Category: New Research Page 4 of 67

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 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.

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.

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.

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.