Category: New Research Page 18 of 67

Above: Faith Deckard presenting her research on how muscle physiology may explain variation in social behavior among Caribbean anoles.

Marathon runners and elite sprinters, like Usain Bolt, have dramatic differences in their muscle physiology that allow them to specialize in their respective track-and-field events. Whereas sprinters have lots of muscle fibers that produce high force but fatigue quickly, marathon runners have lots of muscle fibers that produce less force but allow much longer activity because of their reliance on aerobic respiration. Might this be true for our beloved Caribbean anoles, too? Faith Deckard of Michele Johnson’s lab at Trinity University tried to answer that very question. She studied six species of anoles in the Dominican Republic to test whether anoles that have higher rates of dewlap extension and extend their dewlap for a longer duration have dewlap muscles with a higher proportion of slow-twitch muscle fibers that can be used for endurance. Surprisingly there was no significant correlation between the two behavioral traits and the proportion of slow-twitch fibers! However, this scrutinizing attendee feels pretty strongly that there is a relationship that is just yet to be teased apart statistically. The raw data Faith presented looked very convincing to me, so we’ll see what the future holds for this question. Faith’s results are a very interesting clue to the still-elusive mechanisms that underlie anole behavioral diversity.

Above: Andrew Wang presenting his research on how leptin may be a mechanism underlying life-history trade-offs in green anoles.

All of the gumbo, Po boys, and beignets consumed by attendees of SICB 2017 have to go somewhere after consumption. Much of the energy contained in those delicious foods is used for very important maintenance functions in your body: metabolism, cell repair and replacement, and your immune system. What’s left over after maintenance costs can then be divided amongst other tasks, such as reproduction, movement, and wide variety of other tasks. Unlike humans, anoles do not have unlimited access to gigantic portions of gumbo, so their energetic investments require much harder decisions. Once energy from a cricket, for example, has been put into the immune system, it can no longer be used for making eggs or patrolling a territory a little bit longer. Andrew Wang of Jerry Husak’s lab at the University of St. Thomas was interested in what mechanisms are involved with anoles making these investment “decisions.” He did this by forcing allocation of resources to an energetically expensive trait (endurance running) by exercise training lizards to see what would happen to everything else that they might invest in.

Previous work showed that exercise training and diet restriction results in dramatic trade-offs with reproduction and the immune system. He suspected that what might explain this suppression was the hormone leptin, which is made by fat cells (yours make it, too). Since bigger fat cells means more leptin in the body, leptin can be thought of as a signal to the brain and body of how much resources are available for investment. Indeed, without sufficient leptin, reproduction grinds to a halt from the brain downward. Much like elite athletes, Andrew’s marathon lizards have little to no fat stores in their body, thus suggesting a role for leptin. To address this question, he supplemented half of the lizards with leptin (the rest got only saline as a control) to see if he could “rescue” immune function and reproduction. Interestingly, he found that leptin did rescue his measure of immunity, but it did not rescue reproduction. He attributes this latter finding to either (1) a lack of energetic resources to produce eggs even if there is a leptin signal or (2) the stress of the leptin injections over-rode the leptin signal in the brain where reproduction is controlled. His results suggest some very complex interactions in physiological pathways that can result in the trade-offs observed in many animal species.

Leptin is best known as a satiety hormone, but it has important roles as a signal to the body of adequate energy stores. Image from wiki.brown.edu.

Green anole image from reptilesmagazine.com.

What does it take be a good sprinter? How about a marathon runner? One might think that the traits responsible for such performance traits would be the same in males and females. If you are a green anole, that just isn’t true. Annie Cespedes, working in Simon Lailvaux’s lab at the University of New Orleans, explored the multivariate predictors of seven performance traits (sprint speed, bite force, cling force, exertion, endurance, jump power, and climbing power) in male and female green anoles. Annie explained how animals in nature rely on lots of different performance traits in their daily lives, and the large difference in body size and shape between male and female anoles might mean that the two sexes use different means to be successful in life. To add to this complexity, some individuals are just better overall at ALL performance traits than others (imagine a couch potato versus a very fit athlete), and one must account for this to understand what shapes anole performance.

Multivariate statistics allowed Annie to show that males and females do indeed differ in performance, but only in clinging ability, sprint speed, bite force, and jump power. Even more interesting, the suites of morphlogical traits that explained performance ability differed substantially between the sexes. For example, small females with large leg muscles were better sprinters and jumpers than females who are smaller and are better biters and endurance runners. What is especially important about Annie’s research is her approach. When considering how animals evolve, one must do so by simultaneously looking at a multitude of traits that might impact their survival and reproduction. By knowing how morphology predicts performance, we can begin to better understand how evolution will shape that morphology when selection acts on those performance traits.

(c) OwenMartin12, some rights reserved (CC BY-NC)

The abilities of certain animals to navigate and home on a specific location over long distances are some of the most fascinating behaviors that scientists study. However, studying homing behavior, especially experimentally, can be a major challenge, as many animals home over long distances (thousands of miles), in difficult-to-study environments (underwater, high in the sky), on in ways that are technically difficult or very expensive to monitor. As we know, anoles can be relatively simple (and cheap!) to study. So what if anoles could be developed as a model system for studying homing behavior?

On the surface, the presence of homing behavior in anoles might seem unlikely, as many species are highly territorial and may not travel long distances during their lifetimes. David Steinberg and Manuel Leal showed that, while seeming unlikely at first glance, at least one species of anole, Anolis gundlachi, does indeed show strong homing behavior.

Anolis gundlachi, the yellow-chinned anole, is a denizen of cool, closed forests in Puerto Rico. Because these lizards stick close to their small territories, they likely have little specific knowledge of their surrounding habitats, potentially making navigation through unfamiliar areas difficult. Steinberg displaced anoles 40 and 80 meters from their home territories and then monitored their territories to see how many anoles returned. Surprisingly, 40-60% of females returned and 80% of males returned, even when taken 80 meters from their homes. Simulations of these movements show that it is highly unlikely anoles would be able to return to their territories in this way via random searching. Steinberg then tested whether two common mechanisms that support homing, use of magnet fields and visual detection of polarized light, were responsible for homing, but found that the homing abilities of these anoles do not depend on either of these two senses.

Finally, Steinberg tracked anoles through the Puerto Rican forest using radio transmitters, and found that anoles returned to their home territories with a high degree of accuracy, in some cases making a beeline home within 24 hours! These results suggest that homing ability may be more common in anoles than has previously been considered, and that strong selection for territory ownership in anoles may support spatial memory and navigation in these animals.

Animals frequently compete over resources, and the outcomes of these aggressive interactions depend on a number of factors – one of which is the animals’ previous social experiences. If an animal wins a fight, it may be more likely to win subsequent fights (a “winner effect”), and if it loses, it may be more likely to lose subsequent fights (a “loser effect”).  Garcia et al. (2014, Animal Behavior) previously showed that green anoles exhibit loser effects, but not winner effects. Brittney Ivanov, research technician in Michele Johnson’s lab at Trinity University, wondered whether, since body color in green anoles is associated with social dominance, were color changes in green anoles associated with these loser effects? Could she cause a green anole to be brown if it was forced to lose social contests?

Brittney conducted an experiment using 16 male green anoles. First, in three consecutive days, these focal males interacted with a larger “trainer” male in the trainer male’s home cage for one hour. On the fourth day, the focal males interacted with a size-matched novel male in a cage that was new to both lizards. If the focal males were effectively trained to lose in the first three trials, she predicted that they would lose this fourth trial.

In the series of size-matched trials, 7 of the 16 contests resulted in a clear winner and loser, and 6 of those 7 focal males lost that trial. Further, focal males were less aggressive in the size-matched trial than they were in their previous training trials. These data support the presence of a loser effect in green anoles. Consistent with her previous work, Brittney also found that lizards that were more often green prior to the trials were more likely to win their trials, showing that body color is important in social contests.

This experiment revealed new findings about loser effects and body color. Focal males who lost their size-matched trial were more likely to be brown in the days after the trials – and not only that, they were more likely to become brown after the trials (so, these weren’t just loser males who had been brown all along).

All together, Brittney’s results show that body color can provide important information about a green anole’s fighting ability or motivation, or its recent social experience, and that dynamic body color influences multiple stages of social interaction in this species.

Postdoctoral scientist, Dr. Shane Campbell-Staton, presents his work on CTmax shifts in Anolis cristatellus at SICB 2017.

Greetings from New Orleans, where SICB 2017 is well underway! Kicking off the conference was Dr. Shane Campbell-Staton, currently a postdoctoral researcher at the University of Illinois, Urbana-Champaign. Shane presented some work he has been doing with Kristin Winchell, a graduate student in Liam Revell’s lab at the University of Massachusetts, Boston. Kristin’s work focuses on how the crested anole, Anolis cristatellus, adjusts its biology to life in urban areas. In previous work, Kristin documented adaptive shifts in limb and toepad morphology in these anoles in urban areas, a shift she correlated with the broader perches urban anoles use.

In this neat follow-up study, Shane and Kristin have documented how perch temperatures in urban Puerto Rican habitats are higher than in natural environments on the island. In response, urban Anolis cristatellus have a higher heat tolerance. Results from a common garden experiment indicate that the urban shifts in heat tolerance are primarily due to plasticity. At the moment, Shane is performing genomic analyses to search for signatures of selection on heat tolerance.

Figure 1 from Ng et al. 2016 showing the transect sampling spanning Anolis distichus populations differing in dewlap color (T1-4) as well as control transects (C1-4). Pie charts show dewlap color variation (top row), mitochondrial clade membership (middle row) and nuclear genetic cluster assignments (bottom row).

We sure love dewlaps here on Anole Annals! These flashy signals are incredibly diverse in size, color and pattern, and always make for a gorgeous image (e.g. 1, 2). Yet, we still have much to learn about why there is such a diversity of dewlaps and, furthermore, what are the consequences of such diversity? Previous work by Leal and Fleishman (2002, 2004) suggests that some of this dewlap diversity is due to adaptation for more efficient communication in different habitats. In a recent paper, we sought to identify whether the consequence of such adaptive trait divergence was speciation, or whether locally adapted dewlaps are maintained despite gene flow.

Anolis distichus shows remarkable geographic variation in dewlap color that predictably varies with habitat in a manner consistent with adaptation (Ng et al. 2013). This variation in color across Hispaniola gave us a great opportunity to conduct replicated analyses to identify whether adaptive differences in dewlap color consistently leads to the same genetic outcome.

We sampled populations in the Dominican Republic along five transects that transitioned from populations with orange dewlaps to those with cream or yellow dewlaps. For a comparison, we also sampled four ‘control’ transects where all populations shared a similar dewlap color. If dewlap differences are associated with speciation, we expected to see genetic differentiation between populations at either ends of the transect as this would suggest some level of reproductive isolation. Otherwise, transects showing no evidence of genetic structure would suggest that individuals are freely mating regardless of dewlap color.

Looking at the genetic structure of both nuclear and mitochondrial DNA along each transect, we found that geographic variation in dewlap color is associated with both speciation and gene flow. Three transects showed distinct genetic structure consistent with speciation, with one in particular only showing evidence of hybrids at one site which was a mere 0.89-1.55km away from other sampled sites. On the other hand, the other two transects did not look much different to the control transects, suggesting ongoing gene flow regardless of phenotypic differences.

Considering all transects together, I think there are two main take-aways from our results. First, finding evidence of gene flow across a sharp geographic shift in dewlap color must mean that strong selection is maintaining geographic variation in dewlap color; perhaps due to adaptation to different habitat types. Second, it appears that dewlap divergence does not necessarily lead to speciation. More work, however, is needed along these lines to understand whether the dewlaps we are characterizing as different are actually different from an anole’s perspective or in particular light environments (e.g. 1).

Anolis carolinensis from North Carolina. Photo from Carolina Nature.

One of the most well-known species of anole lizard is Anolis carolinensis, AKA the green anole, which is the only anole native to the continental United States. As a classic model for ecology and behavior, this lizard was the first species of reptile to have a complete genome sequence. Interestingly, only after it became a genomic model, numerous studies (Tollis et al. 2012, Campbell-Staton et al. 2012, Tollis & Boissinot 2014) sought to understand how genetic variation is structured across the geographic range of A. carolinensis,  and to infer historical migration patterns and demographic events to explain the current distribution of green anoles. However, these studies still left many questions unanswered, mostly due to the fact that they were limited in terms of numbers of genetic markers. Now, we have published a new paper in Ecology and Evolution that used a targeted enrichment method to capture more than 500 sequence markers and provide a clearer picture of A. carolinensis historical biogeography.

What we knew about Anolis carolinensis phylogeography

Collecting green anoles for phylogeographic study has been a real hoot, taking us all over the country. Anolis carolinensis ranges across subtropical North America, and consists of five geographically structured genetic clusters supported by both mitochondrial (mtDNA; see Tollis et al. 2012 and Campbell-Staton et al. 2012) and nuclear (nDNA) markers (see Tollis et al. 2012, Tollis & Boissinot 2014). Three of the clusters are found in Florida : one whose distribution primarily hugs the Northwestern coast of the peninsula, another along the Eastern coast of the peninsula, and a third relegated to South Florida. The continental mainland, while making up most of the area of green anole range, harbors only two clusters: one occupying North Carolina and South Carolina, and another from Georgia, west of the Appalachian Mountains and across the Gulf Coastal Plain into Texas.

One confusing result from earlier studies of A. carolinensis molecular phylogeography was the placement of the most basal lineage in NW Florida (Tollis et al. 2012, Campbell-Staton et al. 2012). This didn’t make sense biogeographically, since it is believed that the species dispersed to the continental mainland from western Cuba (Buth et al. 1980, Glor et al. 2005). However, a subsequent nDNA study (Tollis & Boissinot 2014) produced a multi-locus species tree to show that southern Florida harbors the most ancient lineage of A. carolinensis. This discovery of mito-nuclear discordance provided a more satisfying biogeographical explanation that only needs to invoke overwater dispersal to South Florida from Cuba.

Different genetic datasets tell different stories about Anolis carolinensis evolutionary history. (A) Phylogenetic relationships of the major green anole lineages inferred from the ND2 mtDNA locus. (B) Phylogenetic relationships of the major green anole lineages using multi-locus species tree approach (1 mtDNA and 3 nDNA markers). Adapted from Manthey et al. 2016.

From there, things remained unresolved even with nDNA. For instance, while the split between South Florida and the rest of the species received full statistical support in Tollis & Boissinot (2014), the relationships between the other clades were less supported, making it difficult to determine if the A. carolinensis mainland clades arose from separate Floridian sources.

The data used in Manthey et al. 2016

To our knowledge, this is the first Anolis phylogeography study to use targeted enrichment, so I thought I would elaborate on the nature of this kind of dataset. Anchored hybrid enrichment (AHE) relies on probes designed from conserved genomic regions ascertained from a panel of vertebrate genomes – including A. carolinensis – which are flanked by non-conserved regions (the level of conservation in determined by PhastCons scores from the UCSC Genome Browser). DNA samples are pooled, and a set containing thousands of probes is used to enrich libraries that get sequenced on an Illumina platform and assembled into contigs, producing hundreds of homologous loci.

Here’s the breakdown of what we ended up with in the new study: our sample contained 42 individual anoles from 26 localities across eight states, and we were able to obtain 487-512 loci per individual, with an average contig length of 629bp, and an average of 17 SNPs per locus including an average of six parsimony-informative SNPS per locus. Roughly speaking, that’s one parsimony-informative SNP every 100bp for 500 loci, so about 3,000 parsimony-informative SNPS  = not bad! For what it’s worth, the 10 nDNA A. carolinensis markers obtained by more traditional PCR/Sanger sequencing contained about one SNP every 100bp as well (see Tollis et al. 2012 and Tollis & Boissinot 2014). Therefore, AHE produced hundreds more informative loci at a fraction of the cost.

New insights into Anolis carolinensis phylogeography using targeted loci

Using different statistical clustering methods (DAPC and Structure), Manthey et al. supports the same five  genetic clusters as previously described. However, there is now a fully resolved species tree – arrived at using multiple methods. First, the South Florida clade is the most ancient lineage of green anoles, likely splitting off from the rest of the species during the Miocene or Pliocene. However, there is now 100% support for a sister-group relationship between the mainland clades, massively simplifying the story of A. carolinensis. Green anoles likely remained in Florida until the Pleistocene, dispersing northward and onto the mainland where two lineages evolved independently- one along the Atlantic coast in the Carolinas, and another dispersing across the Gulf Coastal Plain.

(A) Map showing geographic localities of 42 green anoles selected for targeted enrichment. (B) Results of species tree analyses. Colored symbols correspond to the five geographic and genetic clusters. Adapted from Manthey et al. (2016).

We also found that despite the best resolution to date for the A. carolinensis species tree, incomplete lineage sorting is rampant across these loci, highlighting the need for these kinds of datasets for phylogeographic studies at this evolutionary distance. For instance, the only clade with any gene trees supporting exclusive ancestry was South Florida: meaning on a given gene tree, pre-defined “clades” are often paraphyletic. The reason the species trees agreed in their topologies is due to fact that they probabilistically invoke the coalescent process, which incorporates incomplete lineage sorting. Previous studies, using ≤10 loci, simply lacked enough statistical power to do this confidently.

More work to be done

As with most scientific endeavors, the new study resolves some outstanding questions but also begs new questions. For instance, although we were able to infer gene flow between the Gulf-Atlantic and NW Florida clades, the degree of allele sharing between populations is still not clear. There seems to be some admixture between the Gulf-Atlantic and Carolinas clades south of the Appalachian Mountains in Georgia, suggesting elevational gradients provide a more effective barrier to gene flow in this species than riverine barriers. Also, the divergence times of the green anole clades are still based only on molecular clock models and could benefit greatly from informative fossils calibrations.

Anolis aquaticus, the semi-aqautic anole. Photo by Lindsey Swierk

From backyard anole enthusiasts to researchers with decades of experience, dewlaps are a favorite topic of discussion here on Anole Annals. We love documenting the diversity of dewlap colors and patterns (1, 2, 3, 4), judging “best/biggest dewlap” contests (1, 2, 3), and noting dewlap oddities across the genus (1, 2, 3, 4). We’re slowly piecing together an answer to the question of what role dewlaps actually serve in signaling and, in particular, what kind of information they might convey. As you might expect, it’s a pretty complex problem, made even more interesting by the fact that dewlap information content probably reflects the unique pressures placed on individual species.

I’ve recently been working on untangling the mystery of dewlaps in a quirky species of anole, Anolis aquaticus. This water-loving anole is found along streams in pockets of southern Costa Rica and northern Panama, and it has the delightful habit of diving into water when startled. Even among the aquatic-specialized anoles, A. aquaticus is different: it tends to live in ultra-close proximity to water, preferring boulders and crevices directly in the “splash zone” instead of streamside vegetation such as other aquatic species like A. oxylophus. There’s also good reason to think that A. aquaticus has a pretty rich social life – male-female, male-male, and female-female pairs can be found within a few centimeters of each other, and often in dense groups on small rocky islands.

In light of their unusual habitat and living arrangements, we decided to explore how dewlaps correlated with multiple morphological parameters in A. aquaticus. In particular, we decided to use this species to explore a long-standing, but recently debated, paradigm that most sexually selected traits (like dewlaps) scale to body size with positive allometry – or, in other words, that they’re disproportionately large in larger individuals. Last year, we captured male and female (who lack the characteristic reddish-orange dewlap) A. aquaticus and measured multiple sexual and non-sexual traits to test this idea. Our results, available in an accepted article in Integrative Zoology, allowed us to contribute our perspective to the greater understanding of the relationship of sexual selection and allometric scaling patterns. Spoiling part of the punchline, our findings do not support the traditional idea that positively allometry is a hallmark of sexual selection.

The dewlap of Anolis aquaticus. Bar represents 1 cm.

But, equally as notable, our results also suggest some interesting features of this species, including the information content of its dewlap and how allometric patterns interact to produce sexual dimorphism. We found that dewlaps are “super-honest” signals in A. aquaticus; they could serve to amplify size differences between males signaling at a distance because of their positive allometric scaling with body size. Consequently, our study and a recent study by Driessens et al. 2015 (on A. sagrei), oppose previous ideas that dewlaps approach an asymptote of optimum size to balance the pressures of signaling with predation. Our findings are also novel in that they suggest that dewlap color (redness) may serve to convey information about male weaponry: anoles with redder dewlaps were found to have head shapes that correspond to producing greater bite force.

By comparing allometric relationships between males and females, we can also begin to identify how sexual differences in proportionality link to sexual dimorphism and ecology. For instance, male hind limb length in A. aquaticus is on average larger than that of females, but becomes disproportionately smaller as male body size increases. This opens the door to the idea that, because males are larger than females, limb length sexual dimorphism might be the result of an optimal limb-body size relationship regardless of sex; A. aquaticus of either sex with overlong limbs would probably be at a disadvantage if they needed to flee over narrower surfaces such as branches or vines.

Scaling relationships of snout-vent length and a) mass, b) limb length, and c) head length for male (closed dots, solid line) and female (open dots, dashed line) Anolis aquaticus. Axes are log scaled.

Finally, our results hint at the existence of two life-stage male morphs in A. aquaticus, as already identified in other anole species. Body scaling relationships show that small males have disproportionately small dewlaps, small heads, and large limbs, whereas large males have bigger dewlaps, bigger heads, and smaller limbs than should be expected for their body size. Taken together, these results provide a foundation for future research into “heavyweight” and “lightweight” male morphs and associated behaviors. With their small home ranges and apparently high tolerance for same-sex home range overlap, this could be an especially exciting avenue of exploration in A. aquaticus. In any case, it’s certain that there will be much to learn from this watery, elusive, semi-aquatic anole.

You can read more about this project in our accepted manuscript published online in Integrative Zoology. My co-author on this study, Maria Petelo, is an undergraduate at the University of Hawaiʻi who was supported by OTS/NSF’s Native American and Pacific Islander Research Experience, a program designed to increase the representation of underrepresented groups in the natural sciences.