Birds are lovely animals. Our avian friends swoop through the air, defecate on field equipment, and consume lizards. What’s not to like?! Well, their shoulder region, for example. Lost interclavicle, reverted muscle pathways, and so many other anatomical adaptations that appear crucial for the modern avian life style, but that are hard to explain in a gradual-evolutionary context. Reconstructing the structural evolution of the avian shoulder remains a challenging task to students of biomechanics and kinematics. When I left my European homestead to enter the Canadian realm of biological sciences, I was hoping to solve the evolutionary mystery of the avian shoulder, at least in part. Alas, the discovery of anoles sent me on a much more convoluted journey.
One of the age old questions in anole morphology is at what point do you stop counting lamellae on the toepad?
Without giving any more information on various techniques or methods, I thought it would be interesting to ask the AA community their personal opinions. Below I have attached a flatbed scan of a toepad. Could people please fill out the corresponding poll below, and I will present the results in a follow up post!
Lamellae numbered 1-51 on the 4th digit of an Anolis lizard hindfoot
Several weeks ago, Anole Annalshighlighted 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.
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.
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.
BEAST estimated phylogeny of anoles. Circles on nodes represent posterior probability, black > 0.95, grey > 0.90, white > 0.70.
In the course of our recent study on sex chromosome evolution in anoles (Gamble et al. in press) [AA post] we assembled a 216-species mitochondrial DNA phylogeny of anoles, the largest published to date (at least that we know of), yet containing only a little more than half of all recognized species. Although we collected new sequences for some species, our dataset is largely built on the hard work of others who collected and published on sequences from across the genus, such as Jackman et al. 1999, Poe 2004, Nicholson et al. 2005, Mahler et al. 2010 [AA post], and Castañeda & de Quieroz 2011 [AA post]. Without access to data from these and other studies, we would have had a far less complete and robust tree for our comparative analyses.
There is a big debate going on now regarding what, where and how much data should be shared in association with publishing academically. I personally feel that providing easy access to those data used and generated during a study serves to accelerate the rate and increase the quality of scientific discovery. I am heartened that more and more journals are making data deposition a requirement for publication, although often this means little more than dumping sequence data to GenBank. Sites like Dryad, Figshare, and GitHub now provide open, permanent, and citable access to raw data, figures and, most importantly in my view, research products like alignments, code and analysis logs. In an effort to make our data as accessible and useful as possible we have archived our alignment, MrBayes and BEAST consensus trees as well as as the BEAST posterior distribution on the digital data repository Dryad [doi link]. It is our hope that other anolologists can use and improve upon these data to ask new, interesting questions and to build a larger, more complete view of the evolution of anoles.
Time honored anole field technique. But since when?
Here at AA, we’ve frequently discussed the art and practice of lizard noosing, such as posts on the best material to use to construct a noose, as well as the variety of suitable poles commercially available. Recently, I was asked a question for which I did not have an answer. To wit, what is the history of lizard noosing? Did our herpetological forebears use nooses? I’m aware that at least some herpetologists in the 70’s were doing so. What about earlier than that? Did Stan Rand noose lizards? Ernest Williams in his younger days? Barbour?
Everyone’s aware that when looking for information, if you can’t find it on Google, it’s not worth knowing. This, however, would seem to be an exception. Wikipedia has no entry on lizard noosing, nor does a Google search on the relevant terms turn up any answers (such a search does, however, turn up a plethora of websites and Youtube videos offering lizard noosing tutorials). So, I put it to you, AA readers: who can enlighten us on the history of anole noosing?
Recently, we had a post on the cool bark anole embryo photographs produced by Catherine May at Arizona State. Catherine has now done this one better by producing a series of photographs, along with explanatory text, detailing the process by which skeletal preparations are made via the old method of clearing-and-staining. As the photo reveals, the resulting products are not only scientifically informative, but quite beautiful. And while on the topic of anole skeletal preparation, check out Thom Sanger’s Halloween-themed post on the same from 2011.
On a recent trip to Grand Cayman I was interested in the UV reflecting dewlap of Anolis conspersus. The dewlaps of these lizards appear blue to our visual system but are maximally reflective in the ultraviolet. While anoles have 4 cone types (ultraviolet, blue, green and red sensitive), humans have only 3 and cannot see UV light so to understand what these lizards look like in the UV, we have to use specialized camera equipment. The photo to the right shows what a displaying A. conspersus looks like to our camera system when imaged in the human visual spectrum as commercially available digital cameras also have only three channels corresponding to the three human cone types. Presumably if we were also able to see in the ultraviolet as many other animals can, our cameras would be designed with a separate channel for ultraviolet.
These images of the lizard in the UV show clearly the regions of the dewlap and that are highly UV reflective and the pattern of UV reflectance in other areas. One somewhat interesting finding is that while the dewlap scales are highly reflective across the human visual spectrum (which is why they appear white to our eyes) they reflect very little UV light. The lower photo is a monochromatic image (both the red and blue channels in this camera are sensitive to UV so the raw image appears purple) that makes it a bit easier to see brighter areas as white. Note how bright the dewlap appears relative to the reflectance standard, when imaged in the human visual spectrum a similar monochromatic image of the dewlap would appear very dark. I believe this shows the potential value of UV photography when studying Anolis dewlap patterns. While the UV nature of the A. conspersus dewlap is uniform, it’s likely that other species have patterns visible in the UV we’ve previously missed. We have also used this UV photography setup in SE Asia to image Draco flying lizards and other species, some of which have patterns that are visible only in the UV band. The goal of this project is to make a camera system with pixel channels similar to the four cone types found in Anolis lizards and birds to image whole organisms and really “see” the patterns organisms experience with their visual system as they would see them. As Anolis visual pigments and their associated oil droplets appear to be fairly conserved, this seems to be achievable.
Another surprise (to me) was the large number of A. conspersus on Grand Cayman using lights at night to feed. I’ve spent many months doing fieldwork in SE Asia and Central America and can’t recall seeing this sort of thing with other diurnal lizard species, but on Grand Cayman it was quite common in A. conspersus. I observed one A. conspersus male chase away a Hemidactylus that got too close to the light, showing that the anoles at least occasionally displaced the group I typically associate with feeding around lights. A check of the literature shows this has occasionally been documented on other Caribbean islands, but as far as I can tell no one has published on this in mainland species. What diurnal lizard species have others observed using lights to feed at night?
Green anole (Anolis carolinensis). Photo courtesy of Karla Moeller.
Ensembl Release 71 includes many updates for Anolis carolinensis, including the addition of the Arizona State University (ASU) Anole Genome Project annotation recently published in BMC Genomics (Eckalbar et al., 2013). This release includes an updated Ensembl gene set and aligned RNA-Seq data from a number of tissues, including embryo, lung, liver, heart, dewlap, skeletal muscle, adrenal gland, ovary, and brain, which have been added to the track viewer. These RNA-Seq data from individual tissues and from the ASU reannotation or the “Anole Genome Project” can be viewed just below the Ensembl gene tracks, as in this example.
Here’s a question for AA readers from Nancy Bunbury, from the Seychelles Island Foundation, who is conducting some exciting work on large gecko interactions, ecological roles, and niche separation in the palm forests of the Seychelles:
“The main species in question is Ailuronyx trachygaster (first field study on this amazing species) and one thing we would love to do is look at movements and territory size (also because we suspect it’s the main pollinator for the coco de mer which has huge conservation and inevitably commercial value). We are looking into GPS tags for the geckos (which are about 150g in weight) but it seems the technology for such a small tag requiring GPS and remote downloading is not yet available. Do you happen to know if such tags have yet been developed and who I might be able to contact for them (I’ve tried the standard larger companies for animal tracking devices)?”
On a recent trip to Toronto, eminent bee-man and pollination biologist James Thomson showed me his lab, including a cage used for bee pollination studies. The cardboard box is a “box of bees” that can be bought commercially and the experiment involves training bees to go to containers with different colors. Despite being fascinated by the research, my mind couldn’t help but wandering to thinking about how useful such a contraption could be to set up in the field for ecological or behavioral anole studies. As you can see, the cage is big enough that it could house a number of anoles at natural densities, and the mesh lets sunlight and rain through. James kindly informed me that the cages can be purchased at Bioquip; the largest they stock is 6′ (h) x 6′ (w) x 12′ (l), but James told me that larger models can be custom-ordered, and that they are very hardy in the field. Someone should try this!
Birds are lovely animals. Our avian friends swoop through the air, defecate on field equipment, and consume lizards. What’s not to like?! Well, their shoulder region, for example. Lost interclavicle, reverted muscle pathways, and so many other anatomical adaptations that appear crucial for the modern avian life style, but that are hard to explain in a gradual-evolutionary context. Reconstructing the structural evolution of the avian shoulder remains a challenging task to students of biomechanics and kinematics. When I left my European homestead to enter the Canadian realm of biological sciences, I was hoping to solve the evolutionary mystery of the avian shoulder, at least in part. Alas, the discovery of anoles sent me on a much more convoluted journey.
Recently, we had a post on the 
