A mourning gecko (Lepidodactylus lugubris) climbing vertically on glass with the help of its impressive toe pads.
I think most people visiting Anole Annals could argue that the adhesive digits of anoles are some of the most fascinating aspects of their biology (or maybe I’m just biased). Digital adhesion is accomplished through toe pads: a collection a broad, modified plantar scales which bear thousands upon thousands of microscopic, hair-like structures (i.e. setae). Through frictional and van der Waals forces, these collections of setae allow toe pad-bearing lizards to easily access vertical surfaces and exploit habitats many lizards cannot. Shockingly, adhesive toe pads have independently evolved several times across lizard evolutionary history (at least 16 times by recent estimates) — once in the common ancestor of anoles, once in a clade of southeast Asian skinks, and 14 times in geckos. Both within and between the different evolutionary origins of toe pads, there is substantial variation in toe pad size, shape, number of scansors/lamellae, and position of the adhesive apparatus.
In our recent study, my collaborators and I took the first steps to characterize how embryonic development is modified to achieve this incredible diversity. Using embryonic material my coauthor Thom Sanger collected as a postdoctoral researcher in Marty Cohn’s lab, in addition to embryonic material I collected over the course of my Ph.D. training in Tony Gamble‘s lab, we aimed to compare embryonic digit development of ancestrally non-padded lizards with that of anoles and padded geckos. We used a model clade approach to broadly sample anoles and geckos, although some species breed more easily in the lab and have more embryological resources than others. All together, we sampled a range of toe pad morphologies in both clades (trunk-ground and trunk-crown Anolis ecomorphs and leaf-toed and basal pads in geckos). To help polarize the developmental changes leading to the origin of toe pads, we also included two ancestrally padless species in our comparisons. After the collection of these diverse embryos, we used scanning electron microscopy (SEM) to characterize scale morphology of the digits throughout embryonic development.
By comparing embryonic material of anoles and geckos, we essentially span the diversity of squamates in a single comparison.
Because of the ~200 million year divergence between anoles and geckos and dramatic differences in adult morphology, we anticipated that we would see stark differences in the developmental origins of toe pads in these species. To our surprise, we found striking similarities in toe pad development between all of the pad-bearing species we examined. We found that toe pads develop after digit webbing recesses. In all pad-bearing species, ridges that become the adhesive scansors and lamellae first form in the distal half of the digit. Throughout development, new ridges begin forming in the proximal direction while the previous ridges begin to grow laterally. Elaborations and derivations in toe pad form, such as bifurcation, occur in the latter-half of embryonic development. The presumably ancestral pattern of plantar scale development we observed in our leopard gecko and fence lizard embryos (both species lacking adhesive digits) demonstrated that scale ridges form all at once along the length of the digit. These differences are similar to those documented between developing non-padded gecko tails and padded tails of crested geckos. This means that anoles and geckos have converged on a similar developmental process! We suggest that toe pads are initially formed through a major repatterning of digital development and then variation is achieved through relatively minor “tinkering,” through either timing or location of developmental patterns.
Scanning electron micrographs (SEMs) of embryonic lizard digit development, progressing from early development (left) to late development (right). The pad-bearing brown anole (Anolis sagrei) and mourning gecko (Lepidodactylus lugubris) have converged on scansor ridges forming in a distal-to-proximal direction, while the paddles leopard gecko (Eublepharis macularius) has scale rows forming all at once along the length of the digit. Lizard photos courtesy of Dr. Stuart Nielsen.
This is by no means the end of this story. We’ve just scratched the surface and there are a several directions to head in. A logical next step is to characterize histological organization through toe pad development. From there, characterizing the genes involved in toe pad morphogenesis, in tandem with the possibilities of new gene editing technologies, would allow us to test mechanisms of toe pad formation and how variation is generated. And, of course, characterizing toe pad development in other species (such as the secondarily padless Anolis onca) may elucidate further conservation or derivation from the trends we found. This is an exciting time to be a toe pad biologist!
Terrestrial animals that venture into the water on a regular basis face a number of challenges not encountered by their strictly terrestrial counterparts. While submerged, they must deal with hydrodynamic drag forces hindering locomotion and with the risk of running out of air. Back on land, the film of water adhering to their body surface may interfere with locomotion and thermoregulation or may increase the risk of bio-fouling. Many semi-aquatic invertebrates (and plants) have developed complex surface microstructures with water-repellent properties to overcome these problems, but equivalent adaptations of the skin have not been reported for vertebrates that encounter similar environmental challenges.
The transition to a semi-aquatic lifestyle has independently occurred multiple times throughout the evolutionary history of Anolis (see Fig. 1A below). In anoles, the skin surface is covered with microscopic hair-like ornaments, and contingent upon its complexity, organization, and length dimensions, these hair-like microstructures may have the potential to generate extreme surface hydrophobicity. Indeed, similar skin surface microstructures have been found in geckos and are shown to be responsible for the highly hydrophobic surface of their skin. The water-resistant properties of anole skin, however, have remained unexamined, but very recent discoveries have provided valuable insight into this matter. Boccia et al. (2021) observed that semi-aquatic Anolis lizards are able to sustain long periods submerged underwater by iteratively expiring and re-inspiring narial air bubbles. As in semi-aquatic insects, a hydrophobic skin is a key requirement for the underwater formation of an air bubble, hence, functional respiration, so a hydrophobic skin in semi-aquatic anoles is implied. However, whether a hydrophobic structured skin surface in anoles has evolved in response to life at the water-land interface is still an open question. Answering this question was the primary goal of our study.
We studied the skin surface morphology of preserved anole specimens using scanning electron microscopy and tested the wettability of the skin surface using contact-angle goniometry (Fig. 1D). We found that the skin surface of semi-aquatic species of Anolis lizards is characterized by a more elaborate microstructural architecture (i.e. longer hair-like structures; Fig 1B,C) and a lower wettability (Fig. 1D,E) relative to closely related terrestrial species. In addition, phylogenetic comparative models revealed repeated independent evolution of enhanced skin hydrophobicity associated with the transition to a semi-aquatic lifestyle, providing evidence of adaptation.
Figure 1 from Baeckens et al. (2021)
We believe our findings bring an additional dimension to the recent biological phenomenon described by Boccia et al. (2021) namely that diving Anolis lizards not only repeatedly and independently evolved a specialized rebreathing behavior with the transitioning to a semi-aquatic lifestyle, but that its evolution presumably also coincided with, or was preceded by, the evolution of a hydrophobic structured skin to successfully do so.
S. Baeckens, M. Temmerman, S. Gorb, C. Neto, M. Whiting & R. Van Damme (2021) Convergent evolution of skin surface microarchitecture and increased skin hydrophobicity in semi-aquatic anole lizards. Journal of Experimental Biology 224(19): jeb242939 (doi: 10.1242/jeb.242939)
Above: Male Anolis bicaorum, endemic to the island of Utila (photo credit Tom Brown).
Why are there more anoles in this plot? This is the question that we continually asked ourselves whilst setting up our 2018 survey plots on the small island of Utila, Honduras, home of the endemic Anolis bicaorum (pictured above). So in 2019 we set out from Heathrow airport, kitted up with A LOT of equipment, for our second field season, based at Kanahau Utila Research and Conservation Facility, with one of the goals being to look into just that (see previous Anole Annals posts on Utila and its anoles).
Above Left: Getting ready to leave Heathrow (photo: Adam Algar). Above Right: Miraculously all the field kit all safely arrived at Houston stopover.
We all know that such factors as the thermal environment, prey resources and structural habitat play important roles in the lives of our beloved anoles. And we also know that you can go to one spot and be overrun with anoles, but go to a seemingly similar spot nearby and find none (usually when the field season is drawing to a close and you still don’t have a large enough sample size). But how are these aspects of anole biology linked? Specifically what determines their abundance, and its variation, at fine scales? While ecological niche theory is well developed, empirical evidence for which factors are most important, and how they interact, is still rare for many taxa, including anoles. Given rapid environmental change, understanding the drivers and mechanisms governing abundance is now more important than ever.
We measured the abundance of the endemic Anolis bicaorum across thirteen 20x20m plots along a tropical habitat gradient, using standard mark-recapture methods, based on Heckel & Roughgarden (1979). Within these plots, we also measured factors relating to the thermal habitat suitability (using 3D printed models fitted with iButtons), structural habitat (perch surveys), canopy cover (leaf area index) and prey availability (arthropod biomass and diversity). We then used N-mixture models and path analysis to disentangle direct and indirect effects of these factors on anole abundance.
Above Left: Emma setting up 3D printed anole thermal models. Above Right: Tom out collecting 3D printed anole replica models (photo credits Adam Algar).
We first decided on several measures for each niche factor which could determine the suitability of the habitat for the anoles. For the thermal environment, we first determined the thermal preference (Tpref) range of A. bicaorum, following Battles and Kolbe (2018). We then calculated two indices to quantify the thermal habitat quality of each plot. The first was the percent of model hours that operative temperatures (from 3D models) were within the Tpref range over the 36-hour study period for each plot. The second was the total number of degrees (°C) that the models deviated from the Tpref range across all models throughout the survey period for each plot, which included the total degrees, the degrees above and degrees below the Tpref range.
Above: Many of the anoles were “side-eye” pros
As a measure of structural microhabitat quality we determined both perch availability by counting surveys and the plot basal area (a measure of stand density), across all tree trunks, palm stems and fence posts in the plot.
Above: A. bicaorum predating on an unidentified spider (Araneae).
For prey availability, we measured arthropod biomass (g) and diversity (Simpson and Shannon’s) from a combination of leaf litter sieving and sweep-net samples taken in each plot. Sweep-net and leaf-litter samples were combined for plot level analyses.
We also measured mean leaf area index (LAI) in each plot using an Accupar LP80 ceptometer. LAI is the one-sided area of leaves per unit ground area and is a measure of canopy density; it is expected to influence thermal environment via the interception of solar radiation (Campbell & Normal 1998; Algar et al 2018).
After we determined reasonable measures of habitat suitability for each factor, we examined univariate relationships between A. bicaorum abundance and each of our habitat variables (percent of time within Tpref, deviation from Tpref, perch number, basal area, arthropod biomass, arthropod diversity and LAI) by including each predictor as a covariate in a multinomial-Poisson mixture model of abundance. The results of the most significant and strongest relationships can be seen in the figure below.
Above: Relationships between Anolis bicaorum abundance and individual niche metrics in forest plots across Utila, Honduras. Relationships were estimated using multinomial Poisson mixture models with a constant detection rate across plots. All variables are scaled to a mean of zero and unit variance; (a) reflects thermal habitat quality, (b) reflects structural habitat quality, (c) reflects prey availability and (d) reflects canopy cover.
We used these models to select a subset of these variables (one representing habitat structure, one prey availability, and one thermal quality) for subsequent path analysis; we also included LAI as the sole measure for canopy cover. We used the path analysis to evaluate the relative strength of direct and indirect effects on abundance. As we could not estimate indirect paths within a single multinomial-Poisson mixture model, we estimated abundance for the path analysis from a multinomial Poisson mixture model that included no environmental covariates, held detection rate constant, and permitted abundance to vary by plot. The results of the path analysis can be seen in the figure below.
Above: Direct and indirect effects of niche axes on A. bicaorum abundance. (a) Values are standardized path coefficients; line width is proportional to the strength of the effect, solid lines indicate statistically significant pathways. ε, unexplained variation. (b) The total effects of covariates on abundance. NP: number of perches; PB: prey biomass; LAI: mean leaf area index; TP: time within Tpref range.
Our results showed that thermal habitat quality and prey biomass both had positive direct effects on anole abundance. However, thermal habitat quality also influenced prey biomass, leading to a strong indirect effect on abundance. Thermal habitat quality was primarily a function of canopy density, measured as leaf area index (LAI). Despite having little direct effect on abundance, LAI had a strong overall effect mediated by thermal quality and prey biomass.
We have demonstrated the interconnectedness of abiotic and biotic components that determine habitat quality and animal abundance. Rather than identify a single strong control on abundance, we found key abiotic factors (canopy cover and thermal environment) affect abundance through multiple pathways and have effects that are mediated by biotic interactions and the niche of the focal species. In particular, our results suggest alignment of thermal niches across multiple trophic levels results in strong indirect effects of thermal environment on anole abundance. Losses of thermal habitat quality, particularly due to canopy loss, may thus have greater effects than appreciated when only direct effects are considered.
Our results demonstrate the role of multidimensional environments and niche interactions in determining animal abundance and highlight the need to consider interactions between thermal niches and trophic interactions to understand variation in abundance, rather than focusing solely on changes in the physical environment. Identifying the factors responsible for population change along habitat gradients will improve our understanding of how multidimensional environments and niches interact to determine population abundance. Which is more important than ever in this ever-changing world.
If you have any questions or just an interest in the work, please feel free to contact me emma.a.higgins@hotmail.com.
I would also just like to thank everyone again who was involved in this project, it was a lot of hard work, but great fun and it couldn’t have been done without the team effort.
Above: Part of the field team, helping process what is certainly not an anole, whilst setting up survey plots (photo credit Adam Algar).
References
Algar, A.C. et al. 2018. Remote sensing restores predictability of ectotherm body temperature in the world’s forests. – Glob. Ecol. Biogeogr. 27: 1412-1425. https://doi.org/10.1111/geb.12811
Campbell, G. S., and J. M. Norman. 1998. An introduction to environmental biophysics. 2nd edition. -Springer-Verlag, New York.
Battles, A.C. and Kolbe, J.J. 2018. Miami heat: Urban heat islands influence the thermal suitability of habitats for ectotherms. – Glob. Change Biol. 25: 562–576. https://doi.org/10.1111/gcb.14509
Heckel, D.G. and Roughgarden, J., 1979. A Technique For estimating the Size of Lizard Populations .Published by : Wiley on behalf of the Ecological Society of America Stable URL : http://www.jstor.org/stable/1936865 References Linked refere 60, 966–975.
Dominican House Wren (Troglodytes aedon rufescens) holding a juvenile Puerto Rican crested anole (Anolis cristatellus). Photo by M.P. van den Burg.
New literature alert!
Predation on the nonnative Puerto Rican crested anole (Anolis cristatellus) by the Dominican House Wren (Troglodytes aedon rufescens) on the Commonwealth of Dominica
In The Wilson Journal of Ornithology
van den Burg & Brisbane
Abstract
Predation on vertebrate species by insect-eating birds is rarely recorded, with only one report for the House Wren (Troglodytes aedon). On 4 January 2019, we observed a Dominican House Wren (T. a. rufescens) consume a juvenile of the nonnative Puerto Rican crested anole (Anolis cristatellus) in Roseau, Commonwealth of Dominica. This observation suggests the Dominican House Wren could additionally prey on the endemic Dominican anole (Anolis oculatus). This record aids our understanding of the ecosystem-wide impact of the A. cristatellus invasion.
Despite being on opposite sides of the world and separated by millions of years of evolution, Draco and Anolis lizards have converged on many common adaptive solutions.
It has long been suspected that the Draco lizards of Southeast Asia were ecological analogues of the Anolis lizards in the Caribbean. But it has only been recently that we’ve started to truly figure out just how similar these two groups actually are. Especially exciting has been the discovery of how lizards in both groups have converged on remarkably similar adaptations: natural selection appears to repeat itself, time and time again.
The classic textbook scenario in anoles is adaptive convergence in ecomorphology, which reflects where lizards tend to hang out in the environment. This is particularly obvious in the adaptation of Anolis limb morphology to different types of perches. I’m not sure we can say whether Draco exhibit the same ecomorphs as Caribbean anoles just yet, but Draco do exhibit the same adaptive changes in limb morphology as the anoles of the Caribbean.
Then there’s the obvious case of the dewlap, which has evolved separately in both Anolis and Draco as a key component of their territorial and courtship displays.
Most Anolis and Draco lizards also rely heavily on elaborate sequences of head-bobs in these displays as well.
This in itself isn’t unusual. Many lizards head-bob and push-up over territory and mates. Up to now, however, Caribbean Anolis lizards were the only ones known to tailor their display movements to ensure detection. For any visually communicating animal, visual “noise” from windblown vegetation and poor ambient light make it difficult to see visual signals. Anolis lizards were exceptional because they actively monitored environmental conditions and exaggerated their body movements when it was visually noisy (e.g., on windy days) or extended the duration of their displays when light levels diminished (e.g., on cloudy days or deep inside a forest).
There are a countless ways lizards might produce a conspicuous signal. Many Australian and Chinese lizards add arm-waves or elaborate tail-flicks or simply rely on colourful ornamentation that stands out well in the environment. Many North American lizards rely on performing lots of head-bobs or positioning themselves to accentuate a colourful badge on their throat or sides. But anoles seemed unique in both the complexity of their visual displays and their capacity to modify their behaviour to the prevailing conditions in the environment.
But after years of studying Anolis on Jamaica and Puerto Rico, and then even more years studying Draco in the Philippines, Borneo and Malaysia, we have now discovered more astonishing parallels between the two lizard groups that extends beyond just morphology.
Southeast Asian Draco lizards exhibit virtually identical strategies for coping with visually difficult environments as do the Caribbean Anolis lizards. Draco use the dewlap in the same way as the anoles, and change the speed (or exaggeration of movements) and the duration of displays in the same way as anoles, and this capacity to tailor displays to the conditions of the environment has also tended to precede what seems to have been adaptive divergences in display behaviour among species.
To discover all this, we had to study many different species of Draco and Anolis (11 and 12 species respectively), including hundreds and hundreds of lizards (727 to be precise), and then conduct thousands and thousands of hours of video analysis (13,310 hours – !!).
To be perfectly honest, what I was hoping to document from all this work was how differences in evolutionary history between the Anolis and Draco had shaped the trajectory of display evolution. Sure, Draco had evolved a dewlap like Anolis, but how that dewlap has been morphologically constructed was quite different between the two groups. I had become quite interested in these so-called “many-to-one, form-to-function” outcomes in evolution, and I was aiming to show something similar for display behaviour.
To be clear, there were differences in how Anolis and Draco lizards responded to environmental conditions, and how plastic changes in behaviour have contributed to display differentiation among species. In fact, the head-bob component of the territorial display has been entirely lost in some Draco species.
But the similarities were stunning and outweighed the differences by a large margin. Even the loss of headbobs in some Draco have intriguing similarities to how some Anolis species have shifted their display effort to the dewlap, which seems to be a more energetically efficient means of producing a conspicuous, complex visual display, than the more tiresome head-bob and push-up movements.
We have also confirmed experimentally in anoles that the manner in which Anolis lizards tailor their displays does actually improve display detection in visually difficult environments. This took a lot of work in itself and required the development of a robotic playback system, but this is now ancient history.
But to complete the loop, a similar type of playback experiment needed confirm the same adaptive benefit in Draco.
Some years ago we had conducted a lengthy field experiment using robot playbacks that were designed to test the response of Draco lizards to different coloured dewlaps. That experiment showed little effect of dewlap colour on detection, but a tangible effect that once lizards saw the dewlap, they used it to evaluate the species identity of the signaller.
I was in the lab one day looking at these old Draco robots to get some inspiration for designing a new system for some other crazy idea I had. As I was fiddling with the mechanism, I noticed that the robots weren’t exactly the same, with the lever controlling the dewlap of one being slightly longer than another. This meant the display probably differed in speed between the robots. These things happen and I didn’t think much of it at the time. The treatments used in the field experiment were systematically inter-changed across the robots to make sure this type of thing didn’t cause any problems.
Later, however, it occurred to me that perhaps this might offer a serendipitous opportunity to confirm the adaptive benefit of at least one of the key convergences exhibited by Draco lizards. I downloaded the data from the original study from its dryad repository, extracted the response times of lizards to the two robots that differed in dewlap speed, and sure enough, detection times were much quicker to the robot with the faster dewlap display.
The top panel (a) shows the differences in dewlap speed between the two robots, while the bottom panel (b) shows the detection time of free living Draco melanopogon.
If you’re interested in a short video introduction to this work, or want to know more about how these findings relate to our general understanding of adaptation and animal communication, you’ll find some answers in this 5 minute video below.
Ever since the seminal papers by Williams and Rand [1,2], the Anolis radiation across the West Indies has increasingly established itself as an alluring example of ecomorphological convergence. Considering an Anolis community on one island, sympatric species have undergone niche partitioning, whereby each species has evolved particular behavioral, morphological, and ecological traits well-adapted for the microhabitat it occupies. Pop over to another island, and voilà, similar sets of ecomorphs can be found— their resemblance so striking and uncanny.
But the Anolis story isn’t clean cut. Studies of mainland anoles have yielded equivocal findings for whether they also conform to the beautiful patterns observed in the Caribbean. Much baseline data on mainland Anolis communities are needed to determine the extent to which convergence occurs and what factors drive differences in community structure. To partly address this gap, Jonathan Losos, Anthony Herrel, Ambika Kamath, and I recently published a paper describing the ecological morphology of anoles in a lowland tropical rainforest in Costa Rica, at La Selva Biological Station.
Accumulating field observations from four field seasons ranging from 2005 to 2017, we draw from over 1000 observations to characterize the habitat use of eight Anolis species that occur at La Selva. These species include Anolis humilis, Anolis limifrons, Anolis lemurinus, Anolis oxylophus, Anolis capito, Anolis carpenteri, Anolis biporcatus, and Anolis pentaprion, and we opted to devote a brief section to the co-occurring Polychrus gutturosus. Our results revealed overlapping niches and substantial variability in habitat use across many species. Furthermore, the morphologies of A. humilis and A. limifrons were at odds with microhabitat use following the predictions of Caribbean anole ecomorphology. Among the two most abundant species, relative hindlimb length was greater for the more arboreal A. limifrons, whereas it was shorter for the more terrestrial A. humilis.
If mainland and island anoles exhibit divergent ecomorphological patterns, this begs the question of how selective pressures differ between mainland and island habitats to drive these differences. Andrews [3] proposed that predation may more strongly influence Anolis diversification on the mainland, because in comparison to islands, predators are far more abundant, anole population densities are lower, and arthropod prey is plentiful. In contrast, Caribbean anoles are thought to be food limited and there may be stronger selection for niche partitioning. Through examining variation in species’ habitat use relative to the abundance of other co-occurring species at La Selva, our data suggests a low level of interspecific competition for this mainland community, corroborating the hypotheses Andrews set forth.
In recent years, the study of mainland anoles has received more attention. We are in great need of ecological, morphological, and life history trait data for Anolis communities throughout Central and South America to further our understanding of the evolutionary trajectories of mainland and island anoles. So, anole biologists, you can throw out your boats and steer clear of the oceanic divide!
[1] Rand, A. S., and E. E. Williams. 1969. The anoles of La Palma: aspects of their ecological relationships. Breviora 327:1–17.
[2] Williams, E. E. 1972. The origin of faunas. Evolution of lizard congeners in a complex island fauna: a trial analysis. Evolutionary Biology 6: 47–89.
[3] Andrews, R. M. 1979. Evolution of life histories: a comparison of Anolis lizards from matched island and mainland habitats. Breviora 454: 1–51.
Brown anole eggs in the field. Photo by Jenna Pruett.
Most oviparous reptiles (excluding birds) bury their eggs in the ground. Usually, after laying, females abandon the eggs and provide no parental care thereafter. As such, non-avian reptiles (henceforth “reptiles”) have often served as model organisms to understand how the environment influences embryo development. Environmental factors of interest are usually temperature and moisture. Indeed, nest temperature can have large effects on development. For example, warm incubation temperatures often result in hatchlings that can run relatively fast while cool temperatures result in hatchlings that run slow. Moisture is also important during development since relatively wet incubation conditions improve the conversion of yolk to body mass resulting in larger hatchlings compared to dry conditions. This process by which the environment has lasting effects on development is known as developmental plasticity. Despite decades of research concerning developmental plasticity in reptiles, there are still many aspects of natural nest environments that are understudied.
One example of such an understudied environmental factor is the type of substrate (i.e. soil) in which females bury eggs. Although many field studies demonstrate that females lay eggs in a diversity of substrates, very few studies have considered exactly how these different substrates might influence development. These few existing studies have focused on turtles. For example, Mitchell and Janzen (2019) buried turtle eggs in three types of substrates in the field: loam, sand, and gravel. Despite all nests experiencing the same prevailing weather conditions, important aspects of the nest environment like moisture available to eggs and temperature differed among the substrates. This resulted in important differences among the hatchling turtles. Indeed, because this species exhibits temperature-dependent sex determination (i.e. the egg temperature determines if hatchlings are male or female), the sex ratios of the hatchlings differed according to the type of substrate in which the eggs were buried.
No study has rigorously considered how substrate types influence development of squamates (lizards and snakes). Therefore, my research associates and I decided to conduct a lab experiment using our good friend the brown anole (Anolis sagrei). This study was recently published in the journal Integrative Zoology (Hall et al. 2021). At our field site in Florida, female anoles lay eggs in two main types of substrates: sand/crushed sea shells and organic debris (Figure 1). We collected male and female lizards from one of our study islands and brought them back to our lab at Auburn University. We also collected a few buckets of the two substrates in which females commonly nest. We collected eggs from the breeding colony and incubated them in each substrate at 4 different moisture concentrations. The goal was to understand if these two substrates had any important effects on development. Moreover, using different moisture concentrations in each substrate allowed us to see if the two substrates might have similar effects on development given particular moisture concentrations.
Figure 1. Representative photos of (a) a female brown anole (Anolis sagrei), (b) aerial view of the substrate collection island, (c) ground view of substrate collection island, (d) organic substrate, and (e) sand/shell substrate. In panel (b), the area inside the red circle is the portion of the island that is most densely populated with lizards. The area within the black line is an example of open canopy habitat where substrate is primarily sand and crushed shell. The area inside the white line is an example of closed canopy habitat with dark, organic substrate. Panel (c) shows the ground view of the same open and closed canopy sites outlined in panel (b).
We measured a variety of traits including water uptake by eggs (eggs absorb water during development), developmental rates of embryos, egg survival, hatchling body size, and hatchling performance (i.e. endurance). The amount of moisture available to eggs provided expected results: greater moisture content resulted in greater water absorption by eggs and larger hatchling body size. We found that the two substrates had little effect on most traits; however, egg survival and developmental rate differed between the substrates: eggs were more likely to die and developed more slowly in the organic substrate than in the sand/crushed shell. Although statistically significant, these effects were not large. The difference in egg survival was about 6% and the difference in developmental rates between the substrates resulted in a one-day difference in the incubation period (i.e. the number of days it takes for the egg to hatch).
It isn’t completely obvious why we observed these differences in egg survival and physiology (i.e. developmental rate). We think the organic substrate might support a greater load of microbes (i.e. fungal spores and bacteria) than the sand/shell substrate. Thus, in the organic substrate, eggs may compete with microorganisms for resources like oxygen during development. Additionally, when exposed to an abundance of microorganisms, eggs may expend energy to fight infection which could slow development and reduce survival. Regardless, other studies have also found that developmental rate can be influenced by the type of incubation substrate, but no mechanism has yet been rigorously tested. Thus, there is still much to learn about how reptile embryos interact with natural nest environments!
In conclusion, the type of incubation substrate can have important effects on embryo physiology and survival, but only a few studies have explored these relationships. What would be most helpful now is a series of studies that consider how microbial communities differ among substrates and how these communities might interact with eggs. Perhaps this work will rest on the shoulders of Kaitlyn Murphy who is currently using microbiology techniques to understand effects of the microbiome on embryo development using brown anoles. If so, the future of this unexplored area of research is in capable hands.
Hall, J. M., Miracle, J., Scruggs, C. D., & Warner, D. A. (2021). Natural nest substrates influence squamate embryo physiology but have little effect on hatchling phenotypes. Integrative Zoology.
Mitchell, T. S., & Janzen, F. J. (2019). Substrate influences turtle nest temperature, incubation period, and offspring sex ratio in the field. Herpetologica, 75(1), 57-62.
Over the years, there has been a lot of discussion on Anole Annals about the large, conspicuous dewlap. And rightly so because it is arguably the most evocative feature of the anoles. Much of this discussion has focussed on its function, such as its role in species recognition, mate choice, and territorial communication. But is there a cost to having such an audacious visual signal?
We needn’t isolate this question to just Anolis lizards. All socially communicating animals need to produce a signal that will be obvious to conspecifics. There’s little point producing a mating or aggressive signal if females or rivals never detect it. But there is a cost to being conspicuous and it can be a matter of life and death: the unintended attraction of predators.
Generally, the assumption has been that animals just incur the potential risk of predation for the sake of successful communication. But just how risky is it? The dewlap is often large and brightly coloured, but when it’s not being used in display, you’d never know anoles even had one.
There are also at least two other independent origins of the dewlap, including in the gliding lizards of Southeast Asia, the Draco. In these lizards, the dewlap is again large and often conspicuously coloured.
For both Anolis and Draco, one of the best ways to find lizards in the wild is by the quick flash of colour as males rapidly extend and retract the dewlap during their territorial displays. In fact, it is often the only way to find Draco, which are camouflaged and extremely difficult to spot, even when you happen to be staring right at them.
I had this crazy idea a few of years ago… Would it be possible to build an army of robotic Draco lizards with plasticine bodies that could retain impressions of predator attacks and measure the risk of predation from performing a conspicuous dewlap display?
It really was a ridiculous thought, but my long-time collaborator Indraneil Das was game.
Robotic lizards compared to the real thing in (a) morphology and (b) behaviour (robots were modelled on Draco sumatranus from Borneo).
It was an awful experiment to do. Building the robot army turned out to be the easy bit. To be clear, it took months of development and manufacture, all of which I did in my garage (long story). It then took years to run the experiment, with multiple replications across two continents because the data was puzzling. There were bushfires, floods, battles with swarming wasps and kamikaze leafcutter ants, chipped teeth, falls from ladders, bogged car rentals, hammered thumbs, and in the end I only just managed to get it finished before the world turned side-ways in 2020.
Left: fresh-faced and optimistic in June 2018; Right: brave-faced but really a little shellshocked with the retrieval of robot 2,120 in February 2020 (NB: batteries have a habit of failing and parts started to corrode so only 1,566 robots were fully functional in the experiment).
It turns out that prey that can produce a signal intermittently — effectively turning their conspicuous display on and off at strategic moments, like the dewlap — can drastically reduce their risk of predation. In fact, attack rates by predators on dewlapping robotic lizards were no different to robots that remained unmoving and cryptic in the environment. Which means there doesn’t really seem to be a large cost from increased predation for animals that perform bouts of conspicuous behaviour.
But this wasn’t the biggest surprise.
The experiment included robotic lizards that kept the large, conspicuously coloured dewlap permanently extended so it was always visible. Think of peacocks with their massive tail trains or other animals that are spectacularly ornamented. These features are always visible and are not signals that can be turned on and off. My assumption was that these robotic lizards would be the hardest hit by predators.
This wasn’t the case at all. Predators actually avoided these robotic prey and to such an extent that the probability of attack was lower than the robotic lizards that remained cryptic and didn’t perform any conspicuous behaviour.
Photo montage of predator attacks left in the plasticine body of the robotic lizards
At first, I found this to be confusing and replicated the experiment over and over again. I even called in my partner Katrina Blazek who is a biostatistician to blind the data and independently perform the analyses (Katrina is also a skilled tailor and made all the robot dewlaps). I also dragged in my colleague Tom White who is an expert on animal colour discrimination to confirm that the dewlap really was as conspicuous to predators as I thought it was.
The data was robust.
This type of predator phobia actually helps explain the evolution of a completely different type of animal signal in nature: aposematic signals or warning signals that some prey evolve to explicitly advertise their location to predators to warn them against attack, usually because they’re toxic. Conspicuous poison dart frogs are an obvious example, so are ladybirds (or ladybugs).
The paradox is how these warning signals could evolve in the first place given the first individuals that tried to advertise their warning would be quickly eaten by predators that had no idea the signal was meant to advertise unprofitably until after the attack.
One of the key hypotheses that has been proposed to resolve this evolutionary paradox is that predators are highly conservative in the types of prey they go for. That is, they tend to avoid prey that look unusual in some way, even if those prey are more easily detected.
This is exactly what happened in this experiment. The robotic lizard with the permanently extended dewlap was ‘weird’ and so predators instead targeted the robotic lizards that either displayed intermittently or remained cryptic, both of which were more typical of their familiar prey.
The take home message is:
Follow your ridiculous idea and call on your friends to help.
(But don’t hold metal tools between your teeth. Your dentist will be very annoyed with you.)
A male A. distichus favillarum (a.k.a. a male Fav) extending it’s dewlap. This animal was photographed in the contact zone between orange- and yellow-dewlapped Favs.
Color and color-pattern research is a powerhouse in the study of evolution. Don’t believe me? I bet that at least one of your top five examples of evolution includes either color or color pattern. It is also very likely that some of the gene names you know by heart are from either color or color-pattern genes. Here’s an exercise: think which were the ‘textbook’ examples of evolution that you were taught in school. I’m sure that at least one of those included either color or color pattern. Here’s a famous example: Peppered moths. Another one? Deer mice. Another one? Heliconius butterflies. Another one? Coral snakes. What about color genes? Does MC1R ring a bell? Another one? ASIP (a.k.a. agouti)? Another one? Well, I should stop here before DJ Khaled sues me for copyright infringement.
Most of these early studies, however – and specially studies that attempted to unveil the genetic basis of color and color pattern – focused on melanin-based traits. The reason for this bias was simple: human color is melanin-based. This means that when these early studies took place, we knew more about the genetic basis of melanin synthesis than any other pigment by a long shot. As a consequence, melanin-based traits were ideal for candidate gene approaches – like the ones implemented in early color and color-pattern studies. As you and I know, though, the world isn’t black and white (see what I did here?). Color is all around us, and it plays all kinds of amazing roles, such as intra-specific communication (think dewlapping anoles), inter-specific communication (think dewlapping anoles), and crypsis (think non-dewlapping anoles). This means that, until recently, we didn’t have the tools to connect the genetic basis of most colorful traits to their phenotypes, specially in non-model organsisms.
Then, something happened: second generation sequencing came around. Illumina, a key player in second generation sequencing, was founded in 1998, the same year that Brazil lost the World Cup Final to France (many Brazilians, like me, think about time in four-year cycles due to the World Cup… and we don’t talk about 2014). With second generation sequencing, we could finally gather loads of data to do all kinds of glorified regressions (sorry generalized linear mixed models), and run computers for very long times so that we could try every feasible parameter combination (sorry Markov Chains) to identify candidate genes for the trait we are interested in. Connecting genotypes to phenotypes and understanding how both genotypes and phenotypes interact and change due to selection is, in fact, one of my main research interests.
I’m particularly interested in understanding the genetic basis of two phenotypes: those associated with color and color pattern, and those associated with the evolution of reproductive isolation.
Semi-aquatic Anolis lizards have some of the most fascinating ecologies, colour patterns, and behavioural strategies in the genus (though I may be biased). Twelve of these neotropical streamside specialists are distributed across much of mainland Latin America and on the two largest islands of the Caribbean. All are rarely found more than a few meters from a stream and some have been observed to consume semi-aquatic prey (or, in the case of A. vermiculatus, even small fish and freshwater crustaceans).
Range map of all 12 semi-aquatic anole species
A riparian lifestyle is also responsible for the signature move that unites all species of semi-aquatics—escape dives! As anyone who has encountered one of these lizards in the wild can attest, semi-aquatics will readily dive underwater when approached. They can stay down for awhile too—up to 18 minutes by my count (Mexico’s A. barkeri currently holds the record). Diving anoles have attracted the attention of tropical biologists for more than half a century now (e.g., Robinson 1962; Brandon et al. 1966; Campbell 1973; González Bermúdez and Rodríguez-Schettino 1982; Birt et al. 2001; Leal et al. 2002; Henderson and Powell 2009; Muñoz et al. 2015; Herrmann 2017) and this work has begun to fill out our natural history knowledge of these enigmatic lizards. However, understandably, most work to date has focused on what these lizards are doing when they’re not in the water. And, as it turns out, there’s a lot to learn if we look below the surface…
In 2009, while studying Anolis eugenegrahami, an endangered semi-aquatic anole from Haiti, Luke Mahler and Rich Glor noticed that an individual they had just released into a clear, shallow stream proceeded to repeatedly exhale and re-inhale an air bubble as it clung to the rocky bottom. Luke and Rich had to move to their next site later that day, so weren’t able to learn more. Sadly, a follow-up field season was cancelled in the aftermath of the 2010 Haiti earthquake.
Years later, when I started my MSc thesis on aquatic anoles in at the University of Toronto, Luke shared this observation with me. When an anole does something once, another anole somewhere else usually does it convergently, so we couldn’t help but wonder whether aquatic anole species elsewhere also exhibited this apparent “rebreathing” behavior. So, when I was planning my first field season in Costa Rica, on a hunch, we purchased an oxygen microsensor, and I set out to establish whether this intriguing behaviour occurred in any other semi-aquatic anoles.
Costa Rica field team, 2017
Colombia field team, 2018
Mexico field team & friends, 2018
The aquatic anoles did not disappoint! During my Master’s, along with an amazing team of colleagues, I visited stream habitats in Costa Rica, Colombia, and Mexico, studying A. oxylophus, A. aquaticus, A. maculigula, and A. barkeri along with the non-aquatic anoles we were able to find at each site. I found that each of these species routinely performed the same behaviour that Luke and Rich had observed in A. eugenegrahami! We named this phenomenon “rebreathing” after the SCUBA apparatus. All of the semi-aquatics we observed performed rebreathing extensively during experimental submersions and are from five phylogenetically distinct lineages, showing a pattern of remarkable behavioural convergence!
As I was conducting these experiments, “rebreathing” was independently discovered in Anolis aquaticus by Lindsey Swierk (see image below, and Lindsey’s 2018 AA post). Lindsey is the world authority on Costa Rica’s diving anoles, and has reams of firsthand knowledge about their ecology and behavior. So we did the obvious thing when we found out about her observation – we invited her to join our project. We managed to deliver our oxygen sensor to Lindsey in Costa Rica via a colleague with overlapping travel plans, and she helped fill out our oxygen use data set for the Costa Rican diving anole species. In addition, Luke tested Anolis lynchi in Ecuador, and various non-aquatic species during fieldwork there and elsewhere (Dominican Republic, Jamaica) to help round out the data set.
A diving A. aquaticus performing rebreathing (Photo: Lindsey Swierk)
Speaking of non-aquatic anoles, what role do they play in this story? An interesting one, as it turns out. Rebreathing clearly seemed fascinating, but one possibility was that it was relatively ubiquitous and that all anoles would rebreathe if you submerged them. To find out, we did just that, carefully dunking aquatic and non-aquatic anoles alike in aquaria or buckets at our field sites.
What we discovered is that most non-aquatic anole species are indeed capable of basic rebreathing, but for the most part, they don’t rebreathe anything like the semi-aquatics do. If they rebreathed at all, non-aquatic species tended to do so only occasionally and irregularly (usually only one or a few re-inhalations). Since semi-aquatic anoles performed rebreathing extensively and consistently, while non-aquatics were capable of the basic components of rebreathing, but did not rebreathe regularly, we think consistent rebreathing may have evolved when natural selection found a new utility for a trait that all anoles possess—hydrophobic skin. The hydrophobicity of anoles’ scales is likely what enables the air bubble to adhere to the diving anoles’ heads (and thereby also enables re-inhalation). All anoles therefore appear to be capable of forming a thin layer (or ‘plastron’) of air along their scales during submersion, but only semi-aquatics appear to make regular use of this ability (see plot below). Hydrophobic skin evolved in anoles long before it was co-opted for rebreathing in stream-dwelling species, and likely had nothing to do with the use of aquatic habitats. In this way, the innovation of underwater rebreathing apparently owes its origins to a fortuitous ‘evolutionary accident.’
Semi-aquatic anoles rebreathed more frequently than non-aquatics (from Boccia et al. 2021)
Although we observed regular rebreathing in all aquatic anole species we studied, we discovered some interesting differences in the way they go about it. There were three main locations along the head to which diving anoles would exhale bubbles (see image below). We noted some variation in the bubble positions used by semi-aquatics, perhaps indicating that are multiple ways to achieve the same rebreathing function.
Bubble positions and use percentages for five semi-aquatic anole species (Drawing credit: Claire Manglicmot)
To determine if ‘rebreathing’ was truly involved in respiration, we used our oxygen sensor to measure the oxygen concentration of the bubbles produced by diving semi-aquatics. This is not as easy as it sounds; bubbles were frequently re-inhaled quickly and diving anoles do not take kindly to being accidentally poked in the nose with a probe. But we persevered, and found that bubble oxygen levels decreased through time, consistent with the respiration hypothesis!
Experimental submersion of an A. maculigula male in Colombia; field assistant James is holding oxygen and temperature sensors ready.
We found some evidence that oxygen decrease followed an exponential decline curve, suggesting either that anoles extract some additional oxygen from the surrounding water by rebreathing (thus slowing the rate of oxygen loss from the bubble), or that metabolic rate (and thus oxygen demand) drops over time during submersion (see figure below). We compared our results to diving insects that use a similar rebreathing apparatus while submerged and found that anole oxygen use matches up well with our expectations for their sizes, and that the metabolic rate of anoles is probably too high for them to remain underwater indefinitely using oxygen captured from the water by the rebreathing bubble (the same is true for the largest diving insects).
Plots A-E show bubble oxygen concentrations through time for five species of semi-aquatic anole. Plot F shows a sham trial (in which I mimicked the bubble movements of diving anoles with a submerged syringe; no oxygen declines were observed). Plot G shows semi-aquatics (blue) and diving insect oxygen consumption rates (black) by mass. The dotted line indicates the theoretical limit of oxygen replenishment per second that could be supported by a bubble gill structure. From Boccia et al. 2021.
The consistency with which unrelated semi-aquatic anoles rebreathed suggests that rebreathing is adaptive for semi-aquatic living; however, our data currently do not allow us to favour a particular physiological functionality for this behaviour. Our top three (not mutually exclusive) hypotheses are: 1) rebreathing allows anoles to access air trapped in their head cavities or within the plastron, which might otherwise not be incorporated into their air supply; 2) the rebreathing bubble functions as a physical gill (as has been observed in diving insects), allowing diving semi-aquatics to extract some oxygen from the surrounding water; and 3) bubble exhalation and re-inhalation allows anoles to remove excess carbon dioxide which builds up during dives. We hope to investigate these possibilities during future work!
We published this work in Current Biology (Boccia et al., Repeated evolution of underwater rebreathing in diving Anolis lizards, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.04.040)
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