Anole Annals

Hello Anolophiles!

Could anyone use a free undergraduate field assistant this summer for any squamate evolution or conservation projects, preferably outside of the U.S.? I am an undergraduate student in Drs. Emily Lemmon and Frank Burbrink’s labs with $5k from a merit based scholarship that I need to spend this summer on my “educational enrichment”. I’m hoping to use my funding to support any living, travel, research, and lost wages expenses associated with being a field assistant for anyone doing squamate research any time between May and the end of July 2024. My current research focuses on phylogeography of North American herps, but I’m eager to assist with any project on any squamate, ideally though not necessarily in the neotropics. I’m looking to learn new field techniques, work with a new biological system, have wonderful discussions on all sorts of herpetological matters with a new research mentor(s), and find inspiration for anticipated upcoming graduate studies.

I have experience with molecular lab methods (DNA extraction, gel electrophoresis, Qubit quantification), museum methods (toe clipping and dissection for tissue extraction, posing and formalin fixing specimens), field methods (lassoing lizards, nocturnal herpetofaunal field surveys, dipnetting, roadcruising), data visualization and statistical analyses in R, and science communication. I’m happy to work in challenging and remote field conditions; the more bugs, venomous snakes, and stinging plants the better, though I would like to come back alive and in one piece. Let me know if you could potentially use a field assistant at povenika@gmail.com , and I would be happy to send along a CV and letters of recommendation.

I woke up after spending the whole night in motion and listening to the engine noise as a crib song. Some years ago, it had been a crab boat in Alaska and it was now equipped to do biological research in the northwest of Mexico. I am here after a few talks with Armando Escobedo; he told me about an amazing project to describe the diet of the lizards living on Maria Cleofas Island. My experience with lizards and trophic niches was scare, but I was motivated to learn about it. “Not every day I have the opportunity to meet the Marías Islands.”

This island with a biblical name, María Cloefas Island, is home to four lizard species. Anolis nebulosus (Clouded Anole), Aspidoscelis communis (Colima Giant Whiptail), Ctenosaura pectinata (Western Spiny-tailed Iguana), and a recently described endemic leaf-toed gecko, Phyllodactylus cleofasensis. After taking  breakfast on the boat deck, Rafael (an undergraduate student like me), Armando and I were taken to the island in a small boat to evaluate the dietary variation of lizard species.

The main goal of our study was to describe the trophic niche of the lizard community, given that the species differ in foraging strategy. We expected to observe higher prey diversity in the active forager (Aspidoscelis communis) compared to the three sit-and-wait foraging species. Also, due to their different habitat use, we expected that the arboreal species (Anolis nebulosus and Ctenosaura pectinata) would share similar dietary niches, and that the terrestrial species (Aspidoscelis communis) might exhibit a partial dietary niche overlap with them. Finally, we expected that the saxicolous and nocturnal species (Phyllodactylus cleofasensis) would have the most distinct prey diversity.

We visited the island during eight weeks between 2017 and 2018. We performed diurnal and nocturnal surveys in all available habitats to manually capture individuals of the four lizards on the island. We obtained stomach contents from a total of 115 individuals using the stomach flushing technique. From this total of samples, 37 belonged to Anolis nebulosus, 11 to Aspidoscelis communis, 36 to Phyllodactylus cleofasensis, and 31 to Ctenosaura pectinata. Despite the movement of the ship, I could check the stomach contents under the stereoscope, and begin to determine the occurrence of each prey item eaten by each lizard species, for later calculatation of their prey diversity and determination of whether the lizards were generalists or specialists, as well as their degree of inter-individual specialization. Furthermore, we looked for similarities within species; therefore, we calculated their food resource overlap and their similarity index. In addition, we performed some analyses to examine differences in each food niche method and to determine if there was a difference in the prey eaten by each species and between years of surveys.

We discovered, surprisingly, a wide variety of arthropods within the stomach contents of the lizards, regardless of their foraging strategy and habitat use! We identified 19 types of prey items such as insects, arachnids, gastropods, and centipedes, with a clear prevalence of beetles, spiders, and vegetation matter in the diets of the lizards. The diet of Anolis nebulosus was the most diverse, composed of 15 items, mostly arthropods, some vegetation matter, and their own skin remains. Aspidoscelis communis consumed 11 prey items, mostly arthropods, while Phyllodactylus cleofasensis consumed 10 prey items, mostly arthropods, some vegetation matter, and their own skin remains. We found nine items for Ctenosaura pectinata; surprisingly, we found a lower amount of vegetation matter, and the rest were arthropods.

The Clouded Anole showed the highest richness of prey items in their stomachs; however, it was not the species with the highest prey diversity. Despite this, Anolis nebulosus exhibited greater prey diversity compared to other insular and continental populations. This expansion of its trophic niche could be attributed to the low predation pressure and high intraspecific competition on the island, which also influenced the phenotypic and behavioral plasticity of the species.

The actively foraging species Aspidoscelis communis showed the highest diversity of prey values. Phyllodactylus cleofasensis showed a significant variety of prey, while Ctenosaura pectinata displayed the lowest values across all three measures of food niche diversity. Thus, Anolis nebulosus, Aspidoscelis communis¸ and Phyllodactylus cleofasensis were generalist species with an increase in inter-individual specialization, while Ctenosaura pectinata remained close to the threshold between specialist-generalist feeding habits and little or no inter-individual specialization.

Based on habitat preferences, we expected that arboreal species (Ctenosaura pectinata and Anolis nebulosus) would exhibit similar prey diversity, and also that the actively foraging species, Aspidoscelis communis, would exhibit a greater prey variety compared to Ctenosaura pectinata. Our results through the different trophic niche approaches aligned with these expectations. Surprisingly, Phyllodactylus cleofasensis, despite being a nocturnal forager, demonstrated similar individual specialization as Anolis nebulosus.

The results from the discriminant functional analysis showed distinctive dietary patterns among lizard species. Aspidoscelis communis exhibited a diet divergence from the other lizard species; part of its diet could potentially be confused with Ctenosaura pectinata’s diet, while the diet of Phyllodactylus cleofasensis showed similarity with the diet of Anolis nebulosus (and vice versa). Finally, the diet of Ctenosaura pectinata had a relatively low overlap with Anolis nebulosus.

Our research on the lizard community of María Cleofas Island has not only demonstrated the wide dietary diversity among species, but has also expanded our understanding of trophic relationships in island ecosystems. Moreover, with this study, we have challenged conventional assumptions about resource partitioning and dietary niche diversity in insular ecosystems.

Don’t forget to take a look at the original paper; you will find some other amazing observations!

This post is an update of one from 2020. Below is the old post based on a presentation by Rachana Bhave at the 2020 SICB meetings. Rachana has now done the genetic parentage studies and published the cool paper in Behavioral Ecology. Here’s the abstract of the paper:

In promiscuous species, fitness estimates obtained from genetic parentage may often reflect both pre- and post-copulatory components of sexual selection. Directly observing copulations can help isolate the role of pre-copulatory selection, but such behavioral data are difficult to obtain in the wild and may also overlook post-copulatory factors that alter the relationship between mating success and reproductive success. To overcome these limitations, we combined genetic parentage analysis with behavioral estimates of sizespecific mating in a wild population of brown anole lizards (Anolis sagrei). Males of this species are twice as large as females and multiple mating among females is common, suggesting the scope for both pre- and post-copulatory processes to shape sexual selection on male body size. Our genetic estimates of reproductive success revealed strong positive directional selection for male size, which was also strongly associated with the number of mates inferred from parentage. In contrast, a male’s size was not associated with the fecundity of his mates or his competitive fertilization success. By simultaneously tracking copulations in the wild via the transfer of colored powder to females by males from different size quartiles, we independently confirmed that large males were more likely to mate than small males. We conclude that body size is primarily under pre-copulatory sexual selection in brown anoles, and that postcopulatory processes do not substantially alter the strength of this selection. Our study also illustrates the utility of combining both behavioral and genetic methods to estimate mating success to disentangle pre- and post-copulatory processes in promiscuous species.

And here’s the post from 2020:

If you’ve ever tried to note how often lizards mate, you’ve likely found yourself staring at an individual for hours at a time, sometimes with little to no movement at all, let alone observing copulations! Further, if you’re unable to catch the animal after your behavioral observations, you may not be able to draw any conclusions about traits that influence how successful an individual is at mating with another.

Rachana Bhave, a fourth year PhD candidate in Bob Cox’s lab at University of Virginia, studies pre- and post-copulatory sexual selection in brown anoles (Anolis sagrei). One of her interests includes estimating mating rates in the wild and, in particular, testing if traits such as body size directly influence these rates. Given the power required to detect selection statistically, using simple behavioral observations can be inefficient. Further, because selection is a measure of covariance between phenotype and fitness, one needs phenotypic values for each individual within her analyses. Thankfully, Rachana was able to come up with a robust technique to estimate mating rates using an island population of brown anoles in Florida: fluorescent powders!

To understand how size affects mating rate in the brown anole, Rachana and colleagues caught 153 adult male lizards in May and 128 adult male lizards in July, weighed them, and then assigned them to one of four fluorescent powder treatments. Each mass quartile was painted with a unique color of fluorescent powder on their cloaca and released to their initial capture location. After two days, all females on the island were captured and their cloaca were examined under UV light to look for the presence and color of fluorescent powder, which would suggest that she mated with a painted male. Using this technique, Rachana found that within two days, 24% of the captured females had mated in May and 48% had mated in July. These rates were shockingly high for such a short time frame!

Further, she found that both larger males and larger females mated significantly more than smaller males and females across the two sampling periods. Interestingly, 2% of females had multiple colors on their cloacas, which suggests they mated multiple times with males from different size classes in the two-day span. Because multiple matings within the same size class would be undetectable, this is likely an underestimation of multiple matings in the wild.

Next, Rachana plans to quantify male reproductive success using genetic parentage analysis to begin to tease apart how pre- and post-copulatory selection influences selection. We are all looking forward to her results next year! Meanwhile, you can take a look at her poster to find out more on her website.

We recently published (together with Alex Tinius and Luke Mahler) a paper in Evolution in which we explore how the strength of the femur of Anolis lizards might be attained by two independent mechanisms, and how the interaction between them represents a previously unexplored axis of phenotypic diversity! I will try to summarize the main ideas here.

Since the beginning of my PhD, I’ve had almost unlimited access to the CT-scanner of the lab (the Mahler Lab at the University of Toronto) and to hundreds of anole specimens which we borrowed from many herpetological collections (we are very grateful to them!). The scanner allowed me to enter a world otherwise inaccessible: that of muscles, internal organs, fat, and bones. These are traits we are not used to thinking, but their characteristics can be described as well as we do with more familiar traits like the length of a limb or the color of a dewlap.

In the beginnings of this project, I focused on the bone mineral density (BMD) of the femur. BMD is positively associated with the bending strength of the bone and, given the classical ecomorphological relationships shown by anoles which link limb morphology and habitat use, I expected the BMD of the femur to be relevant to their evolution. With this in mind, I started collecting BMD data from as many anole species as I could.

At some point while reading papers, I came across the fact that not only mineral density, but also the cross-section shape of the femur (or any other long bone) influences its bending strength (Currey, 2003; Dumont, 2010; Jepsen, 2011). In theory, if you have a cylindrical hollow bone (like a femur) with a given mass and density, the best way to make it stronger is to redistribute the bone tissue such that the walls are as far as possible from the center of the cylinder (i.e., to make the walls thinner). In other words, if r is the radius of the inner ‘hollow’ volume, and R is the total radius of the femur, increasing r/R increases the bending strength of the bone. This, in consequence, results in a bone with a larger diameter and a relatively larger ‘hollow’ volume in its center:

However, you cannot go on increasing BMD and the hollowness of the bone forever. One reason for this is that the bone eventually becomes too heavy to be functionally viable. Higher density increases weight for obvious reasons, but increasing the hollowness of a long bone eventually increases its weight because the ‘hollow’ part of the bone is actually not hollow in live animals, but is full of fat which, of course, contributes to the total mass of the bone.

Bone mineral density and “hollowness” then represent two independent ways to increase the bending strength of a long bone. But since these two determinants of bone bending strength are limited by their cost to fitness (e.g., due to excessive weight), we expected a certain balance between the two. Some sort of trade-off in which a species either has high-density, thick-walled bones or low-density, thin-walled bones, or any intermediate strategy between these two extremes:

Excited by this new idea, we started complementing the BMD measurements with shape measurements (r/R). Would we find the expected trade-off? As we started plotting species-level data for femur BMD and shape, it started to be clear that a negative association was there. Eventually, our results indicated that there is a strong evolutionary correlation between the hollowness of the femur (represented by r/R) and BMD, both considering only males or only females:

This was very exciting because the association was clear and strong! The construction of anole femora seemed to be limited to combinations found in a narrow band in phenotypic space, as expected. But this apparent constraint can also be understood as an opportunity for phenotypic diversification. If both variables, BMD and shape, are important enough to influence bone bending strength and simultaneously represent costs to fitness, a hypothetical trade-off between them would result in a spectrum of viable strategies to build a femur. In other words, there would be more than a single way to build a strong, viable femur!

However, we still had to test whether all these strategies were resulting in equivalent levels of bone strength. For this we calculated a bone strength index (BSI), a variable that depends on both BMD and shape and which has been shown to be consistent with experimentally measured bone strength. If the different combinations along the spectrum of available strategies can really provide equivalent levels of performance, the negative relationship between BMD and shape should align with performance isolines in that same phenotypic space (i.e., the negative relationship should be parallel to isolines of equivalent performance).

That’s roughly what we found! After accounting for size, the spectrum of existing strategies seemed to align with isolines of performance, meaning that a femur with a particular strength could be obtained through different shape-BMD combinations. This is similar to a many-to-one mapping pattern, except that here we are not talking only about morphological traits, but the interaction between architectural design and the material properties of a structure.

 

Finally, we tested whether anole species under different selection pressures had actually evolved to use different parts of the spectrum, taking advantage of this evolutionary flexibility. To do this, we compared the strategies used by island and mainland anole species.

Specifically, we hypothesized that mainland anoles, short-lived species with faster life paces (Andrews, 1976, 1979; Lister and Aguayo, 1992), would not have enough time to reach high mineralization levels in their bones (it’s been shown in other species that bone mineralization is a lengthy process whose peak is often reached way after sexual maturity; Bala et al., 2010; Bonjour et al., 1994) and thus would tend to evolve strategies based on higher r/R values (i.e., mainland species should evolve hollower bones to compensate for low mineral density). We expected island species to show, on average, the opposite strategy. Different pieces of evidence (detailed in the paper) supported this hypothesis!:

Previous medical papers had proposed and, to certain extent, demonstrated the compensatory relationship between BMD and bone architecture in the bones of humans, mice, and others. However, a macroevolutionary relationship between both variables had not been tested yet. Our results show a species-level pattern consistent with this compensatory mechanism.

Overall these results show how a many-to-one system of form to function can include not only morphological traits, but also the material properties of biological structures. This suggests that the way phenotypes evolve is, as usual, more complex than we previously thought, especially when certain traits are not so easy to study.

This many-to-one system has not only resulted in the evolution of phenotypic diversity in a single trait, but might have also favored the diversification of anoles under contrasting selection pressures. Although femur evolution is constrained by the need to achieve a minimum performance level, plus the physical and viability limits imposed on its structure, the possibility that its performance has two independent determinants (BMD and shape) represents an opportunity for the evolution of alternative phenotypes. This flexibility might have facilitated anole evolution across environments where one determinant is constrained, which could have been the case for mainland anoles as hypothesized in our paper…

I invite you to read the original paper for more details!

 

References

Andrews, R. M. (1976). Growth rate in island and mainland anoline liz- ards. Copeia, 1976(3), 477–482. https://doi.org/10.2307/1443362

Andrews, R. M. (1979). Evolution of life histories: A comparison of Anolis lizards from matched island and mainland habitats. Breviora, 454, 1–51.

Bala, Y., Farlay, D., Delmas, P. D., Meunier, P. J., & Boivin, G. (2010). Time sequence of secondary mineralization and microhardness in cortical and cancellous bone from ewes. Bone, 46(4), 1204–1212. https://doi.org/10.1016/j.bone.2009.11.032

Bonjour, J. P., Theintz, G., Law, F., Slosman, D., & Rizzoli, R. (1994). Peak bone mass. Osteoporosis International, 4, S7–S13.

Currey, J. D. (2003). The many adaptations of bone. Journal of Bio- mechanics, 36(10), 1487–1495. https://doi.org/10.1016/s0021- 9290(03)00124-6

Dumont, E. R. (2010). Bone density and the lightweight skeletons of birds. Proceedings of the Royal Society of London. Series B: Biological Sciences, 277(1691), 2193–2198. https://doi.org/10.1098/rspb.2010.0117

Jepsen, K. J. (2011). Functional interactions among morphologic and tissue quality traits define bone quality. Clinical Orthopaedics and Related Research, 469(8), 2150–2159. https://doi.org/10.1007/ s11999-010-1706-9

Lister, B. C., & Aguayo, A. G. (1992). Seasonality, predation, and the behaviour of a tropical mainland anole. Journal of Animal Ecology, 61(3), 717–733. https://doi.org/10.2307/5626

Toyama, K. S., Tinius, A., & Mahler, D. L. (2023). Evidence supporting an evolutionary trade-off between material properties and architectural design in Anolis lizard long bones. Evolution, qpad208.

We recently published a chromosome-scale assembly of the slender anole (Anolis apletophallus) genome, a species that has been studied for decades at the Smithsonian Tropical Research Institute in Panama.

Here is the abstract: The slender anole, Anolis apletophallus, is a small arboreal lizard of the rainforest understory of central and eastern Panama. This species has been the subject of numerous ecological and evolutionary studies over the past 60 years as a result of attributes that make it especially amenable to field and laboratory science. Slender anoles are highly abundant, short-lived (nearly 100% annual turnover), easy to manipulate in both the lab and field, and are ubiquitous in the forests surrounding the Smithsonian Tropical Research Institute in Panama, where researchers have access to high-quality laboratory facilities. Here, we present a high-quality genome for the slender anole, which is an important new resource for studying this model species. We assembled and annotated the slender anole genome by combining three technologies; Oxford Nanopore, 10X Genomics linked-reads, and Dovetail Omni-C. We compared this genome with the recently published brown anole (Anolis sagrei) and the canonical green anole (Anolis carolinensis) genomes. Our genome is the first assembled for an Anolis lizard from mainland Central or South America, the regions that host the majority of diversity in the genus. This new reference genome is one of the most complete genomes of any anole assembled to date and should facilitate deeper studies of slender anole evolution, as well as broader scale comparative genomic studies of both mainland and island species. In turn, such studies will further our understanding of the well-known adaptive radiation of Anolis lizards.

And here is a slightly longer summary of what we did (and some results): We used a hybrid genome assembly by combining three technologies: Oxford Nanopore, 10X Genomics linked-reads, and Dovetail Omni-C. We annotated our slender anole genome using the Dovetail Genomics annotation pipeline and compared our genome with the recently published brown anole (Anolis sagrei) and the canonical green anole (Anolis carolinensis) genomes. We also estimated the repeat elements composition and repetitive landscape using the RepeatModeler and RepeatMasker pipelines.

After several rounds of improvement, our final genome assembly for the slender anole was ~2.4 Gbp in size with with a scaffold N50 of 154.6 Kbp and a GC content of 43.8%. The slender anole genome was thus substantially larger than both the green anole (1.89 Gbp) and brown anole (1.93 Gbp) genomes. Our annotation using the Dovetail pipeline identified a total of 46,763,836 bp coding regions and a total of 33,912 gene models. The number of gene models identified for the slender anole was higher than that of both the green anole (22,292) and brown anole (20,033).

Authors: Renata M. Pirani^1,2*†, Carlos F. Arias^2,3, Kristin Charles¹, Albert K. Chung^2,4, John David Curlis^2,5, Daniel J. Nicholson^2,6, Marta Vargas², Christian L. Cox^2,7, W. Owen McMillan², Michael L. Logan^1,2

 

Affiliation:

(1) Department of Biology and program in Ecology, Evolution, and Conservation Biology, University of Nevada, Reno, Reno, 89557, United States

(2) Smithsonian Tropical Research Institute, Panama City, Panama

(3) Data Science Lab, Office of the Chief Information Officer, Smithsonian Institution, Washington, 20013, United States

(4) Department of Ecology and Evolutionary Biology, Princeton University, Princeton, 08544-2016, United States

(5) Department of Ecology and Evolution, University of Michigan, Ann Arbor, 48109-1085, United States

(6) University of Texas, Arlington, Arlington, 76019, United States

(7) Florida International University, Miami, 33199, United States

*Corresponding author: renatampirani@gmail.com

† Present address: Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 90095, USAFigure 1 Figure_2

This week, anoles are in the genomic spotlight for three papers– Pirani et al. (2023), Taft et al. (2023), and Farleigh et al. (2023). I’ve briefly highlighted each below, but check em’ all out!

New literature alert!

 

Pirani et al. (2023) usher in a new age of Anolis lizard biology by publishing the first mainland anole reference genome– a Panamanian species, Anolis apletophallus. It’s a great assembly (scaffold N50 of 154 Mb with an estimated 2.4 Gbp genome), and will be an excellent resource for the community as we continue to expand our genomic stockpile for this group. Give their new paper a read in G3: Genes, Genomes, and Genetics.

 

Taft et al. (2023) provide the first reference genomes for two species of Bradypodion, the dwarf chameleons. Synteny analysis (looking at gene order conservation across chromosomes) between the two chameleons and Anolis sagrei demonstrates relatively conserved genomic structure across greater than 150 million years of divergence!

 

Farleigh et al. (2023) investigate the natural hybridization of two Puerto Rican grass anoles–A. pulchellus andA. krugi–using a ddRAD approach (genome-wide SNPs) to understand the directionality of introgression, and how this pattern of introgression is differentially reflected in the genomes of populations across the island.

reprinted with permission

How Do You Create a Book about All the Reptiles of a Mega-diverse Country?

Reptiles of Ecuador | Story of the book

By Alejandro Arteaga. August 2023.

 

Everyone grasps the fundamental concept of a field guide: a book designed to aid in the identification of species in the wild while offering pertinent information about them. Unlike encyclopedias, field guides strive to provide information about EVERY species within a specific animal or plant group in a given geographical area. Field guides can be comprehensive when the number of species covered is limited. For instance, a field guide centered on the crocodiles of the Americas would include only ten species.

However, how do you create a comprehensive field guide about a species-rich animal group in a mega-diverse country?

To answer this question, I will tell you the story of the Reptiles of Ecuador book, a meticulously created field guide that has gained attention due to its expansive scope, novel photographic style, open-access nature, and funding strategy.

The idea for a Reptiles of Ecuador book emerged in 2010 from a casual conversation with a wildlife photographer friend. I posed a question:

Why are there field guides for birds and mammals of Ecuador, but none dedicated to reptiles?

His response was candid: “I’m not sure… you should consider writing one.”

Andrea Ferrari, editor in chief of Anima Mundi, enjoys reading the Amphibians and Reptiles of Mindo book. Photo by Lucas Bustamante.

His suggestion caught me off guard. I had never contemplated writing a book and, even though I found the concept intriguing, at 18 years old and just commencing my studies in biology, I did not feel qualified for such a task.

Throughout my childhood, I had drawn inspiration from field guides spanning diverse animal groups, ranging from insects to birds, and more recently, amphibians and reptiles. Consequently, I had a general idea of how a field guide about herpetofauna should look like.

But I had no idea how write one.

How could I possibly compile a book encompassing ALL reptile species within a country as biodiverse as Ecuador? The nation boasts a staggering 500 reptile species!

There are 500 species of reptiles in Ecuador, including the Emerald Tree-Boa (Corallus batesii), a snake that lives in the canopy of the Amazon rainforest and is seen no more than once every few years. Photo by Jose Vieira.

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