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Examples of embryos with normal and abnormal craniofacial development

Embryos are not just little organisms encapsulated within their eggs. Embryonic development is dynamic; the embryo transitions from one to a few undifferentiated cells to a stage where the various parts like arms, legs, and faces become apparent to a form that resembles the species that will eventually emerge. A panoply of signaling events and rapid rates of cell division are all tightly choreographed to make sure that development proceeds in a predictable, species-specific fashion.

But this dynamism of development also makes the embryo susceptible to environmental perturbation. Heat, chemical exposure, and pathogens can all disrupt normal embryonic development, sending the embryo down paths that may lead to fatality or reduced fitness. In our recently published study, my colleagues, students, and I demonstrate that heat stress, paralleling what will likely be experienced during the 21st century, can induce structural malformations to the brain and face of lizard embryos.

In 2014 and 2015 I was a post-doc in the Cohn Lab at the University of Florida. At the time, I had been dissecting and observing anole embryos for approximately 14 years. Throughout those 14 years I had observed only a handful of malformed embryos, maybe on the order of 10-20 embryos after collecting thousands of embryos from numerous anole species. Yet, in the summers of 2014 and 2015, while working alongside then graduate student Bonnie Kircher, I collected more malformed Anolis sagrei embryos than I had in all of my previous years. I certainly didn’t realize it in the moment as malformed embryos were still relatively rare compared to the total number of embryos we were collecting. But, by the end of my time in Florida the number jumped out at me. Sometimes the rate of development seemed to depart from the normal sequence of development. Other times the embryo was clearly not well and would likely not survive to hatching. Even as other projects accelerated, my interest in these malformed embryos remained piqued.  When I began my faculty position Loyola University Chicago, I decided to invest the lab’s time and resources into determining whether this pattern was real or just a chance observation.

In spite of other options­–unique genetic mutation running rampant in Gainesville populations of brown anoles seems highly unlikely–I decided to investigate the effects of heat stress on embryonic development. The effects of global warming had been widely discussed as a threat to ecotherm populations and anoles have been at the center of both field and lab observations since the outset. A number of studies have also shown reduced hatching success from lizards incubated at relatively high temperatures. Relating this back to my observations in Florida, it also occurred to me that Bonnie and I used a different collection strategy for our breeding colony those years; we would regularly replenish or add to the colony from field-caught lizards throughout the summer. This raised the possibility that the embryos developing within the gravid females were exposed to the environmental heat stress before being deposited into our waiting hands. Luckily, brown anoles are prolific egg producers, providing my lab with the ability to test whether heat stress induces embryonic malformations under different incubation regimes.

Embryos incubated under conditions reflecting those observed in shady nest sites exhibit malformations in only one to two embryos out of every 100 live embryos. These nest sites tend to be relatively stable in temperature, rarely rising about 30 degrees Celsius. However, embryonic heat stress induces malformations in 10-30% of embryos exposed to incubation conditions that parallel nest sites that would be located in sunny locations. These putative nest sites reach temperatures above 36 degrees Celsius, the critical thermal temperature for the embryos, for up to eight hours per days. The malformations we observed were not evenly spread across the body. Instead, we saw the greatest concentration in the brain and face of the developing lizards. Most malformations included a change in facial proportion, from subtle changes in facial length to pronounced brachycephaly and/or clefting. In one case, the entire face and forebrain were ablated in the embryo! When it comes to the induction of structural malformations, the most sensitive period of development is around oviposition, including the time that the egg is still within the female. Although we do not yet know how many of these embryos would successfully hatch, our experiments do raise concerns about the long-term impacts of global warming on ectotherm development.

Examples of extreme, but rare malformations observed in eggs reared at elevated temperatures.

The consistent pattern of thermal-induced neural and facial anomalies made us think that there may be a common underlying cause of these changes, leading us to create and test a new model of embryonic thermal stress. Based on our understanding of amniote craniofacial development, we predicted that disruption to Hedgehog signaling, one of the earliest signaling pathways needed for facial development, could create the full spectrum of observed malformations. After measuring processes up and downstream of Hedgehog signaling (e.g., cell death and signaling within the presumptive facial cells respectively), it does, in fact, appear that Hedgehog signaling is disrupted in the face of embryos experiencing thermal stress. Depending on the degree of response by a particular embryo, everything from normal to extremely malformed embryo could be induced. At this time, it appears that our model holds for brown anoles and may be applicable in species far beyond anoles and lizards.

In our proposed model, heat stress affects Hedgehog signaling, causing a disruption to normal facial morphogenesis.

There remains much to learn about normal and abnormal facial development in lizards. We do not yet know what other signaling pathways are equally disrupted during thermal stress or whether there are endogenous buffering mechanisms that help to maintain normal development in the face of external stress. Perhaps one of the most important discussions that needs to occur is how we study rare events. These events could be uncommon, but extreme heat events that exceed the “normal” conditions typically observed in the wild. These are increasing in regularity and may have significant impacts on ectotherms later in the 21^st century. Alternatively, the rare events could be the emergence of malformed embryos which occur in only a fraction of individuals, even when the average phenotype is not dramatically altered. For species such as the brown anole, this may not be alarming. But for species with relatively few viable hatchings each season, embryonic heat stress could have dramatic impacts on their long-term viability. These developmental perspectives  are needed to fully understand the ways that global change will affect the lives and longevity of lizards and other ectotherms.

Embryos are impacted by a range of challenges associated with anthropogenic change. See more in Sanger 2021, Integrative developmental biology in the age of anthropogenic change

An illustration from Gosse (1851) depicting Jamaica’s crown-giant, Anolis garmani (note that Gosse used the old name, “Dactyloa Edwardsii”), adjacent to a West Indian boid (Chilabothrus).

Jamaica’s six endemic Anolis species–A. garmani, A. reconditus, A. valencienni, A. lineatopus, A. grahami, and A. opalinus–have long captured the fascination of Caribbean naturalists. And how could they not? Although only six, these lizards are among the Caribbean’s most eye-grabbing. Take it from Phillip Henry Gosse, who recounted his experience catching one of these six species using a twine lasso in A Naturalist’s Sojourn in Jamaica (1851):

“The mode in which I formed an acquaintance with the species may be worthy of being related. One day in February, having ascended the ridge with a companion, my attention was arrested by a Lizard about a foot long, and of a lively green colour, on the trunk of a small tree, head downward, intently watching our motions as we stood near. My young friend suggested the possibility of capturing it by slipping a noose over its head, while its attention was engaged by whistling. I laughingly proceeded to try the spell; and having made a noose of small twine, which I tied to the end of a switch, I gently walked towards him, whistling a lively tune. To my astonishment he allowed me to slip the noose over his head, merely glancing his bright eye at the string as it passed. I jerked the switch; the music ceased; and the green-coated forester was sprawling in the air, dangling, greatly to his annoyance, at the end of my string. He was very savage, biting at every thing near; presently his colour began to change from green to blackish, till it was of an uniform bluish black with darker bands on the body, and a brownish black on the tail: the only trace of green.”

Since Gosse’s work on West Indian reptiles (including formal designation of the Jamaican anole radiation as Placopsis), much has changed in our understanding of Jamaica’s Anolis diversity and evolution. In 2002, Todd Jackman and colleagues published a study aiming to estimate the phylogenetic relationships among Jamaica’s anoles.

The authors found support for the following topology (Jackman et al. 2002; Fig. 7): A. lineaotopus and A. reconditus form a clade that is sister to the remaining Jamaican anoles, followed by A. valencienni, A. garmani, and, ultimately, A. grahami + A. opalinus. Jackman et al. would continue in the same paper to discuss phylogeography and intraspecific divergences. Notably, a single A. opalinus from Hardwar Gap (Blue Mountains) appeared to be more closely allied to A. valencienni than to other sampled A. opalinus individuals. This diverse array of mitochondrial haplotypes recovered from A. opalinus then raises the question, does the history of Anolis on Jamaica involve hybridization, or is it merely a result of incomplete lineage sorting we may expect from a rapid adaptive radiation?

Nearly twenty years went by without an answer. Was the phylogenetic hypothesis posited by Jackman et al. correct? What’s going on with A. opalinus? Separately, in the two decades since the work by Jackman and colleagues, a revolution has occurred in phylogenetic biology. In the study of phylogeny, if introgression (e.g., ancient mitochondrial capture as a result of hybridization) has played a role in shaping the evolutionary history of the relevant group, it’s important that we test if such events have occurred, and subsequently be able to disentangle and ultimately account for them in our phylogenetic estimations. Failure to do so can lead to misleading topologies, inaccurate reconstructions of evolutionary histories and parameter estimates, as well as shortcomings in accurately describing and recognizing biodiversity.

Luckily, resolution to this issue has been afforded by several folks (Claudia Solís-Lemus and Cécile Ané, to name just two) who have developed statistical methods to detect and account for both incomplete lineage sorting (ILS) and reticulate patterns of evolution (introgression). So, how does this relate to those oh-so-cool Jamaican anoles? This is where Myers et al. (2021) come in.

In a new study in the Spotlight section of Systematic Biology, Myers and colleagues re-open the investigation into Jamaican Anolis phylogeny using the aforementioned suite of novel methods. For those in attendance at the Joint Meeting of Herpetologists and Ichthyologists in 2018, the early stages of this work were presented by students conducting research at the American Museum of Natural History, previously featured on Anole Annals. Using genotyping-by-sequencing (an approach that has been applied elsewhere in Anolis research), the authors generate swaths of SNP data from the nuclear genome. To be exact, the total dataset comprised 257,317 base pairs (just over 2,900 loci). Restriction-site-associated approaches (i.e., RAD-Seq) to sequencing DNA have made it feasible to capture large, representative samples of the nuclear genome at low cost.

Armed with data from both the nuclear and mitochondrial genomes, Myers et al. (2021) found dramatically different relationships and evolutionary histories among species between the two sources. Figure 3 demonstrates this disparity well, and further hammers home the general dangers of violating model assumptions in phylogenetic inference (in this case, the multi-species coalescent [MSC] model, which assumes a lack of gene flow among sampled taxa).

First, when using solely the mtDNA data in a MSC framework, the authors find A. opalinus from the Blue Mountains to be polyphyletic, a result concordant with the inference of Jackman et al. (2002). Specifically, one A. opalinus lineage (Blue Mountains) is sister to A. valencienni, whereas the other A. opalinus lineage is sister to A. grahami. Notably, the time calibrated mtDNA gene-tree suggests the two A. opalinus mtDNA genomes are more than 30 million years divergent!

The authors then infer a species tree using the GBS data in an MSC framework (one that assumes gene-tree species-tree discordance can be attributed to incomplete lineage sorting and accounts for such). With this tree, the two A. opalinus lineages form a clade sister to A. grahami and A. garmani. Finally, with SnaQ (a modeling program developed by Solís-Lemus and colleagues to infer reticulation events across a tree), the authors recovered an identical topology to the nuclear DNA based species-tree, but with high support for a single introgression event (and hence, reticulate evolution) between A. opalinus and A. grahami. Using simulations, Myers and colleagues provide evidence against ILS as the causative agent for gene-tree species-tree discordance, favoring hybridization as the culprit.

I won’t spoil the Discussion, but much of Jamaican Anolis evolution remains open-ended (including the role of adaptive introgression in shaping Anolis communities). It’s probably safe to say Gosse wasn’t pondering the possibility of adaptive introgression in shaping Jamaican Anolis phylogeny. However, resolution of the outstanding questions identified by Myers et al. (2021) will come primarily through Gosse’s philosophy–getting out in the field, catching lizards, and getting a more-fine scale, phylogeographic picture of the variation over space and time in Jamaican Anolis.

I thank Ed Myers and Kevin de Queiroz for feedback on this blog post.

Literature Cited:

Jackman, T. R., Irschick, D. J., De Queiroz, K., Losos, J. B., & Larson, A. 2002. Molecular phylogenetic perspective on evolution of lizards of the Anolis grahami series. Journal of Experimental Zoology 294(1) 1-16.

New literature alert!

Interspecific Gene Flow and Mitochondrial Genome Capture During the Radiation of Jamaican Anolis Lizards (Squamata; Iguanidae)

In Systematic Biology

Myers, Mulcahy, Falk, Johnson, Carbi, de Queiroz

Gene flow and reticulation are increasingly recognized as important processes in the diversification of many taxonomic groups. With the increasing ease of collecting genomic data and the development of multispecies coalescent network approaches, such reticulations can be accounted for when inferring phylogeny and diversification. Caribbean Anolis lizards are a classic example of an adaptive radiation in which species have independently radiated on the islands of the Greater Antilles into the same ecomorph classes. Within the Jamaican radiation at least one species, A. opalinus, has been documented to be polyphyletic in its mitochondrial DNA, which could be the result of an ancient reticulation event or incomplete lineage sorting. Here we generate mtDNA and genotyping-by-sequencing (GBS) data and implement gene-tree, species-tree, and multispecies coalescent network methods to infer the diversification of this group. Our mtDNA gene-tree recovers the same relationships previously inferred for this group, which is strikingly different from the species-tree inferred from our GBS data. Posterior predictive simulations suggest that our genomic data violate commonly adopted assumptions of the multispecies coalescent model, so we use network approaches to infer phylogenetic relationships. The inferred network topology contains a reticulation event but does not explain the mtDNA polyphyly observed in this group, however coalescent simulations suggest that the observed mtDNA topology is likely the result of past introgression. How common a signature of gene flow and reticulation is across the radiation of Anolis is unknown; however, the reticulation events that we demonstrate here may have allowed for adaptive evolution, as has been suggested in other, more recent adaptive radiations.

In a recent study in the Proceedings of the National Academy of Sciences, Bock et al. (2021) conduct a genomic and phenotypic appraisal of adaptive evolution and invasion biology of Anolis sagrei (Wikimedia Commons).

In 2004, Jason Kolbe and colleagues published a now-classic invasion biology study in Nature, mapping out the population genetics of invasive Anolis sagrei populations. Using approximately 1,200 bp of mtDNA sequence data (ND2 and adjacent tRNAs), Kolbe et al. examined the evolutionary origins of the Brown Anole in its journey out of its ancestral area (Cuba), and into a broad invasive range. In several out-of-Cuba dispersal events that formed the collective invasive Brown Anole in Florida, Kolbe et al. (2004) found that invasion had actually increased genetic diversity within these populations, far greater than that observed in the native range. Furthermore, many of the global invasive populations of A. sagrei (e.g., Taiwan, Grenada), sourced from Florida, had maintained comparably high levels of genetic diversity. Hence, the work by Kolbe et al. demonstrated that the repeated introduction of multiple, evolutionarily diverse lineages derived from the native range and subsequently injected into a novel range may be a key force in driving successful invasion.

If we fast-forward five years to 2009, two years prior to the release of the A. carolinensis genome, Chris Schneider publishes a paper in Integrative and Comparative Biology (Schneider 2009) titled “Exploiting genomic resources in studies of speciation and adaptive radiation of lizards in the genus Anolis.” In his paper, Schneider discusses the unique opportunity that lies ahead in understanding evolutionary theory through the lens of Anolis genomic resources. Schneider’s vision—as I perceive it—was one that sought to excite the evolutionary ecology community about the wonderful opportunities ahead that Anolis lizards present in understanding, most broadly, the genetic basis of adaptation.

Now, in 2021, more than 15 years after the study by Kolbe et al., Bock et al. (2021) return for an integrative evolutionary investigation of A. sagrei throughout Florida using a recently generated reference genome and corresponding morphological data. In line with Schneider’s (2009) perspective, Bock et al. (2021) tell a captivating story of adaptation, genome biology, and invasion. Wielding the power of one of the most contiguous and complete squamate genomes assembled to date (more on that another time!), the authors identified a large-effect locus posited to be responsible for adaptive shifts in limb length, which in turn provides insight into how natural selection can modulate hybridization during the course of biological invasion.

New literature alert!

Changes in selection pressure can facilitate hybridization during biological invasion in a Cuban lizard

In PNAS

Bock, Baeckens, Pita-Aquino, Chejanovski, Michaelides, Muralidhar, Lapiedra, Park, Menke, Geneva, Losos, and Kolbe

Abstract

Literature cited:

Kolbe, J. J., Glor, R. E., Schettino, L. R., Lara, A. C., Larson, A., & Losos, J. B. 2004. Genetic variation increases during biological invasion by a Cuban lizard. Nature 431(7005): 177-181.

Schneider, C. J. 2008. Exploiting genomic resources in studies of speciation and adaptive radiation of lizards in the genus Anolis. Integrative and Comparative Biology 48(4): 520-526.

From the pages of the Gainesville Sun , referring to a recent paper in Oecologia.

From April to September of 2017, Jesse Borden was climbing trees and counting lizards around Alachua County.

Sometimes, he could be found in branches on the University of Florida campus or in people’s backyards. Other times, his distinctive red helmet popped in and out of leaves in nearby forests.

The 34-year-old UF student is in his fourth year pursuing a doctoral degree in interdisciplinary ecology, and much of his work has focused on Gainesville’s native green anole lizards and their responses to two threats: development and invasive brown anoles.

He found that in the presence of brown anoles, the green natives moved about 17 times higher in trees, or about 8.3 meters in median perch height, to coexist. But the shift did not allow the lizards to overcome their habitat loss from human development.

These findings were recently published on Oct. 7 in the journal Oecologia.

“The extent to which [green anoles] were shifting was pretty fascinating,” Borden said. “That appears to allow them to coexist with the threat of an invasive species, the brown anole that is competing with it, but it doesn’t make them immune to other effects like urban development. And so it seemed like urbanization was the strongest driver of their decline across the landscape.”

Brown anoles were first introduced to the mainland southeastern U.S. in the early 1900s and were well established by the 1940s, according to the study. In Alachua County, they have been established for decades and appear to thrive in urban environments, Borden said. It is not known exactly how they came to the area, but it could have been via cargo and boats.

To study the green anoles’ response to both development and brown anoles at the same time, he and other helpers surveyed 61 trees and the ground around them for lizards twice each, once per day and once after dark. They then statistically analyzed the data for metrics like abundance, perch height and urbanization.

“It was a lot of fun,” Borden said. “Many thanks to so many kind people who let us use their backyard trees.”

The student said his findings raised many questions and topics for future research, such as how much time the green anoles spend higher in the trees. He is currently working on a project looking at evidence for change in body shape across the urban to natural gradient in lizard species here and change in their temperature tolerance to cold.

For Gainesville residents who miss seeing the little green lizards, Borden said, there are a few things people can do to help bolster their habitat space in the city. Planting native vegetation of varying heights in yard space can benefit the green anoles. Protecting and preserving forest patches and trees also supports them and lots of other wildlife.

“I just hope people are noticing the green anoles,” Borden said. “I find them so beautiful. They’re super cool. They’re a really fun and special part of the southeast U.S. and Florida.”

Contact Borden online via Twitter at @JesseBBorden or Instagram at @borden_ecology_adventures.

green anole basking under UVB, photo credit: r/anoles user, u/BMKMNC

Every so often I notice some of my anoles hanging from the screen lid of their enclosure, directly underneath the UVB light. At first I thought that it was just a fluke, but I have since observed them doing it on multiple occasions. Moreover, this UVB basking behavior has been reported by another green anole owner on the r/anoles subreddit (see pictures below). There must be something that compels them to do this.

I considered that maybe they are UVB basking to compensate for a deficiency. I changed my anoles’ UVB bulbs, but they still sporadically engage in the basking behavior. So, I am looking into other lighting options because I have concerns that the lizards aren’t getting enough UVB light through the screen, or that the lights aren’t strong enough. I do not have a UVB meter to confirm these suspicions. Regardless, my anoles all seem healthy and active.

Although I have not been particularly scientific with my observations, I have noticed that UVB basking is engaged in more frequently by my females and my elderly male anole. Perhaps they require more vitamin D than the average male green anole due to fertility and senescence, respectively. I have not noticed a trend regarding when this behavior occurs; it seems to happen at random. More careful observation is required.

What are your thoughts regarding the cause of UVB basking? Have you witnessed your anoles engaging in this behavior as well?

Green anole basking under UVB, photo credit: r/anoles user, u/BMKMNC

My senior green anole, tanning under UVB light

In a new study published in the Proceedings of the National Academy of Sciences, Patton et al. (2021) examine island and mainland radiations of Anolis lizards in an effort to understand what occurs when “adaptive radiations collide.” Discussion of mainland anoles merits featuring one of the oddest mainland species, the Ecuadorian Anolis proboscis (female and male pictured here; credit Santiago Ron [Wikimedia Commons]).

New literature alert!

Upon hearing “anole lizards,” those in the evolution and ecology community familiar with the outstanding diversity of Anolis lizards may immediately reflect on the replicative adaptive radiations that have occurred in the Greater Antilles, painting a portrait of adaptation, convergence, and ecological character displacement that has served as the basis of research among Caribbean biologists for decades. But, perhaps, what is less generally appreciated is that the vast bulk of Anolis lizard diversity (currently sitting at 436 species, per the ReptileDatabase) actually occurs on mainland Central and South America! Indeed, if we were to zoom out on the Anolis Tree of Life, we could pick out three major clades that represent independent adaptive radiations– one in the Greater and Lesser Antilles, and then two on the Mainland. Hence, as has been appreciated by many Anolis biologists before (most recently, Huie et al. 2021), the multiple radiations of these lizards provides the substrate to examine not only convergence, but, additionally, what happens when these clades come into contact? What happens when adaptive radiations collide?

This question forms the basis (and title) for a recently published study by Patton et al. (2021), who attempt to untangle themes of adaptation, historical biogeography, convergence and divergence in ecology and morphology, and the diversification dynamics of the three major Anolis radiations.

When adaptive radiations collide: Different evolutionary trajectories between and within island and mainland lizard clades

In PNAS

Patton, Harmon, Castañeda, Frank, Donihue, Herrel, and Losos

Abstract:

Oceanic islands are known as test tubes of evolution. Isolated and colonized by relatively few species, islands are home to many of nature’s most renowned radiations from the finches of the Galápagos to the silverswords of the Hawaiian Islands. Despite the evolutionary exuberance of insular life, island occupation has long been thought to be irreversible. In particular, the presumed much tougher competitive and predatory milieu in continental settings prevents colonization, much less evolutionary diversification, from islands back to mainlands. To test these predictions, we examined the ecological and morphological diversity of neotropical Anolis lizards, which originated in South America, colonized and radiated on various islands in the Caribbean, and then returned and diversified on the mainland. We focus in particular on what happens when mainland and island evolutionary radiations collide. We show that extensive continental radiations can result from island ancestors and that the incumbent and invading mainland clades achieve their ecological and morphological disparity in very different ways. Moreover, we show that when a mainland radiation derived from island ancestors comes into contact with an incumbent mainland radiation the ensuing interactions favor the island-derived clade.