using calipers or a ruler. But in some situations a finer-scale analysis may be necessary, such as when differences in growth rate may be subtle, within the range of error associated with those manual methods. Fluorescent calcium chelators provide the precision needed to measure differences on the order of microns per day. In the recent paper, this technique was used to measure facial elongation in sexually mature green anoles, which was only ~8um per day in males and ~4um per day in females. These compounds are stable, are not highly toxic to animals, are relatively inexpensive, and can be easily used in the field or the lab. They can also be applied to adults or hatchlings with little modification to the protocol as injection volumes are typically 10-20ul depending on size. Ultimately, there is a lot of versatility to the way in which this method can be applied.

Dimorphism in facial growth rates between male and female A. carolinensis. Modified from Sanger et al. 2014.

Dimorphism in facial growth rates between male and female A. carolinensis. Modified from Sanger et al. 2014.

While new to herpetology, this technique was adopted from the biomedical literature on fracture repair where precise spatiotemporal measure of bone deposition is required. The general experimental framework is that pulses of chelators with different fluorescent properties are delivered at distinct intervals, the skeleton prepared, and the distance between the labels recorded from digital photographs. Calcium chelators are available that fluoresce under many of the standard filters used in modern microscopy – including green (calcein), red (alizarin complexone), orange (xylenol orange), and blue (calcein blue and oxytetracycline) – offering great experimental flexibility. Once incorporated into the bone, their signature remains strong for at least 30-45 days, until it is remodeled away as the living skeleton continues to grow and reshape itself. In the recent paper on craniofacial dimorphism, fluorescence in the facial skeleton could be observed following simple removal of the skin because the face has little to no overlying connective tissue. Measuring growth of the vertebrae or limbs is also possible, but may require careful sectioning of the bone using either plastic or paraffin protocols. Ultimately I think that there is a lot of potential with this method that has yet to be explored in the context of organismal biology. I hope that by highlighting this method here more people become aware of its utility and give it a try.