Over at Lizards and Friends, Amy Payne from Michele Johnson’s lab reports on her first field experience studying green anoles. Fear not–they kept an eagle eye out for snakes.

The spectacular new anole species, Anolis eladio, named after Hispaniolan nature photographer extraordinaire Eladio Fernandez, who first discovered the species.

We have been remiss here at AA in not reporting on a recent monograph in Novitates Caribaea (the journal of the Museo Nacional de Historia Natural of the Dominican Republic) by Köhler and Hedges dividing the Hispaniolan green anoles into sixteen species, up from the previously recognized four. Specifically, Anolis chlorocyanus is split into four species, A. coelestinus into five species, and A. aliniger is subdivided into six species. Poor A. singularis remains as it is.

The analysis is based on mitochondrial DNA and morphological characters. The monograph is available online and should be consulted for the fine details. Appended below are the abstract and the heart of the methods.

Say what you may about the proliferation of new species (and word on the street is that this will not be the last word on green anole species diversity), some of the new species are spectacular in appearance and certainly there is more variation in this group than many may have realized.

Abstract:

We revise the species of green anoles (i.e., the species related to Anolis aliniger, A. chlorocyanus, and A. coelestinus) occuring on Hispaniola. Based on our analyses of morphological and molecular genetic data we recognize 16 species of green anoles, eight of which we describe as new species (A. apletolepis sp. nov., A. chlorodius sp. nov., A. divius sp. nov., A. eladioi sp. nov., A. gonavensis sp. nov., A. leucodera sp. nov., A. prasinorius sp. nov. and A. viridius sp. nov.) and three of which are raised from subspecific to species level (A. cyanostictus, A. demissus and A. pecuarius) and one is resurrected from synonymy with A. chlorocyanus (A. peynadoi). Because the six syntypes of A. chlorocyanus (MNHN 785, 787, 2007.2066–09) are conspecific with the only available syntype of A. coelestinus (i.e., MCZ 3347), we have petitioned the International Commission of Zoological Nomenclature (ICZN) to use its plenary power to set aside the type status of the syntypes of Anolis chlorocyanus and to allow the designation of a neotype in order to stabilize the current and long established usage of the names A. chlorocyanus and A. coelestinus. For each species we provide a standardized description of external morphology, color descriptions in life, color photographs in life, description and illustration of hemipenis morphology (if available), distribution maps based on the specimens examined, comments on the conservation status, and natural history notes. Finally, we provide a dichotomous key for the identification of the 16 species of green anoles occuring on Hispaniola.

And here’s how they did it:

For this study, we have examined a total of 787 specimens of green anoles from Hispaniola. Head length was measured from the tip of the snout to the anterior margin of the ear opening. Snout length was measured from the tip of the snout to the anterior border of the orbit. Head width was determined with the broad tips of the calipers aligned with the levels of posterior margin of eye and supralabial scales, respectively, with the calipers held in a vertical position relative to the head. Dorsal and ventral scales were counted at midbody along the midline. Tail height and width were measured at the point reached by the heel of the extended hind leg. Subdigital lamellae were counted on Phalanges II to IV of Toe IV of the hind limbs, and separately on distal phalanx. We considered the scale directly anterior to the circumnasal to be a prenasal. Abbreviations used are AGD (axilla–groin distance), dorsAG (number of medial dorsal scales between levels of axilla and groin), dorsHL (number of medial dorsal scales in one head length), HDT (horizontal diameter of tail), HL (head length), HW (head width), IFL (infralabials), IP (interparietal plate), SAM (scales around midbody), ShL (shank length), SL (snout length), SO (subocular scales), SPL (supralabial scales), SS (supraorbital semicircles), SVL (snout–vent length), TL (tail length),VDT (vertical diameter of tail), ventrAG (number of medial ventral scales between levels of axilla and groin), and ventrHL (number of medial ventral scales in one head length). In reporting the frequencies of character states, we used the following terminology (Köhler submitted): if a character state was present in more than 65% of the examined specimens, we coded it as “usually”; <65% but >20% “commonly”; <20% but >5% “occasionally”; and <5% “exceptionally”. The use of size categories also follows Köhler (2014): (1) small: <50 mm SVL; (2) moderate-sized: 50–60 mm SVL; (3) moderately large: 60–80 mm SVL; (4) large: 80–110 mm SVL; (5) giant: >110 mm SVL.

As lines of evidence for species delimitation, we apply a phenotypic criterion (external morphology: coloration, morphometrics, and pholidosis) and a criterion for reproductive isolation (genetic distinctness of the cytochrome B and ND2 genes). Sequences from 77 ingroup and two outgroup taxa were analyzed (a total of 2217 aligned sites). Alignments (MUSCLE) and best-fit model selection were performed in MEGA 6.06 (Tamura et al., 2013). A maximum likelihood (ML) analysis was performed using MEGA 6.06), unpartitioned, using the evolutionary model GTR + I + Γ. Gaps were treated as missing data. All parameters for the ML analyses were estimated by the program during the run. Branch support in the trees was provided by standard bootstrap analysis (2,000 replicates). A Bayesian phylogenetic analysis using MrBayes 3.2.2 (Ronquist et al., 2012) also was performed, also using the GTR + I + Γ model. The Bayesian analysis was set to two parallel runs for five million generations, sampled every 100 generations, each run employed three heated and one cold chain, with a temperature parameter of 0.10. The first 10% of samples were discarded as burn-in. Convergence was assessed by the standard deviation of split frequencies (< 0.01 in all cases).

Congratulations, Kristin Winchell and co-authors!

And for those of you keeping track, that’s five anoles on the cover of Evolution in the last six years (ending a three-year drought).

Incidentally, the paper  continues to attract attention, most recently in the pages of IFL Science:

photo credit: Robert Eastman/Shutterstock

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Small tropical lizards called anoles have adapted to life in the urban jungle by evolving stickier hands and feet as well as longer arms and legs, according to a recent Evolutionstudy. These help them cling to concrete walls, walk across slippery windows, and perch on metal fences with as much ease as their forest-dwelling cousins.

Urbanization is rapidly increasing around the world, with humans living in nearly two-thirds of the planet’s terrestrial areas. As a result, animals are being confronted with new habitats – from decorative, non-native plants to impervious surfaces and artificial lights. And with these come novel selection pressures. While many wildlife species can survive in cities, relatively little research has been done on whether these populations have adapted (in an evolutionary sense) to their newfound environments.

Crested anoles (Anolis cristatellus) are trunk-ground specialists; they use their long limbs and stocky build to navigate across broad surfaces like tree trunks or the forest floor. The species is native to Puerto Rico, which has been utilized intensively for agricultural cash cops like sugar cane, tobacco, and coffee throughout the 19th and 20th centuries. This has led to massive declines in native wildlife and tree cover. Around the same time, the island underwent major industrialization: 94 percent of the 3.7 million citizens now live in urban areas.

To see if the lizards have adapted to urbanization, a team led by Kristin Winchell from the University of Massachusetts Boston compared the ecology, morphology, and DNA of hundreds of male crested anoles living in three high-density Puerto Rican cities – Mayagüez, Ponce, and San Juan – with anoles living in three subtropical forests nearby.

As predicted, the temperature, humidity, and substrate availability varied a lot between urban sites and their neighboring natural areas. Additionally, urban lizards often used artificial substrates, which were generally broader than the substrates in forests. However, city anoles had longer forelimbs and hindlimbs relative to their body size, and they also had more lamellae – tiny scales on the undersides of their toes that help them “stick” to surfaces.

The team also reared the hatchlings of wild-caught adult pairs from one urban and one natural population: 25 males and 25 females from each of the two populations. They found that the differences between urban and natural wild populations were maintained in their captive-reared offspring – which means these differences are likely genetically based.

The artist says:

Photo from http://www.exoticpetvet.com/breeds/Green%20Anole.htm

Cornerstone recently reported abstracts from an undergraduate research symposium at the University of Minnesota Mankato. Included in the event were four projects from the laboratory of Rachel Cohen.

Seasonal Effects on Kisspeptin Concentration in the Green Anole Lizard, Anolis carolinensis

Nicholas Booker, Minnesota State University Mankato
Hyejoo Kang, Minnesota State University Mankato

Gonadal steroid hormones are responsible for reproductive behaviors; disruption in production of these hormones is also linked to fertility issues. The hypothalamic-pituitary- gonadal (HPG) axis controls the production of sex steroid hormones, testosterone and estradiol. A peptide, kisspeptin, stimulates this axis by acting on neurons in the hypothalamus. The green anole lizard, Anolis carolinensis, is a seasonally breeding animal that shows drastic changes in behavior and physiology between the breeding and non- breeding seasons. One such change is a large increase in testosterone levels in the breeding season compared to the non-breeding season. These fluctuations in testosterone concentration in green anoles allows for a great opportunity to study the HPG axis. In the current study, we used brain tissue from breeding and non-breeding season green anoles to perform western blot analysis on kisspeptin concentration. Due to the increase in testosterone in the breeding season, we hypothesized that an increase in kisspeptin concentrations will be observed in breeding season compared to the non-breeding season lizards. These results would suggest that kisspeptin does indeed play a role in stimulating the HPG axis and that kisspeptin could potentially be used as a treatment for infertility.

The Effect of Steroid Hormones on Neuronal Size and Number in Two Brain Regions Important for Reproduction

Jaeyoung Son, Minnesota State University Mankato

Steroid hormones, such as testosterone (T) and its metabolites (estradiol, E2, and dihydrotestosterone, DHT), are critical for the production of reproductive behavior. These hormones play a role in neural plasticity, such as changes in neuronal size change and brain region volume. Our study is examining the role of steroid hormones in maintaining the morphology of brain areas involved in reproduction, such as the ventromedial hypothalamus (VMH) and preoptic area (POA). We are using the green anole lizard (Anolis carolinensis) as a model because they are seasonally dimorphic, with more reproductive behaviors and higher steroid hormones in the breeding compared to non-breeding season. We treated our animals with different steroid hormones: T, DHT, E2, and blank capsules as a control. We collected the brains, sectioned the tissue and measured neuron size, number and density in the VMH and POA. We are expecting to find smaller and increased numbers of neurons in the animals treated with steroid hormones compared to the controls. This result would support the idea that steroid hormones are critical for the maintenance of brain areas important for reproduction.

Seasonal Variation in the Dorsolateral and Medial Cortex of Green Anole Lizards

Amber Day, Minnesota State University Mankato
Abdi Abdilahi, Minnesota State University Mankato

The hippocampus is a region of the brain involved in spatial learning and memory, and has been shown to add new neurons in adult animals. Steroid hormones, specifically testosterone

(T) and its metabolites (estradiol, E2, and dihydrotestosterone, DHT), have been shown to play a role in the addition of adult-born neurons to the brain. The green anole lizard, Anolis carolinensis, is a seasonally breeding animal that exhibits seasonally dimorphic behaviors, as well as seasonal anatomical differences in the brain. The pronounced differences between the breeding and non-breeding seasons make this lizard an excellent model for the study of how steroid hormone differences impact the brain. We examined the volume of and addition of new adult-born neurons to the dorsolateral and medial cortex in the lizard, which is analogous to the mammalian hippocampus. We sectioned brain tissue from breeding and non-breeding animals, performed a Nissl stain, and are measuring volume of the regions. We expect that the region will be larger in the breeding season due to the increase of territorial and courtship behaviors. We also treated animals with T, DHT, E2 or nothing as a control and performed an immunohistochemistry to examine how steroid hormones impact neurogenesis. We expect to see significantly more neurogenesis in the dorsolateral and medial cortex of T, DHT, E treated animals in comparison to the untreated group. Our experimental results may provide a greater understanding of the mechanisms that regulate the neural control of reproduction and territorial behaviors.

Amygdala Morphology and Neurogenesis in the Green Anole Lizard

Jadden Roddick, Minnesota State University Mankato
Nicholas Booker, Minnesota State University Mankato
Abodalrahman Algamdy, Minnesota State University Mankato

Steroid hormones and their derivatives play a major role in the reproductive system. One region in the brain that is involved in reproduction is the amygdala. We are examining the relationship between steroid hormones and neuron size, number and neurogenesis in the amygdala of the green anole lizard (Anolis carolinensis). Green anoles are exceptionally good models to examine the neural control of reproductive behaviors because they are seasonally breeding animals and exhibit unique behavioral and physiological differences in the breeding season compared to the non-breeding season. These behavioral differences are likely caused by seasonal changes in circulating steroid hormone levels. For our project, breeding green anole males were gonadectomized and a capsule containing testosterone, estradiol, dihydrotestosterone or left empty was inserted under the anole’s skin. The animals were injected with bromodeoxyuridine (BrdU; a new cell marker) for three days after the treatment. After one month, brains were collected, sectioned, and placed on slides. An immunohistochemistry for BrdU and Hu (neuronal marker) was conducted to examine the presence of new neurons in the amygdala. Alternate sections were Nissl stained and used to count cell number and measure soma size. We expect to see a decrease in neuron number, soma size, and neurogenesis in the animals treated with hormones compared to the animals treated with the blank capsule because we see this pattern in breeding season animals. This work will help provide more insight into the neural control of reproduction.

Anole egg from http://www.anoleimaging.com/Anoles/ag_16_egg2.html

A concerned Anole Annals reader writes in:

My dog just violently chomped  a female alone. Along with her entrails protruding from her body we two eggs. One was small, under-developed the other was the size they are laid. I have at the time done my best to put it into a container and emulate the  same conditions outside ( I live in Florida) with  dirt, leaf litter (small) moisture and heat. I removed the placental outer membrane which would have been separated if she had laid. I feel terrible my young and excitable dog did this. Is there any hope?

Can anyone advise?

A noble beast and mighty warrior.

Read all about it in Rodríguez-Cabrera et al.’s new paper in IRCF Reptiles & Amphibians.

Egret and maybe anole

We’ve come to realize, sadly, that just about everything will eat anoles. Birds are particular culprits and we’ve seen some horrifying examples of egrets downing the little green and brown fellows. Now comes a report that a whooping crane, of all things, will also indulge.

Vladimir Dinets–he of crocodilian behavior fame (check out his awesome book, Dragon Songs )–reported on dietary observations a reintroduced population of cranes in Louisiana. The anolivory represents the first instance of whooping crane predation on a lizard (but not on a squamate, as the photo above attests).

Photo by Pat Shipman

We’ve often commented on the interactions between the green anole, Anolis carolinensis, and the brown anole, Anolis sagrei. We’ve also had periodic posts from Pat Shipman on Little Cayman, who moonlights as an anthropologist and science and history writer when not watching anoles.

Here’s further evidence that greens and browns can coexist: A. maynardi (a relative of A. carolinensis) and A. sagrei side-by-side, ten feet up on a wall.