Category: New Research Page 3 of 66

Exercise and the Immune System in Green Anoles

Female Green Anole

Exercise has many effects on your body, most of which are good, and is why we humans do it to stay healthy. However, some of those changes, especially under very intense regimens, can have unseen consequences that might be bad. Your immune system, for example, responds to different types of exercise (aerobic endurance versus anaerobic resistance) by altering which branch of your immune system is dominant at that time. Both kinds of exercise tend to increase the more specific ‘humoral immunity’ (B-cell immunity below) over the more general ‘cell-mediated immunity (T-cell immunity below), though the routes to get there are very different for the two kinds of exercise. However, most of what we know about exercise-immunity tradeoffs is from humans and rodents. What about in other animals that have limited access to resources? Might simple energy limitation cause overall immunity suppression when energy is diverted to athletic performance?

My former student Andrew Wang and I studied this experimentally with green anoles. We trained lizards for endurance on a treadmill, or for resistance with weights on a racetrack, for 9 weeks, and compared those to a sedentary control group. Both of these types of locomotion are important to anoles in the wild, and the training schedule was meant to simulate the high end of movement patterns in nature. We then subjected them to three immune challenges: (1) swelling response to phytohemagglutinin (cell-mediated immunity), (2) antibody response to sheep red blood cells (humoral immunity), and (3) wound healing ability (integrated response across all parts). We expected that if simple energy limitation explained tradeoffs, all immune measures would decrease, with endurance-trained suffering the most. If protein limitation was the reason for tradeoffs, then we expected all immune measures to decrease, with sprint-trained suffering the worst. Finally, if the response is due to changes in molecular pathways specific to type of exercise, we expected humoral immunity to be favored over cell-mediated in both trained groups.

Figure 1 from Wang and Husak (2020)

Our results did not support only one of our hypotheses. Endurance-trained lizards had the lowest cell-mediated immunity, whereas sprint-trained had the lowest wound healing ability. Antibody production did not differ among treatments. Our hypothesis of sprint-trained lizards (or even endurance-trained) having the lowest overall immune function was not supported, suggesting that energy limitation alone does not explain immune system alteration. For sprint-trained lizards, energy was likely important, since wound healing, an expensive task, went down the most in that group. For endurance-trained lizards, though, the change in T helper cell production favored humoral over cell-mediated immunity. Since both types of exercise favor humoral immunity, it was not too surprising that antibody production did not differ among treatments. Lots of questions remain to be answered, though!

What does this all mean? In nature, individuals vary dramatically in how much, and for how long, they move around their environment. Those that are more active, thus likely have different immune capabilities compared to more sedentary individuals. It would be very interesting to see how natural variation in survival strategies, high-performance versus high-immunity, affected success in nature. This is a wide-open field for anoles and other reptiles!

Source: Wang, A. Z. and J. F. Husak. 2020. Endurance and sprint training affect immune function differently in green anole lizards (Anolis carolinensis). Journal of Experimental Biology

Clouded Anoles: How Islands Affect Morphology

Ecogeographical rules attempt to simplify ecological and evolutionary processes that shape morphology. In a cool study published this summer in Current Zoology, Anaya-Meraz and Escobedo-Galván (2020) examine the combined effect of Rensch’s Rule and van Valen’s Island Rule in Clouded Anoles. Specifically:

Rensch’s Rule: within lineages, sexual dimorphism decreases in magnitude with increased body size when females are the larger sex but increases in magnitude when males are the larger sex.

The center black line indicates 1:1 male to female size, the top line and bottom lines indicate male- and female-biased size dimorphism, respectively. *Adapted from Piross et al. 2019.

van Valen’s Island Rule: describes the tendency of diminutive and large mainland species to trend toward gigantism or dwarfism on islands, respectively, due to competitive factors.

*Adapted from Lomolino, 2005

In their paper, Anaya-Meraz and Escobedo-Galván ask, how does Clouded Anole (Anolis nebulosus) sexual size dimorphism change when the Island Rule could be in effect?

Using 305 Clouded Anole museum specimens, they found that sexual size dimorphism differs between the mainland and island populations. While all populations revealed variation in the degree of sexual size dimorphism, populations on the Islas Tres Marías uniformly possess male-body size bias. But on the mainland, 40% of the populations had the opposite pattern, female-body size bias.

Intriguingly, Anaya-Meraz and Escobedo-Galván note that in the Clouded Anole, island males spend almost 50% more of their waking period engaged in some form of social interaction (Siliceo-Cantero et al. 2016). This is offered as an explanation for why male Clouded Anoles also have larger dewlaps among the Tres Marías populations.

In lizards, the Island Rule may not necessarily stand out as a trend (Meiri, 2007), but we see from Anaya-Meraz and Escobedo-Galván’s study that male Clouded Anoles are larger on islands. On the Antillean Islands, the magnitude of sexual size and shape dimorphism of anoles decreases with increased anole species diversity (Butler et al., 2007). The Islas Tres Marías populations follow this pattern in having increased sexual size dimorphism when not competing with other anole species.

*Adapted from Poe et al. 2017.

Overall, Clouded Anole body and dewlap sizes are larger in insular populations while Rensch’s Rule does not show a clean pattern in this species. However, as noted by the authors, it is important to consider the adaptive force of being on an island versus the ancestral condition. To truly understand morphological evolution within a species and across the genus we need to know body size trends of closely related species. Moreover, some researchers are discouraging studies that determine the universality of ecogeographical rules in favor of integrative approaches based around hypothesis testing (Lomolino et al. 2006, Lokatis & Jeschke, 2018).

What do you think? Is there room for using ecogeographical rules within an integrative framework (See Benítez-López et al. 2020)? Or do ecogeographical rules obscure true drivers of adaptation?

References:

Anaya-Meraz, Z. A., and A. H. Escobedo-Galván. 2020. Insular effect on sexual size dimorphism in the Clouded Anole Anolis nebulosus: when Rensch meets Van Valen. Current Zoology, doi: 10.1093/cz/zoaa034.

Benítez-López, A., L. Santini, J. Gallego-Zamorano, B. Milá, P. Walkden, M. A. J. Huijbregts, and J. A. Tobias. 2020. The island rule explains consistent patterns of body size evolution across terrestrial vertebrates. bioRxiv 2020.05.25.114835. Cold Spring Harbor Laboratory.

Butler, M. A., S. A. Sawyer, and J. B. Losos. 2007. Sexual dimorphism and adaptive radiation in Anolis lizards. Nature 447:202–205. Nature Publishing Group.

Lokatis, S., and J. M. Jeschke. 2018. The island rule: an assessment of biases and research trends. Journal of Biogeography 45:289–303. Wiley Online Library.

Lomolino, M. V. 2005. Body size evolution in insular vertebrates: generality of the island rule. Journal of Biogeography 32:1683–1699.

Lomolino, M. V., D. F. Sax, B. R. Riddle, and J. H. Brown. 2006. The island rule and a research agenda for studying ecogeographical patterns. Journal of Biogeography 33:1503–1510.

Meiri, S. 2007. Size evolution in island lizards. Global Ecology and Biogeography, 16:702-708.

Poe, S., A. Nieto-montes de Oca, O. Torres-Carvajal, K. De Queiroz, J. A. Velasco, B. Truett, L. N. Gray, M. J. Ryan, G. Köhler, F. Ayala-Varela, and I. Latella. 2017. A Phylogenetic, Biogeographic, and Taxonomic study of all Extant Species of Anolis (Squamata; Iguanidae). Systematic Biology 66:663–697.

Piross, I. S., A. Harnos, and L. Rózsa. 2019. Rensch’s rule in avian lice: contradictory allometric trends for sexual size dimorphism. Scientific Reports 9:7908. Nature Publishing Group.

Siliceo-Cantero, H. H., A. García, R. G. Reynolds, G. Pacheco, and B. C, Lister. 2016). Dimorphism and divergence in island and mainland Anoles. Biological Journal of the Linnean Society, 118:852–872.

This post was originally published on biomh.wordpress.com.

Riding the Ups and Downs: Naturally Fluctuating Nest Temperatures Are Important for Proper Development in Brown Anoles

A cartoon of a brown anole hatching from the egg. This cartoon was created by Francesca Luisi for Inside JEB.

A common challenge facing biologists is measuring environmental conditions in the field and appropriately replicating these conditions in a controlled experiment. What makes this particularly hard is that natural environments are always changing. For example, most lizards lay eggs in nests in the ground and then abandon them, providing no parental care during development. While eggs develop, nest temperatures are not constant; they fluctuate on a daily, weekly, and seasonal basis along with weather conditions. Think, for example, about how temperatures fluctuate every day due to the rise and fall of the sun. Most egg incubation experiments, however, fail to capture the true variation in nest temperatures when they design experimental treatments. For example, they might incubate eggs at a constant temperature or at temperatures that repeat the same daily change in temperature over and over again. Real nest temperatures, however, rise and fall by different degrees each day. Over a long incubation period (e.g. 40-60 days), eggs can experience a lot of different temperatures! This can result in lots of important effects on development because nest temperatures can influence the body size, running speed, and even learning ability of hatchling lizards.

In this study, we incubated brown anole eggs under incubation treatments that differed in how closely they match real nest temperatures. We found that natural temperature fluctuations improved hatchling lizards’ endurance and survival compared to simpler approximations (e.g. constant temperatures, repeated daily fluctuations). This paper was featured in the Journal of Experimental Biology‘s Inside JEB; therefore, Kathryn Knight has written a summary of our study for a general audience, and the cartoon above was created by Francesca Luisi to illustrate the main findings of our study.

HallJ. M. and WarnerD. A. (2020). Ecologically relevant thermal fluctuations enhance offspring fitness: biological and methodological implications for studies of thermal developmental plasticityJ. Exp. Biol. 223jeb231902. doi:10.1242/jeb.231902

How Do We Deal with Non-Confirmatory Results?

Fig 1. Photo of some members of the research team on one of our experimental small islands by J. Losos

Thanks to Nick for doing a new research post when our paper “Consumer responses to experimental pulsed subsidies in isolated vs. connected habitats” first came out. Here I want to give some backstory on the road to publication (all views are my own).

This was an epic experiment overall: 52 experimental units, 4+ years, thousands of person-minutes of lizard surveys, thousands of food web stable isotope samples, several tons of seaweed, and one hurricane that washed it all away.

I think the most interesting thing about this paper is that we did not find what we expected.

For some biological background, a meta-analysis (Yang et al. 2010) of largely observational studies found that populations increase the most and the fastest when consumers respond to resource pulses (brief, unpredictable periods of resource superabundance) via both aggregation and reproduction. To test the prediction that without aggregation the numerical response would be slower and smaller, in the current study we manipulated seaweed on mainlands (as in our previous study, e.g., Spiller et al. 2010, Wright et al. 2013) and also on very small islands (Fig. 1) where aggregation on ecological time scales is not possible.

Despite a bigger N this time around, we did not replicate the numerical response on mainlands that we saw in Spiller et al. (2010). In other words, more seaweed did not result in more lizards on mainlands. Conversely, we saw fast and large population gains on small islands. We did replicate the timing and magnitude of the diet shifts, indicating that lizards were consuming the subsidized resources. So whether resource pulses translate into more individuals is context-dependent, even with the same researchers using the same methods with the same species. In the discussion we talk about what could be driving these differences.

Now to my main story with this post: what happens when you have un-tidy, non-confirmatory results? The first reviews at a top tier ecology journal were very positive about the generality of the questions and the realistic temporal and spatial scale of the experiment. We were rejected for not being able to explain the mechanisms; fair enough. However, this same critique would be true even if we had confirmatory results. I don’t think we would have drawn that critique, or at least it would not have had such a large impact on the editorial decision, with confirmatory results. We next tried at a second-tier ecology journal, and were rejected without review.

I was up for the tenure the year this paper was going through the review process. Pretty much the only way the paper would be accepted pre-dossier would be to go back to the first journal and accept their original offer to shunt to their online-only sister journal. I have happily made that call in the past given different trade-offs. In this case, I felt rejection was largely being driven by the non-confirmatory results, which I stubbornly believed did not compromise the quality of the paper. To me, good science is asking good questions (i.e., rooted in theory) with good design; the value of the paper is not predicated on the outcome of the study. I asked some senior profs in my department for advice and got both, “a published paper is better than no paper” and “do what you would have done regardless of tenure.” I went with the latter because at that point I felt one paper was not going to make or break my diverse contributions over the prior five years.

I decided to try next at The American Naturalist for a couple reasons. One, their checklist for authors signals similar values to mine, such as indicating whether the study was pre-registered. Another was that by chance, Dan Bolnick, current EIC, was in my session at the ESA meeting. Dan announced that he would be holding “office hours” to promote submission to AmNat. I had never pitched a paper to an editor before, but this was made easier since (also by chance) I know Dan from grad school.

I gave Dan my 2-minute pitch, emphasizing that we had unexpected results that we couldn’t fully explain. He opened his response with, “I sympathize…”, and I braced for the polite rejection. But he meant that he literally sympathized, because he had a study with confirmatory results published in a high profile journal, but a later replication with more data was non-confirmatory and ended up several tiers down. He encouraged me to submit (with no guarantees of course), and I leaned in hard to our unexpected results and lack of replication, from the cover letter through supplemental material, being as transparent as possible. It was still a tough and long review process, and the paper has several real limitations, but I am gratified that it got into a top journal on its merits as planned, warts and all, without spin.

We haven’t seen the replication crisis in ecology I think for two main reasons. One is that big field experiments like our pulsed subsidies studies are rarely repeated (for lots of reasons), and two because ecologists are very comfortable with context-dependency. But how often is a lack of replication due to real biological differences that are useful to understand (as I argue was the case in our paper) vs. the statistical issues that plague other disciplines? Ecologists are often taught to cope with non-confirmatory results by reframing to “tell the story you have,” which runs the risk of HARKing, one of the four horsemen of the reproducibility crisis. Preferences for confirmatory results help drive these practices. In our study, the questions, hypotheses, and design were essentially pre-registered in the grant that funded the work, and staying committed to a plan regardless of the results is the best defense against the garden of forking paths. 

As for studies rarely being repeated in the first place, I am haunted by a review of restoration studies by Vaughn and Young (2010) that found fewer than 5% of studies were initiated in more than one year, and 76% of studies that did use multiple years found different results in different years. To me this means that we should not inhale too deeply on single studies, we should focus more on replication and less on novelty, and that our inability to replicate some of the results of Spiller et al. 2010 is a feature, not a bug!

If you are interested in learning more about this system, check out Piovia-Scott et al. 2019 which shows that the strength of top-down control by lizards varies predictably over the course of the pulse.

 

 

 

 

Muscle map on scapulocoracoid of Anolis insolitus

Morphology of the Scapulocoracoid of Anolis Ecomorphs

 

From the onset of my scientific career I have been fascinated by the pectoral girdle. In its structural and functional diversity it is barely rivaled by any other skeletal part of the tetrapod body. Anoles, in particular, employ their forelimbs not only in locomotion, but also in various routines of display, grooming, feeding, or mating. It is likely that the different functional roles fulfilled by the pectoral limb and girdle impose varying, and potentially opposing, selective pressures onto the evolution of its structural form.

Jane Peterson briefly alluded to the structural variance displayed by the different anole ecomorphs, relating them to specific locomotor requirements by providing brief descriptions in her thesis (1973) and the First Anolis Newsletter (1974). However, beyond this initial work, and a few qualitative assessments in papers regarding phylogenetically informative characters, very little is known about the variability of the anole pectoral girdle.

right scauplocoracoid of Jamaican anoles

Right scapulocoracoid of three anole species, representative of the Jamaican lineage. The arrow denotes anterior. (via Tinius et al. 2020)

In many ways, our recent publication in the Annals of Anatomy (Tinius et al. 2020) is a dream come true (at least for me), as it allowed us to finally visualise the patterns of morphological variation that Peterson (1974) could only communicate in descriptions. Because the shoulder girdle is comprised of multiple elements that are mobile with respect to one another, this paper only investigated one of its moieties: the scapulocoracoid. This paired structure spans the entire height of the body wall, is comprised of developmentally very different compounds, and directly connects the forelimb to a midline element, the presternal plate. These attributes made it a great starting point for our investigations of the pectoral girdle.

In describing the scapulocoracoid of two non-anoline iguanids, Polychrus and Pristidactylus, we anchored our comparisons in two well-studied and closely related lizards. We then expanded on this anatomical framework by comparing all representatives of the monophyletic Jamaican anole radiation to their respective ecomorph representatives on Puerto Rico and Hispaniola. We tried to take full account of the variability of the scapulocoracoid by examining it both qualitatively, in images and comparative description, and quantitatively, through geometric morphometric analysis.

CVA of the right scapulocoracoid of Anolis ecomorphs

Canonical Variate Analysis (CVA) of the right scapulocoracoid of Greater Antillean anoles, including warp image of the scapulocoracoid denoting shape changes along CV1 and CV2. (via Tinius et al. 2020)

We found that regardless of potential phylogenetic constraints on skeletal morphology, morphospatial occupancy differs markedly between ecomorph groups. Unexpectedly, twig anoles show the most distinctive shape of the scapulocoracoid, with a relatively tall scapula and anteroposteriorly short coracoid, similar to the situation found in chameleons (Fischer et al. 2010). But despite a significant overlap in morphospatial occupancy, the other three ecomorphs examined (trunk-ground, trunk-crown, and crown-giant) also exhibit trends towards a specialized scapulocoracoid morphology, such as a relatively wide/cylindrical scapulocoracoid in trunk-ground anoles.

These variations in form likely impact the size and vectors of muscles attaching to the scapulocoracoid. One muscle group that is likely particularly impacted by the differences in scapulocoracoid form is the M. serratus anterior. This muscle group originates laterally on the cervical ribs and inserts on the medial aspect of the suprascapula. The M. serratus anterior group stabilizes the scapulocoracoid during locomotion and protracts/retracts it along the body wall. The anteroposteriorly more extensive suprascapula of crown-giant anoles likely facilitates more forceful scapular retraction, through the relatively greater attachment area for this muscle and the anterior disposition of its insertion area. Contrastingly, the relatively tall scapula of twig forms likely allows for a greater moment arm acting through this muscle group, while the anteroposteriorly short suprascapula facilitates more precise protraction/retraction of the scapulocoracoid.

Muscle map on scapulocoracoid of Anolis insolitus

Right scapulocoracoid of Anolis insolitus in a) lateral, and b) medial view, showing the attachment sites of major muscle groups that act upon the scapulocoracoid. (via Tinius et al. 2020)

My only regret about this project is the exclusion of Cuban anoles, which markedly limited our ability to compare patterns in a wider phylogenetic context. Most of the crown-giant and trunk-crown anoles examined belong to their own ecologically homogenous clade, making it impossible to discern ecological from morphological signal.

The Jamaican Anolis clade provides a glimpse into what might be achieved with a phylogenetically broader sample, as it represents four major ecomorph groups (five, if you attribute A. opalinus to the trunk group) plus two non-ecomorph species within a seven-species radiation. Despite the relatively young age of the Jamaican clade, its ecomorph representatives exhibit a push towards specialized morphologies of the scapulocoracoid, even if this level of specialization is markedly smaller than in their Puerto-Rican and Hispaniolan relatives. A future widening of our sample should allow us to answer some intriguing questions regarding the retention and diversification of ecomorphologically specialized forms within distinct phylogenetic lineages.

Literature cited

Fischer, M.S, Krause, C. & Lilje, K.E. (2010): Evolution of chameleon locomotion, or how to become arboreal as a reptile.─ Zoology, 113:67-74.

Peterson, J.A. (1973): Adaptation for arboreal locomotion in the shoulder region of lizards.─ Ph.D. thesis, University of Chicago.

Peterson, J.A. (1974) [In:] Williams, E.E. (ed.) The First Anolis Newsletter. Cambridge, Massachusetts: Museum of Comparative Zoology, Harvard University.

Tinius, A., Russell, A.P., Jamniczky, H.A. & Anderson, J.S. (2020): Ecomorphological associations of scapulocoracoid form in Greater Antillean Anolis lizards.─ Annals of Anatomy, 231; doi.org/10.1016/j.aanat.2020.151527.

Sleeping Behavior of the Puerto Rican Twig Anole, Anolis occultus

In August, we published a paper in the Caribbean Journal of Science entitled, “Sleeping Behavior of the Secretive Puerto Rican Twig Anole, Anolis occultus.” Check out our new post on the Chipojo Lab blog about the paper!

Levi Storks, Manuel Leal. 2020. Sleeping Behavior of the Secretive Puerto Rican Twig Anole, Anolis occultus. Caribbean Journal of Science 50(1):178–87.

Morphology Does Not Distinguish Candidate Species of Anolis distichus

Photo by Richard Glor

Anolis distichus, the North Caribbean bark anole, is probably best known by readers of Anole Annals for its striking variability in dewlap color among populations. Primarily based on these differences in dewlap color, Albert Schwartz published a monograph with descriptions for 18 subspecies distributed across Hispaniola and the Bahamas (1968). The large number of subspecies and the question of their origin has helped establish Anolis distichus as one of the most intriguing cases for the study of speciation in anoles. Do the subspecies of Anolis distichus represent geographic patterns in dewlap color variation? Or, are the subspecies evolutionarily isolated lineages worthy of species status?

Molecular genetic data have revealed several genetically distinct populations of Anolis distichus that appear to be at varying stages of the speciation process. However, with the exception of A. d. ignigularis and A. d. favillarum, these genetically distinct groups did not align with the subspecies described by Schwartz, corresponding better to geography than patterns of dewlap color variation (Geneva et al. 2015). Most recently, using genome wide markers and multicoalescent species delimitation methods, MacGuigan et al. (2017) identified six candidate species within A. distichus. The authors, however, did not update the taxonomy because the boundaries among the candidate species remained unclear.

To clear up those boundaries, we tested if the candidate species identified by MacGuigan could be distinguished by morphological characters. Because Schwartz considered scale counts along with dewlap and body color pattern in his monograph and was not able to recover any diagnostic differences, we focused on morphometric characters and measured 13 traits from more than 500 animals (available on Dryad). We conducted univariate and multivariate analyses to test if (a) any of the individual characters distinguished candidate species; and (b) if characters considered in aggregate could distinguish the candidate species. Because the candidate species are parapatrically distributed across Hispaniola, locality information has the potential to aid diagnosis. To account for this, we carried out comparisons of all candidate species together and only the pairs of candidate species that potentially come into contact. 

Although ANOVAs recovered significant differences in mean character values, visual examination of violin plots showed that none of them were diagnostic for any of the candidate species.  Discriminant Function Analysis (DFA) revealed clustering of some of the candidate species, but there was still substantial overlap in multivariate space and candidate species were diagnosed with poor accuracy. DFA did prove to be more accurate when asked to classify individuals only between the pairs of potentially co-occurring candidate species instead of all of the candidate species together. Ultimately, we still rejected the hypothesis that candidate species could be distinguished on the basis of our morphometric dataset due to gaps in our sampling and overall similarity among the candidate species.

Univariate and multivariate results from Myers et al. (2020). (A) Example violin plots for two characters, snout-vent length and head width. (B) Multivariate plots of DFA results.

Because both univariate and multivariate analyses did not recover support for the hypothesis that the candidate species could be distinguished by morphometric characters, we decided to test how many species were supported by the data.  We tested alternative species delimitation scenarios with a model fitting approach that uses normal mixture models (Cadena et al. 2018). Normal mixture models consider morphological variation to consist of a mix of different normal distributions and, unlike DFA, does not require individuals to be assigned to different groups a priori. We tested the support for models specifying up to as many as 12 species and compared the support for these generic models to models specifying MacGuigan’s candidate species and Schwartz’s subspecies. The best supported normal mixture model specified two groups; however, the model specified a group containing 489 individuals and another with 24 individuals and followed no clear geographic trend. We scrutinized the principal components used to estimate these models and determined that longitudinal and vertical ear opening diameter were driving this result. We removed them and conducted the normal mixture model analysis with the reduced dataset and recovered a single morphological group.

Is Anolis distichus only one species? Do the candidate species lack distinguishing features? We weren’t comfortable making either conclusion. Our dataset of linear morphometric characters was not capable of distinguishing candidate species, but future datasets featuring other aspects of phenotypic variation e.g., geometric morphometrics, might. Larger, genome-scale datasets with more comprehensive geographic sampling than previous molecular genetic studies will also help address the question of species boundaries in A. distichus. We also discuss some possibilities for why we were unable to recover distinguishing morphological differences, including that local adaptation across Hispaniola’s environmentally heterogeneous landscapes has resulted in morphometric variation that does not align with candidate species boundaries. Ng et al. (2013) found that dewlap color in A. distichus is correlated with local environmental signaling conditions, which would explain why dewlap color does not correspond with putative evolutionary lineages in this group of lizards. Many of the morphometric traits we considered (e.g., limb length) affect ecological performance and may have responded similarly to selective pressures. 

We were not able to resolve the confounding taxonomy of Anolis distichus in this paper, but I enjoyed the project and found it to be a very rewarding first publication. I was able to travel to the Dominican Republic to catch lizards twice as an undergraduate for this project and we were able to amass a large and geographically comprehensive dataset. Working on the taxonomy of this confounding group of lizards helped me realize my interest in speciation, which I plan to pursue further in my PhD. A lot of friends and collaborators helped with this project by assisting with fieldwork and providing input on manuscript drafts. And, of course, this work wouldn’t have been possible without my co-authors and mentors, Pietro de Mello and Rich Glor

References

Cadena, C. D., F. Zapata, and I. Jiménez. 2018. Issues and perspectives in species delimitation using phenotypic data: Atlantean evolution in Darwin’s finches. Syst. Biol. 67:181–194.

Geneva, A. J., J. Hilton, S. Noll, and R. E. Glor. 2015. Multilocus phylogenetic analyses of Hispaniolan and Bahamian trunk anoles (distichus species group). Mol. Phylogenet. Evol. 87:105–117.

MacGuigan, D. J., A. J. Geneva, and R. E. Glor. 2017. A genomic assessment of species boundaries and hybridization in a group of highly polymorphic anoles (distichus species complex). Ecol. Evol. 7:3657–3671.

Myers, T. C., P. L. H. de Mello, and R. E. Glor. 2020. A morphometric assessment of species boundaries in a widespread anole lizard (Squamata: Dactyloidae). Biol. J. Linn. Soc. 130:813-825.

Ng, J., E. L. Landeen, R. M. Logsdon, and R. E. Glor. 2013. Correlation between Anolis lizard dewlap phenotype and environmental variation indicates adaptive divergence of a signal important to sexual selection and species recognition. Evolution 67:573–582.

Schwartz, A. 1968. Geographic variation in Anolis distichus Cope (Lacertilia, Iguanidae) in the Bahama Islands and Hispaniola. Bull. Mus. Comp. Zool. 137:255–309.

Anole Genomes Webinar

Tomorrow (30 June 2020) I will be presenting a webinar on our ongoing work assembling Anolis genomes. The webinar is hosted by Dovetail Genomics who provided the core technology we used to generate high quality genome sequences. The talk is at 11AM EST. If you want to watch live and have the chance to ask questions, you can register here. If you can’t make it but still want to hear what we are up to, Dovetail will post the video on their website alongside other speakers in the series.

Vasotocin and Chemical Communication in Anolis carolinensis

 

A male green anole basking on my porch in Atlanta, Georgia. (Photo source S. M. Campos)

Growing up in Texas, I often found Anolis carolinensis green anoles (my first love) basking on my front porch and developed an early obsession with studying their natural behavior. Green anoles are the only anole endemic to North America (but see Wegener et al. (2019) suggesting the Cuban green anole, Anolis porcatus, is the same species). Anolis carolinensis are often referred to as American Chameleons due to their ability to switch between green and brown skin colors, despite being a completely different family than true chameleons. In graduate school, I worked with a different lizard genus, Sceloporus (from Greek “skelos” meaning leg, “poros” meaning hole), named for the scent producing glands on their inner thighs called femoral glands. The realization that some lizards modulate their social behavior based on chemical information that is deposited by other lizards was pivotal in my research career. Here, I’ll discuss chemical communication in A. carolinensis and the serendipitous discovery that the neuropeptide arginine vasotocin (AVT) plays some role in stimulating this chemical communication.

Anolis is an important animal model for studying the neuroendocrine control of visual communication due to their hormonal modulation of  vibrant color displays and conspicuous push up displays. Large eyes and brain areas dedicated to processing visual information suggest that detecting and responding to the visual environment is very important to anole survival and fitness. In contrast, anoles do not have the femoral or precloacal glands described in other lizards, which are used to deposit scent marks. Their olfactory bulbs (the portion of their brains that responds to volatile and non-volatile chemical signals) are tiny structures that are nestled in front of their eyes and behind their nares, attached to the rest of the brain by a long narrow nerve tract. Therefore, it is not all that surprising that anoles have long been considered microsmatic, relying very little on their sense of smell.

A CT scan of Anolis sagrei showing the main and accessory olfactory bulbs (yellow and blue arrows, respectively). The main olfactory bulb responds to volatile chemicals detected by the olfactory epithelium in the nose (such as odors in the air) and the accessory olfactory bulb responds to non-volatile chemicals detected by the vomeronasal organ. (Source Photo by Ed Stanley, arrows added by S. M. Campos)

So what is the deal with this peptide hormone, AVT? AVT and its mammalian homologue vasopressin (AVP) regulate social behavior in animals and decades of research has shown that AVT works within the visual sensory system of green anoles to modulate competitive and reproductive interactions. In non-reptilian animals like fish and mammals, AVT/AVP plays a similar role in modulating social interactions through the chemosensory system. Whether AVT influences chemosensory behavior in reptiles is unknown, representing an important gap in our understanding of the evolution of social behavior.

Now, the serendipitous part of the story. My postdoc advisor, Walter Wilczynski, built his career studying how AVT impacts visual communication in social interactions of frogs and green anoles. Previous work showed that green anoles can differentiate between AVT-treated and saline-treated males during live social interactions, but found no obvious differences in visual display rates between AVT-treated and saline-treated males, suggesting differences in behavior may be due to available chemical information. In the present study, we asked  whether an untreated lizard responds to a live AVT-treated male by altering its rate of chemosensory behavior, which we would expect if AVT-males and saline-males emit different chemical signals.

From left to right: Study authors Stephanie M. Campos, Walter Wilczynski, and Valentina Rojas. (Photo source S. M. Campos)

While lizards breathe in odors in a manner similar to humans (olfaction), they also have a secondary sense of “smell” called vomerolfaction. The latter involves using their tongues to bring chemicals from the outside environment into their mouths and deliver those chemicals to the vomerolfactory organ (often referred to as Jacobson’s organ in snakes) located in the roof of their mouths. We can easily quantify chemosensory behavior involving the tongue by counting the number of licks (tongue touches to a substrate, such as a rock), tongue flicks (tongue extrusions into the air), and lip smacks (draws odors into the mouth) a lizard performs. Use of these behaviors give us an estimate of a lizard’s level of interest in the chemical information available in their immediate environment. Chin wiping, or jaw rubbing, is another chemosensory behavior that may either deposit chemical signals or help to detect chemical signals already on a substrate. We provide short video clips in the online version of our article to show each of these behaviors.

In our experiment, we gave adult male green anoles an intraperitoneal injection of either an AVT or saline (control) solution, then introduced an untreated lizard (male or female) into the home tank of the treated lizard for a filmed 30-minute interaction. We measured rates of chemosensory behavior and the latency to perform these different behaviors. Since lizards tend to use higher rates of tongue flick behavior for exploratory purposes as they move around their environments (Cooper et al. 1994; Mason 1992), we also counted short bouts of locomotion.

Untreated males that interacted with AVT-males performed more tongue flicks and lip smacks compared to males that interacted with saline-males. Interestingly, lizards that interacted with AVT-males tended to move around less compared to lizards that interacted with saline-males. This suggests that the higher rates of chemosensory behavior by untreated males that interacted with AVT-males was not simply due to an increase in locomotion. We also found that untreated males moved around more than untreated females, regardless of treatment, demonstrating a general sex difference in locomotion among green anoles. We found no significant differences between treatments in chemosensory behavior performed by untreated females.

Lizards in their home tanks. (Photo source S. M. Campos)

When we examined the behavior of treatment males (which received injections), we found that AVT-males were faster than saline-males to perform a chemical display and, more specifically, a tongue flick toward untreated males. This suggests that AVT increases the level of initial interest in chemical information that is available during asocial encounters.

What about visual displays? We found no significant differences in visual display rates of untreated lizards, but did find that as treatment males performed more visual displays, untreated lizards moved around more (Supplementary Materials).

These results collectively suggest that AVT impacts chemosensory behavior during social interactions in green anoles, even in untreated social partners. More broadly, the mechanisms used by AVT to impact chemosensory behavior may be evolutionarily conserved. Our results are consistent with previous work linking AVP in mammals and AVT in fish to chemosensory-mediated interactions, such that AVT in reptiles deserves more research attention in the future. Furthermore, even in microsmatic lizards like Anolis, the impact of chemical communication on social dynamics should not be ignored. This study examined social dynamics between two live lizards and did not isolate the chemical signal. Thus, further work is necessary to determine whether similar changes in chemosensory displays occur when isolated chemical stimuli are presented to untreated lizards.

Tolerance to Urbanization is Widespread in Anoles

From Winchell et al. (2020): Anoles throughout the Caribbean differ in their tolerance to urbanization. Red colors = urban tolerant, blue colors = intermediate tolerance, green colors = urban intolerant.

Seven years ago I asked for the help of Anole Annals readers as I started to think about how different species of anoles throughout the Caribbean tolerate urbanization. This question, it turned out, was a lot more complex than I had originally anticipated! The idea was simple, find out which species are in urban areas and to what extent they use urban habitat elements, then determine if there is an evolutionary signal in urban tolerance and what traits are correlated with urban tolerance. Many hours of troubleshooting and brainstorming with my coauthors Klaus Schliep, Luke Mahler, and Liam Revell (and years later) and this study is finally out in the journal Evolution: Phylogenetic signal and evolutionary correlates of urban tolerance in a widespread neotropical lizard clade.

Anolis lineatopus, one of many urban tolerant anoles (photo K. Winchell)

Inventorying urban species

To figure out which anole species are tolerant of urbanization, my initial plan was to survey researchers and the literature to score each of the 100+ Caribbean species based on their presence in different types of urban habitats and their habitat use. Although I got a lot of great feedback from this original survey, it left a lot of gaps in the dataset. I needed to find a more objective way to assess urban tolerance.

With the help of Klaus Schliep and Luke Mahler, we decided to examine location records in museum collections (via GBIF) to determine which species had been observed (collected) in urban environments. Because we suspected museum records might be biased towards non-urban habitats, we also examined location records from the citizen science database iNaturalist, which we suspected might be biased in the opposite direction (i.e., people photograph things where they live). For each record, we looked at satellite imagery and scored the observation as urban or non-urban, then tallied the total number of observations and the total number of urban observations per species.

Even with these two data sources, we noticed gaps in our data for some species. So we included a third source, Henderson & Powell’s (2009) book on the Natural History of West Indian Amphibians and Reptiles. This fantastic reference (highly recommended!) gives detailed natural history information and summarizes key features of every anole (and other Caribbean herps) in the Caribbean. Of course, this is more subjective than the location-based data, so Luke and I came up with a scoring system that assigned a set number of urban tolerant or avoid “points” based on key descriptors. For example, if a species was described as being common around houses and often observed on buildings, it would get points for being tolerant of urbanization. In contrast, a species described as having a restricted range and intolerance of anthropogenic disturbance, it would get points for being intolerant.

Analyzing urban tolerance in a phylogenetic framework

We combined these disparate data sources into a logistic model with parameters we set based on the number of urban observations we would need to be certain of urban tolerance and how many total observations we would need to be certain of our species assessment. This resulted in a probability of being an urban avoider or urban tolerant for each species, which we used as our prior probabilities for these states in our phylogenetic model. We then reconstructed ancestral states and missing tip states for urban tolerance in 131 species of Caribbean anoles.

Of course, we don’t mean to say that we attempted to reconstruct the evolution of urban habitat use — anoles are far older than urbanization! Instead, we wanted to understand the evolution of the behavioral, physiological, ecological, and morphological traits traits that influence whether a species will exploit or avoid urban habitat when it arises. The threshold model is well-suited for this type of complex trait. The threshold model assumes that a discrete trait is determined by a combination of continuously valued characteristics. These characteristics may be measurable, unmeasurable, or even unknown. As a taxon accumulates specific trait changes, the species is pushed incrementally closer and closer to the discrete state change (in this case urban tolerance), and the more recently this discrete character state has flipped, the more likely a reversal to the previous state could occur. From this model we can extract a single continuously valued trait, the liability, that underlies the complex trait of urban tolerance.

Urban tolerance in Caribbean anoles, from Winchell et al. (2020).

Traits of urban species

So what did we find? To start, urban tolerance appears to be widespread in Caribbean anoles and has a strong phylogenetic signal. Because of that, we suggest that our approach may be used to predict urban tolerance of species that either have yet to encounter urbanization or for which we are lacking information. This application could be particularly useful for determining which species are likely to be intolerant of urbanization and thus should be prioritized in conservation efforts. At the other end of the urban tolerance scale, we caution that our approach should not be used to predict species that are robust to anthropogenic habitat loss, but rather that it might be useful to identify species that are promising for future urban ecology and evolution studies.

Finally, we used the liability score for each species to try to get a better understanding of what those traits underlying urban tolerance are exactly. Using PGLS we looked for correlations between the liability and a suite of ecological and phenotypic traits. We found that species that are more tolerant of urbanization had higher field body temperatures, fewer ventral scales, more rear lamellae, shorter hindlimbs, and experience warmer and drier climates within their native range. These traits may be key “pre-adaptations” enabling species to colonize urban habitats as they arise and to take advantage of anthropogenic niche space (i.e., on and around buildings). For example, urban habitats tend to be hotter and drier than nearby forest sites, so it makes sense that species with larger ventral scales, higher field body temperatures, and which experience hotter and drier temperatures in their non-urban range would be predisposed to tolerate urban habitats. Similarly, lamellae are important for clinging to smooth surfaces, which may be particularly beneficial in urban habitats dominated by smooth anthropogenic surfaces.

Lastly, we found, somewhat to our surprise, that no one ecomorph seems to be best suited for urban environments. Based on our experience, we had thought that trunk-ground anoles would be more likely to tolerate urbanization, but it turns out that there are a lot of trunk-ground anoles that are intolerant of urbanization and a lot of species from other ecomorphs that are tolerant (think A. equestris or A. distichus)!

Page 3 of 66

Powered by WordPress & Theme by Anders Norén