Incubation temperature is an important factor in development for anoles (and other ectotherms). Thom Sanger, a professor at Loyola University in Chicago, IL, presented his research on how high temperatures affect brain formation in developing anole embyros. With the help of undergraduates and a high school summer intern, Dr. Sanger found that when developing eggs were heat-shocked, many embryos were lost (75%), but for those that survived, forebrains became smaller (In the figure, A is normal and B is deformed). Interestingly, malformation of the forebrain affects the size and shape of the face, and so surviving heat-shocked embryos exhibit cranial malformations. As Sanger continues his research, he will follow a neural degeneration hypothesis, which boils down to (no pun intended) the idea that thermal stress increases the rate of cell death, and the amount of cell death affects facial shape. While the effects of high temperature may seem alarming, Sanger notes that this does not happen very often in nature; females are generally pretty good at selecting suitable nest sites. But, because development is similar across reptile taxa, anoles can be an excellent model system to inform predictions about what may happen to species that are in danger.
Anolis morazani. Photo by Josiah Townsend from iNaturalist.
The Anolis crassulus subgroup contains ten morphologically-conserved highland anole species found throughout Nuclear Central America. Its members have long been a source of headache for region’s systematists. To quote Meyer & Wilson (1971): “…specimens of the crassulus group from Guatemala and Mexico have a bewildering array of admixtures of the distinctive characters observed in Honduras… The inter-relationships of the populations… [of the crassulus group] are exceedingly complex, and… we are unable to suggest a satisfactory arrangement.” This was followed up 21 years later by McCranie, Wilson, & Williams (1992): “Clearly, a thorough analysis of crassulus-like specimens from throughout their range… is sorely needed”, and repeated by McCranie & Köhler (2015) 13 years after that.
Despite the need for a thorough investigation into this group, our understanding of the relationships and validity of these taxa has not improved much. This is partly because this subgroup has been poorly represented without broad sampling in larger-scale molecular phylogenies. Two samples in particular, an A. crassulus (from Chiapas, Mexico) and an A. sminthus (from Olancho, Honduras), have been continuously utilized, without additional samples from these species. Most recently, Nicholson et al. (2017; six species) and Poe et al. (2017; three) expanded the molecular sampling for this group, using single exemplars as part of broader analyses.
In a study published last month in BMC Evolutionary Biology, Josiah Townsend and I examined the evolutionary relationships of the majority of this subgroup, in order to provide a starting point for resolving some of the confusion surrounding these taxa.
Fig. 4 from Hofmann & Townsend, 2017: Species tree of the Anolis crassulus subgroup. Black nodes indicate PP > 0.95; PP < 50 not shown. Inset photo: Anolis heteropholidotus (2) by JHT.
The results of our multilocus phylogenetic investigation gave us some interesting new insights into the subgroup. We found support for the monophyly of the A. crassulus subgroup relative to other Anolis (as opposed to its paraphyly, as recovered in Poe et al. 2017), and the validity of all of its species (excepting the two we could not sample). Additionally, we recovered considerable overlooked diversity within this subgroup. Anolis crassulus itself represents at least four lineages corresponding to distribution: the Chortis Highlands of Honduras, the Salvadoran Cordillera, Guatemala, and Chiapas, Mexico. Surprisingly, the sample from Chiapas previously used in many phylogenies was recovered as an undescribed lineage sister to A. anisolepis, not conspecific with any of the four A. crassulus lineages, including another Chiapan lineage. We also recovered the widely-used “A. sminthus” sample (which was previously hypothesized by McCranie and Kohler (2015) as representing an undescribed lineage more closely related to A. crassulus) as an undescribed lineage sister to A. morazani, and found additional mitochondrial lineages within A. heteropholidotus and A. rubribarbaris. Diversification within the group was estimated to have started in the early Miocene in the Chortis Highlands (supporting the results of Nicholson et al. 2017), with the Honduran population of A. crassulus diverging from the other three lineages approximately 13 MYA.
Fig. 5 from Hofmann & Townsend, 2017: Chronogram showing results from divergence dating and ancestral area reconstruction analyses. PP shown when < 1.
Given the relatively deep divergence times within this group when compared with the apparent lack ecological and morphological diversification, we hypothesized that this subgroup represents a non-adaptive radiation, though extensive study is necessary to determine if these traits are as conserved as they appear. A taxonomic revision of the Chortis Highland population of Anolis crassulus is being finalized, but a great deal of work remains in order to improve our understanding of these highland anoles.
One more update from the SICB conference in San Francisco last week!
Across vertebrates, the ratio of lengths of the second and fourth digits of the hand are influenced by testosterone and estrogen. This could be of particular importance in species such as anoles, in which the fourth digits of the hindlimbs are extremely long and critically important in locomotion, but previous studies of the 2D:4D ratio in anoles have produced varying results. In the final poster session at SICB, undergraduate Griffin McNamara, working with Bonnie Kircher in Marty Cohn’s lab at the University of Florida, presented preliminary results from a study of cleared and stained brown anole (Anolis sagrei) hind feet. Griffin has big plans for continuing this work, so watch for future publications with these findings!
A, wattsi is one of five introduced species on the islands. A new report in Living World: the Journal of the Trinidad and Tobago Field Naturalists’ Club, indicates that it’s on the move.
Dan Warner (left) and Tim Mitchell (right) beside their poster on impacts of population density and time of hatching on survival and early life phenotypes of Anolis sagrei
Tim Mitchell a post-doctoral researcher at University of Minnesota with Emilie Snell-Rood presented his work from his prerious post doc in Dan Warner’s lab where he investigated the impacts of density and timing of hatching on the survival and growth of Anolis sagrei hatchings. Seeking to specifically address these questions:
How does investment in offspring size and number shift seasonally?
Does the timing of hatching influence survival or growth in the field?
And does adult density influence survival or growth of hatchlings in the field?
Adult anoles were brought into the lab on three different dates and breeding was split into three corresponding windows of time: Cohort 1 (February 23rd – April 27th), Cohort 2 (June 18th – July 30th), and Cohort 3 (September 5th – October 15th). On experimental islands, adult densities were manipulated to create high and low lizard densities. Hatchlings from cohorts 1, 2, and 3 were released onto high and low adult density islands in June, August, and October, respectively, and researchers returned the following spring to recapture the marked lizards.
Breeding in the lab revealed a seasonal shift from producing more smaller offspring early to producing fewer larger offspring later in the season. Adult densities on the islands did not affect hatchling survival, but there was a substantial survival advantage to being an early-hatched lizard. Size and growth of hatchlings were influenced both by timing of hatching and the adult densities. So happy to catch up with my academic family and see the cool research they are doing!
Figure 1. Differences in claw clipping used in Bloch and Irschick (2005) and our study. (A) Bloch and Irschick (2005) clipped the entire claw after the distal end of the toe pad. (B) In our study, we partially clipped the distalmost portion of the claw.
Toe and claw clipping are common techniques used to identify individuals in mark and recapture studies, but their impacts on whole organism performance are unclear (Dunham et al., 1988). Anoles have not only developed subdigital adhesive toe pads to promote adhesion on relatively smooth substrates, but have also retained claws to enhance attachment to rough substrates (Irschick et al., 1996; Zani, 2000). Thus, clipping entire toes or claws may have drastic effects on the clinging ability of anoles or other adhesive pad-bearing lizards. In our recent article published in Acta Herpetologica, my co-authors and I investigated how partially removing the claws of brown anoles affects their adhesive performance.
Figure 2. Mean maximum clinging force of Anolis sagrei with intact and partially clipped claws. Overall, partial claw clipping had no significant effect on maximum clinging ability.
Bloch and Irschick (2005) removed entire claws from Anolis carolinensis (Fig. 1A) and measured its impact on their clinging ability. Not surprisingly, claw removal resulted in a significant decrease in the clinging ability of A. carolinensis, likely a consequence of the severing of flexor tendons that are critical in adhesive toe pad engagement. In an effort to test this hypothesis and preserve these tendons, we used a motorized force sensor (Niewiarowski et al., 2008) to measure the maximum clinging ability of 19 Anolis sagrei before and after their claws were partially clipped (Fig. 1B).
Overall, we found that partial claw clipping did not significantly impact maximum clinging ability (Figure 2). This suggests that clipping the entire claws of anoles may indeed sever the flexor tendons crucial to toe pad engagement. Furthermore, we expected clinging ability to increase after partial claw clipping because claws should theoretically interfere with the contact the subdigital adhesive pads are capable of producing. However, this did not appear to be the case, suggesting that claws may not inhibit the engagement of subdigital pads or that morphological features and/or behavioral traits reduce the effect of this interaction.
Anolis sagrei
Although permanent marking solutions would be most beneficial for mark and recapture studies, partial claw clipping may be a useful alternative for shorter-term studies because it does not appear to reduce adhesive performance on smooth substrates. Future work should further consider the interactions between subdigital adhesive toe pads and claws, and determine the possible ramifications for adhesion and adhesive locomotion, particularly on rough substrates. Be sure to check out our full article for more details!
References
Bloch, N., Irschick, D.J. (2005): Toe-clipping dramatically reduces clinging performance in a pad-bearing lizard (Anolis carolinensis). J. Herpetol. 39: 288-293.
Dunham, A.E., Morin, P.J., Wilbur, H.M. (1988): Methods for the study of reptile populations. In: Biology of the Reptilia, pp. 331-386. Gans, C. Huey, R.B., Eds, Alan R. Liss, Inc., New York.
Irschick, D.J., Austin, C.C., Petren, K., Fisher, R.N., Losos, J.B., Ellers, O. (1996): A comparative analysis of clinging ability among pad-bearing lizards. Biol. J. Linn. Soc. 59: 21-35.
Niewiarowski, P.H., Lopez, S., Ge, L., Hagan, E., Dhinojwala, A. (2008): Sticky gecko feet: the role of temperature and humidity. PLoS ONE 3: e2192.
Zani, P. (2000): The comparative evolution of lizard claw and toe morphology and clinging performance. J. Evol. Biol. 13: 316-325.
In the face of mate competition, sperm morphology can vary in a way that can enhance an individual’s chances of siring offspring of females. Studies in the past have attributed increased relative testis size as an approximate measure of an individual’s response to sperm competition. However, this does not take into account the internal architecture of the male testes that may contribute to changes in sperm morphology.
This was the focus of a poster presented by Hanna Hall titled “The evolution of sperm and testis morphology in Anolis lizards” in collaboration with Ariel Kahrl and Michelle Johnson. The authors sampled 2-20 individuals of different species of anoles in Puerto Rico and the Dominican Republic. They compared body size, sperm length ( 15 cells per individual), and the composition and size of various layers of the testis, by conducting a phylogenetic least squares regression on the average values obtained for each species.
The authors found that larger body size was associated with a larger testis size, which was in turn correlated with presence of large seminiferous tubules and a larger luminal area, where mature sperm are stored. Contrary to their expectation though, none of these aspects were associated with producing longer sperm. Further the Gonado-Somatic index (GSI), a common metric that serves as an indicator of relative testis size, was not correlated with any aspects of the internal testis architecture.
An interesting finding in this study was that species with a higher proportion of epithelial cells in the testis produced longer sperm. This result was surprising because larger number of epidermal cells may be associated with smaller spermatogonal cells, which would be predicted to form shorter sperm. The authors suggest that the correlation between lumen area and testis size may result because investment in sperm storage is more important, and that species may be producing large number of sperm which may be longer in length. Nevertheless, more data is needed to understand how changes in sperm morphology affect fertilization success and, further, under what circumstances does size and count of epithelial cells vary. The lack of correspondence of these results with that shown in birds by Lupold et al. 2008 suggests that the mechanisms underlying sperm competition may be taxa or species-specific. We will be eyeing the Johnson Lab for more details on the same in the coming years.
An example of an aggressive display by a lizard. Photo Credits: Neil Losin Photography
Testosterone has long been though to influence male aggression behaviors. But can this same hormone influence aggressive behaviors in females too? Ellee Cook addressed this question in her talk titled “Investigating the potential for testosterone to mediate territorial aggression in female Anolis lizards.”
Ellee focused on studying a population of Anolis gundlachi in the forests of Puerto Rico. Ellee studied the response of focal females to a staged territorial intrusion by another female who was placed on a cage lid, and compared it to a scenario where she directly approached the lizard. She captured the focal females after twenty minutes of the trial and measured their size and took a blood sample to estimate the circulating levels of testosterone. Her prediction was that higher levels of aggression would be correlated with higher levels of testosterone.
Her data showed that females were indeed aggressive towards intruding females and had much higher displays of aggression in comparison to when they were presented only a lid or were directly approached. Surprisingly (or not so surprisingly), testosterone was not a significant predictor of female aggression. In fact, none of the hormonal measures corresponded to female aggression. This finding could have resulted for several reasons: A) the amount of testosterone detected in females was much lower than that found in males, making variation in testosterone almost impossible to detect; B) High aggression may be caused by spontaneous spikes in testosterone that may be hard to detect; C) Female aggression may be governed by a completely different mechanism.
This study raises an important question about the relevance and drawbacks of existing paradigms which are male-centric and thus cloud our understanding when it comes to female behaviors. Cheers to more feminist paradigms in biology!
This post was written by Brittney Ivanov, research technician in the Johnson Lab.
PhD candidate, Abby Beatty, from Auburn University presented a poster entitled Integrating research into the classroom: causal effects of IGF1 and IGF2 on growth in the brown anole. The poster focused on an enhanced method of teaching science, particularly labs. The program, called C.U.R.E (Course-based Undergraduate Research Experience), allows students to experience teaching labs in a way that is more authentic and typical of the research experiences of graduate students. In most science labs, students are provided with different protocols and methods as well as a predetermined set of goals and results that explains how the experiment should turn out. The teaching method Abby proposed gives students the opportunity to learn from their failed attempts, before receiving the correct answers. Here we can draw a parallel with the approach used by the chemistry tutor.
The course lasted for 2 semesters, consisted of undergraduate and graduate students, and began with a pre-survey that assessed student’s current knowledge as well as their ability in certain cognitive skills: analyzing, applying, creativity, evaluating, understanding, and memory. The students then chose a topic (related to Abby’s dissertation work) to be the focus of the labs. From this, they were able to develop methods and design their labs.
Specifically, the first semester class cloned and expressed IGF1 and IGF2 (insulin-like growth factors) using a bacterial vector. Similarly, the second semester class cloned IGFBP2. Abby then used these proteins to optimize methods for studying the growth rate of eggs and hatchling brown anoles. Hatchlings were monitored for 10 weeks following an injection with either IGF1, IGF2, or vehicle (NaCl + 15% Gelatin). Two trials were performed on the hatchlings and one on the eggs. In the first hatchling trial, IGF1 and IGF2 treatments had significantly higher death rates than control groups, but there was no association with body size. In the second trial, which used refined and updated methods, there was no significant effect on survival or body size, when compared to control groups. Finally, egg treatment did not correlate with survival or body size.
As the class completed each step in this process, they reviewed their work and if their methods were unsuccessful, discussed a better approach. Following completion of the course, the students received a post-survey assessing the same skills and knowledge as the pre-survey.
Abby found the class gained significantly in these skills, particularly receiving higher survey scores in the areas of creativity and understanding. She also found that the average score on the knowledge assessment was higher in the classes post-assessment survey than in the pre-assessment, indicating that the students may be gaining from this method of teaching. Control surveys from a class taught using a typical lab curriculum are not available, but there are plans to include this over the course of coming school semester.
These data, while still preliminary, highlight the benefit of implementing this kind of teaching strategy. When students are able to explore the process of asking and answering questions they generally become more engaged in their work and better prepared for more authentic research experiences.
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