Category: New Research Page 9 of 67

SICB 2019: Insulin-like Signaling across Life Stages in Brown Anoles

The somatropic axis regulates growth in vertebrates

Our growth during development is controlled by a complex brain-body axis called the somatotropic axis. Put simply as in the photo below, the hypothalamus in the brain signals the pituitary to release growth hormone into the body where it stimulates the liver to produce two forms of insulin-like growth facts (IGF1 and IGF2). Both growth hormone and IGF have different effects on growth of muscle and bone. While we rely on mouse models to study how IGF might impact human development, it turns out that the relative secretion of IGF1 and IGF2 over the course of life is quite different in the two species. In fact, we know little about IGF production and signaling in non-mammals.

Expanded view of the somatotropic axis to show receptors and binding proteins. From Yakar et al. (2018).

Abby Beatty, from Tonia Schwartz’s lab at Auburn University, set out to determine the developmental pattern of the somatotropic axis in brown anoles. Of course, the axis is much more complex than the diagram above, including receptors in various tissues, binding proteins that carry the signals around the body (see below), and proteins in cells that cause responses (IRSs). Abby studied expression of IGF1, IGF2, and five binding proteins during brown anole development, from embryo to hatchling to adult. She expected to find that IGF1 and 2 would be expressed differentially and that the expression patterns would differ across life stages. That’s exactly what she found. IGF1 and 2 were both low and similar in expression early in development, but at hatching IGF increased with IGF2 expressed more than IGF in adults. Surprisingly this is more like a human pattern than a mouse is!

As for the binding proteins, expression was similar for all of them in the brain, gonads, and liver, but BP3 was expressed less in the heart. It’s still unclear what these patterns in binding proteins mean for brown anole development, but they make for some excellent future research questions! Indeed, these results add to the already-long list of things that makes anoles good model systems.

SICB 2019: Large Immune Challenges Do Not Decrease Performance

Christine Rohlf from the University of St. Thomas presents her research on immune-performance tradeoffs.

Traveling to SICB is always exciting, but like any trip through crowded airports, hotels, and convention centers, you’re more likely to get sick during your travel if you’re not careful. As we all know, getting a travel cold (or worse) makes you feel terrible and certainly doesn’t make you want to run on a treadmill! The same is likely true in wild animals, including anoles. Mounting immune responses is energetically expensive, but so are other things that lizards have to do, like forage, escape predators, and process food. So, does an increasingly large immune challenge decrease a lizard’s ability to perform? Christine Rohlf, an undergraduate student in Jerry Husak’s lab at the University of St. Thomas, wanted to find out in green anoles.

Christine designed a laboratory experiment to determine whether two types of immune challenge, alone and in combination, decreased bite-force performance, sprint speed, or endurance capacity compared to controls. Some lizards received two sequential injections of lipopolysaccharide (LPS), some received a skin wound with a biopsy punch, and some received both. LPS is a signal on gram-negative bacteria that, when injected, tricks the body into thinking it is infected with bacteria. So, you get an immune response, but you don’t actually get an infection.

Surprisingly, none of the immune challenges affected sprint speed or endurance compared to controls. Although the lizards were not calorie-restricted, they were on a modest diet, meaning that energy was limited, but clearly not enough to make a difference. Apparently these two immune challenges aren’t as costly as we thought. The only effect that Christine found was that the second LPS injection significantly decreased bite force. Because bite force is likely the least energetically expensive trait of those measured (imagine running until you’re exhausted versus biting into a hard piece of French bread), Christine suspects that the decrease in bite force was due to a lack of motivation while feeling sick. Future work with calorie-restricted lizards should tell us if mounting an immune challenge is a significant cost to anoles.

SICB 2019: Tail Autotomy Happens More When the Tail Stores More Energy

Amy Payne of Trinity University presents her research on tail autotomy in 7 lizard species.

One of the most interesting features of many lizards, including anoles, is that they can willingly, and actively, lose their tails to escape predators. While it might seem counterintuitive to lose a large body part, it’s better than being eaten! Despite the obvious benefit of surviving another day, there are some costs associated with tail autotomy.

Amy Payne, a student in Michele Johnson’s lab at Trinity University of San Antonio, wanted to know whether the frequency of tail loss across seven species was associated with predatory and social use of the tail as well as energetic content of the tail. For those that are anole-inclined (which is why you’re here), Amy included A. cristatellus and A. carolinensis. She caught and measured hundreds of lizards, and made behavioral observations on them as well. She was then able to quantify how many lizards of each species had a lost/regenerated tail, as well as what proportion of each tail was lost.

Surprisingly, frequency of tail loss was not associated with using the tail in a social or predatory context. However, there was an association between these two functions of the tail: species that more often used their tail for predatory use also used their tail in social contexts more. There was no relationship between the frequency of tail loss and the proportion of the tail that was lost on average across species. But she did find some really cool results when looking at energetic content of the tail. Amy found that there was a significant positive relationship between frequency of tail loss and tail energy content. That is, the more energy that lizards have in their tails, the more frequently individuals in that species will have a lost/regenerated tail. While this seems opposite to what one might casually predict, Amy hypothesizes that the predator-distraction to survive function of tail autotomy is more likely to succeed if the tail is larger and more beneficial to the predator. In other words, if a lizard has a scrawny tail and drops it off for a predator, it is more advantageous for the predator to ignore the low-cal tail and just eat the lizard. This would put selection on species with low-energy content tails to be more prudent about when they drop their tails. These really interesting results open up some exciting areas for future research on the costs and benefits of tail autotomy!

SICB 2019: Does a Tropical Anole Evolve When Colonizing a Novel Habitat?

Anolis apletophallus from Panama, a well-studied species from the Panama mainland.

Over the past 15 to 20 years, the study of evolution has undergone something of a paradigm shift. Whereas scientists used to believe that evolution in most animals was a slow process, only observable over longer timescales, we now know that evolution is fast. Meaningful change can occur in many types of traits, including morphology and physiology, in just a handful of generations of a given organism. With this shift in our understanding, many biologists have begun conducting experiments which attempt to observe evolutionary processes in action, and shed light on how evolutionary mechanisms play out in the real world.

Dan Nicholson, a student in Rob Knell’s lab at Queen Mary University of London, worked with Mike Logan and a team of researchers to do just this in a tropical anole, Anolis apletophallus. Dan and his colleagues caught over 400 individual anoles from the mainland and introduced them to a novel environment: four small, anole-free islands formed when the Panama Canal was created. Two of these islands were similar to mainland habitats, while two had wider types of vegetation. Prior to placement on these islands, Dan measured a suite of characters of these individuals, including perch height, size, leg length, head, and toe morphology, enabling him to observe any changes in the distribution of these traits over time.

After leaving the anoles on their new tropical island homes for a year, Dan returned to recapture the survivors and measure both them and their offspring. By comparing the traits of the surviving lizards and their young with those of the population founders, Dan could observe changes in traits as well as measure natural selection on them. At SICB 2019, Dan reported that he found that anoles on islands with wider vegetation did indeed use these broader perches and that anoles also perched closer to the ground. Correspondingly, he found that toe pad size decreased and that hindlimb lengths were longer on some islands, potentially allowing lizards to better exploit lower, broader perches. 

Anoles on all islands also showed a reduction in head depth. The reason is unclear, but Dan is looking into whether differences in competition or the prey community are potentially driving this pattern. Finally, measuring selection was very difficult and analyses proved problematic, though in some cases selection estimates do seem to match with observed changes in morphological characters. Dan and his team are hoping that adding data from another generation of anoles will clarify these effects, so stay tuned!

Keep track of the latest from Dan on Twitter: @DanJNicholson

Natural Selection on Morphology in a Tropical Lizard After a Rapid Shift in Habitat Structure NICHOLSON, DJ*; LOGAN, ML; COX, C; CHUNG, A; DEGON, Z; DUBOIS, M; NEEL, L; CURLIS, JD; MCMILLAN, WO; GARNER, T; KNELL, RJ; Queen Mary University London

SICB 2019: The Life and Death of an Extralimital Population of Invasive Brown Anoles

Brown anoles are invasive throughout the southeastern United States and are often transported via the nursery trade.

As invasive species expand across landscapes, they may engage in new interactions including with native competitors and prey as well as encountering novel environmental conditions such as different temperatures or patterns of rainfall. It is often difficult to observe the process of how invasive species which are dispersing across landscapes are affected by these novel conditions, because it may be difficult to find edge populations of invaders, and those extralimital populations which do not survive may have disappeared before scientists can observe them.

In southern Florida, many anole species have been introduced and are expanding their ranges, perhaps none more prolifically so than the brown anole (Anolis sagrei). In the past 75 years or so, brown anoles have occupied all of peninsular Florida, the eastern seaboard of Georgia, and Gulf Coast habitats through Louisiana. Many of these expansions are thought to occur via hitchhikers on cars or via the nursery trade, in which potted plants with adults or eggs are transported to new areas. These introductions may fail for many reasons (e.g., inhospitable environments, low numbers of colonizers, intentional extirpation by humans), but these processes of dispersal, establishment, and extirpation are difficult to study. Dan Warner, a professor at Auburn University, took advantage of a known extralimital population of brown anoles in a greenhouse in central Alabama to study the survival of a population created through this type of dispersal.

This population of anoles existed well north of its continuous invasive range in the United States and was exposed to much colder winter conditions than other studied populations.  It was present at the greenhouse from at least 2006, and so survived for at least 10 generations, long enough for adaptation to these novel thermal conditions to potentially occur. Working with a team of undergraduates, graduate students, and post-docs, Dan assessed the thermal conditions in the greenhouse environment, conducted mark-recapture studies of the population, and measured thermal tolerances of lizards.

Dr. Amélie Fargevieille and Jenna Pruett representing the Warner Lab at SICB 2019.

At SICB 2019, Dr. Amélie Fargevieille and Jenna Pruett presented results from the study, showing that the greenhouse population included all life stages of lizards and reached a total size of >1000 individuals. While one might expect that these northern lizards would have altered critical thermal limits, the Warner lab showed that both the upper and lower thermal limits of these lizards (the temperatures at which their movements became uncoordinated), were the same as those found in lizards from warmer, southern populations. These results indicate that existence in a colder northern climate for >10 years did not lead to adaptive changes in thermal limits, perhaps due to the population occupying a thermally-buffered habitat, i.e., the greenhouse.

While hurricanes have facilitated several fascinating studies of anole adaptation (e.g., Schoener et al., 2017, Donihue et al., 2018), they may also take these opportunities away. In the case of this population, Hurricane Irma blew off the greenhouse roof in 2017 (which remained unrepaired), exposing this population to the rigors of a central Alabama winter. Multiple surveys in 2018 confirmed that there were no survivors of this previously robust population. Dataloggers confirmed that, even in the most sheltered microhabitats that remained, temperatures dropped below the critical thermal minima of brown anoles, presumably extirpating the entire population.

Recent Extinction of a Viable Tropical Lizard Population from a Temperate Area WARNER, DA*; HALL, JM; HULBERT, A; TIATRAGUL, S; PRUETT, J; MITCHELL, TS; Auburn University.

SICB 2019: Do Bark Anoles Show Behavioral Syndromes?

Daisy Horr, an undergraduate researcher at Trinity University, discusses how bark anole behavior varies across several different social contexts.

Animals often use diverse behavioral repertoires to adjust to new, unexpected, or changing conditions very quickly. While it may seem like individuals could always use the best behavior for any given situation, we know that instead behaviors are often related within an individual. In other words, an individual’s behaviors are not always independent and may represent an underlying “behavioral syndrome” or correlated set of behavioral responses to related environmental conditions. These behavioral syndromes are also sometimes called “personalities” (though application of this word to animals can be a bit controversial!). So, for instance, an individual that has a “bold” behavioral syndrome might take little time to explore a new habitat or consume a novel food item more quickly, but also be more likely to stay active in the presence of a predator rather than hiding (the safer option!).

While anoles have been the focus of much behavioral research, we still lack an  understanding of the diverse behavioral phenotypes, including behavioral syndromes, which are displayed by a variety of anole species. The bark anole, Anolis distichus, is native to Hispaniola but also found in southern Florida where it has been introduced. While small, bark anoles can be quite feisty, and are known for their dramatic display behavior in the presence of male and female conspecifics.

Male bark anoles demonstrate pushup displays prior to engaging in combat.

Taking advantage of the bark anole’s willingness to put on a show, Daisy Horr, an undergraduate student and McNair Scholar in Michele Johnson’s lab at Trinity University, led a group of fellow researchers in assessing whether these anoles show behavioral syndromes. To do this, Daisy and her collaborators measured the degree to which male bark anole behaviors were repeatable across three different contexts: trials with another male present, trials with a female present, and solo trials in which no other anoles were present. They measured variables quantifying movement as well as display behaviors such as pushups and dewlap extensions during these trials. The team also wanted to see whether the measured behavioral traits were linked to morphological and physiological variables.

Daisy and colleagues found no support for the idea that behavior was linked to morphology, including size of the body, head, and dewlap, and mass of the whole body, the liver, and fat pads (structures holding fat as energy reserves), or the hematocrit of the lizards, a metric quantifying how many red cells are present in the blood. Bark anoles did show some level of behavioral consistency, however. Movement behaviors were quite repeatable even between trials with and without conspecifics. Display behaviors, however, including pushups and displays, were repeatable within, but not across contexts. This work suggests that bark anoles have consistent behavioral syndromes in some contexts. Looking forward, research into behavioral syndromes in anoles could offer insights into how behavior may vary with habitat use, ability to invade novel environments, or selection on behavior itself!

SICB 2019: Sexual Differences in Relative Lengths of Toes

Today I had the pleasure of attending an excellent talk by University of Florida undergraduate Griffin McNamara. I was really impressed with the work he presented, especially for an undergraduate.

McNamara was investigating the ratio in digit length between the 2nd and 4th digits. This is interesting because in mammals, especially humans, this ratio is sex specific, with men typically having longer ring fingers than pointer fingers. A lot of research has looked into the developmental reasons for this, with a likely relationship to hormone exposure of the developing fetus. Applying these ideas to anoles makes sense because the toe of anoles are somewhat unique in lizards, as we all know here. McNamara is looking into if sexual dimorphism in 2nd and 4th digit length was also present for anoles.

McNamara wasn’t the first person to measure this in anoles, but he was the first to use cleared and stained specimens, which likely greatly improved his ability to accurately measure digit length. Interestingly, he found that the pattern was reversed, with males having longer 2nd digits, not longer 4th digits as in mammals. In addition, this pattern didn’t show up until a lizard’s teenage years, with juvenile anoles not showing a difference between the sexes. Using cell staining to visualize dividing cells, he was able to narrow down the digit discrepancy to growth in the 2nd phalanx during sexual maturity.

Suspecting that this late onset dimorphism might still be related to hormone exposure, McNamara got his fingers on some testosterone-treated female anoles from collaborators and found that they had “masculinized” digit ratios, although not as much as true males.

I thought this was a great study, combining old school cleared-and-stained approaches with cell biology and experimental endocrinology. It also opens up lots of interesting questions. Is there an adaptive reason that mammals and lizards have sexually dimorphic digit lengths? Is it just a quirk of development? Does this digit length reversal have anything to do with the fact that the shape of anoles’ rear feet is already kind of mirrored as compared to our hands?

Local Adaptation in Mainland Anole Lizards: Integrating Population History and Genome-Environment Associations

Figure 1. Anolis punctatus, South America’s coolest lowland anole – literally. Picture by Renato Recoder.

In ectothermic organisms, environmental factors such as temperature and water availability constrain physiological and behavioral performance. Therefore, the occurrence of species in varied environments may be associated with local adaptation. On the other hand, experimental studies have shown that physiological function can be highly conserved within species over broad environmental gradients, which may be associated with the homogenizing effects of population gene flow. In a recently published study, we focus on widespread South American anoles to investigate whether the occurrence of species in distinct environments is linked to local adaptation and whether population structure and history have constrained adaptive differentiation.

Based on molecular data, my collaborators and I have previously found that arboreal lizard species have independently colonized the Atlantic Forest from Amazonia, subsequently expanding southward towards subtropical regions. This is the case of Anolis ortonii and Anolis punctatus (Fig. 1), whose ranges now encompass a climatic gradient from warm and wet conditions in Amazonia to cooler and less rainy settings in the Atlantic Forest. Our new study investigates whether species establishment in distinct climates is associated with potentially adaptive genetic differentiation between populations. To this purpose, we implement genome-environment association analyses on the basis of thousands of restriction site-associated DNA markers. Moreover, to estimate levels of gene flow – a force that could oppose adaptive differentiation – we perform historical demographic inference under a genetic coalescent framework. Lastly, to characterize the climatic gradients presently occupied by A. ortonii and A. punctatus, we estimate climatic space occupancy over their ranges.

Analyses of genetic structure inferred distinct populations in Amazonia and the Atlantic Forest in both anole species (Fig. 2), suggesting that separation of these forests following a period of contact in the past has favored genetic divergence. In the two species, historical demographic analyses inferred large effective population sizes, mid-Pleistocene colonizations of the Atlantic Forest from Amazonia, and post-divergence population gene flow (Fig. 3). These results support the hypothesis of recurrent rainforest expansions that connected presently disjunct biomes in northern South America.

Figure 2. Genetic clustering based on all SNPs from Anolis ortonii (A) as well as all SNPs (B) and candidate SNPs only (C) from A. punctatus. Proportions in pie charts on maps correspond to ancestry coefficients estimated by genetic clustering analyses. Grey areas on map indicate South American rainforests. Red arrows indicate A. punctatus sample MTR 20798 from Pacaraima on the Brazil-Venezuela border in the Guiana Shield region, a locality that is climatically similar to Atlantic Forest sites (see Fig. 4); this sample is genetically more similar to eastern Amazonian samples based in the entire SNP dataset, yet more similar to Atlantic Forest samples based on the candidate SNPs only.

Figure 3. Population history (from SNAPP) and historical demographic parameters (from G-PhoCS) inferred for Anolis ortonii (A) and A. punctatus (B). Parameters are the time of coalescence between populations (in millions of years, Mya), effective population sizes (in millions of individuals, M), and migration rates (in migrants per generation, m/g). Colors of terminals correspond to genetic clusters in Fig. 2.

Genome-environment association analyses found allele frequencies of 86 SNPs in 39 loci to be significantly associated with climatic gradients in A. punctatus. Among the candidate loci, eleven uniquely mapped to known protein-coding genes in the reference genome of Anolis carolinensis; two mapped non-specifically to more than four genes; and the remaining mapped against non-coding regions, which may correspond to regions that regulate gene expression or that are physically linked to genes that underwent selection. In the case of A. ortonii, no SNPs were associated with temperature and precipitation variation across space. Constraints related to population structure and history do not seem sufficient to explain discrepant signatures of adaptation between the two anole species; instead, this discrepancy may be related to species differences in climatic space occupancy over their ranges (Fig. 4).

Figure 4. Environmental space occupancy along latitude based on climatic PC1 for Anolis ortonii and A. punctatus. Samples used in genetic analyses are indicated with a black dot. Higher PC scores correspond to drier and colder sites. Dashed line indicates the approximate region of a pronounced north-south climatic turnover in the Atlantic Forest identified by previous studies. Red arrow indicates A. punctatus sample MTR 20798 from Pacaraima, a mid-elevation site (820 m above sea level) in the Guiana Shield region that overlaps climatically with Atlantic Forest sites (horizontal axis). Note that the two species experience largely similar climates in Amazonia and the northern Atlantic Forest, yet A. punctatus occupies cooler and less humid localities that are not occupied by A. ortonii in the southern Atlantic Forest.

The candidate genes identified in A. punctatus play essential roles in energy metabolism, immunity, development, and cell signaling, providing insights about the physiological processes that may have experienced selection in response to climatic regimes. Similar to our study, other investigations of anole lizards found differences in the frequency of alleles that underlie ecologically relevant physiological processes between populations that inhabit contrasting habitats. These examples support the hypothesis that adaptation to colder climates has played an essential role in range expansions across anole taxa, including mainland and Caribbean forms that span altitudinal and latitudinal gradients.

Anolis ortonii. Spotting this cryptically colored rainforest anole is quite challenging indeed. Picture by Miguel T. Rodrigues.

This investigation illustrates how studies of adaptation on the basis of genome-environment association analyses can benefit from knowledge about the history of landscape occupation by the species under investigation. Data on population structure and history can provide insight into how gene flow and natural selection interact and shape population genetic differentiation. Moreover, information about the direction and routes of colonization of new habitats can support spatial sampling design, help to characterize landscape gradients, and support the formulation of hypotheses about how organisms have responded to environmental variation in space.

To know more:

Prates I., Penna A., Rodrigues M. T., Carnaval A. C. (2018). Local adaptation in mainland anole lizards: Integrating population history and genome-environment associations. Ecology and Evolution, early view online.

Nomenclature of Dactyloidae: Revisit and Opinions Wanted

Hi everyone. I recently received and have to determine what to do with the following paper (editor’s note, for background, see this recent post):

As an administrator and bureaucrat at Wikispecies I have to decide how to proceed with this group of reptiles. I have made a tentative start here but please realize this is a simple start easily undone.

I recall the last time this came up, in 2012. I joined the discussion at the time. However, despite my comments at the time, I did not follow splitting the genus up then.  In the end, my view is for stability and consensus. By stability, I mean the actual meaning of stability under the ICZN code, which does not apply here. But consensus could.

Why is this paper different? Well, first up, last time it was a PhyloCode paper and as such is relatively easy to ignore, as it does not submit to the rules of nomenclature. However, this time it is an ICZN compliant paper so you cannot ignore it. As stated many times, names are to considered as valid on publication or refuted–there is no ignore. So the above paper may be refuted, but not ignored.

Last time, many argued that the genus is monophyletic. This is not really an argument against splitting. It’s a position statement. The order Testudines is also monophyletic, should every turtle species (275 living species) all go back into the genus Testudo? The current genera or lack of them present are only a reference to the history of research. It does not mean it is the most suitable arrangement.

More importantly is diagnosibility. Can the new proposed genera and their inherent species be adequately diagnosed? This is a more important question.

Note that a genus with some 500 species is generally considered too big. Many writers over the years have deemed between 100-200 species about the maximum size wanted. However, this does still need to address the previous point on diagnosibility.

Another point people brought up last time was stability. Well, stability actually refers to the mononomial and whether a name can be replaced by a forgotten name. It is used as a reason to reverse priority. This is the code purpose of stability. Note that the combination first up does not have to be stable, and second is a taxonomic decision, not a nomenclatural one. Hence outside the code.

So what I am after: Basically I want to see through any commentary if the people who work on anole’s are likely to use this new nomenclature. If they are, I will adopt it at Wikispecies. That will require the moving and reorganisation of some 550 pages. I do not take that on lightly. Hence I am asking you, the people who work on anoles, first. My decision will be based on the answers I get. I do not work on anoles. I am a turtle and tortoise specialist. But I do have a job to do at Wikispecies.

For your information, I have discussed this briefly with Peter Uetz at Reptile Database also. He also was not sure what to do, but remembered the last time it came up here. So I am reaching out to all of you on this issue. I am after consensus, not stability. As I said, stability does not apply here. But I will say that to reject the nomenclatural proposals of Nicholson et al. (2018) does require a refutation. They have presented to science in good faith in a very good journal, Zootaxa. We cannot ignore this and as a taxonomist, I will not.

In advance, I thank everyone for their comments. I think this issue needs to be openly debated.

 

 

A Second Caribbean Anole Species Introduced to Brazil

 

The brown or festive anole, Anolis sagrei, is an invasive species in several countries in the Americas and Asia. This species is native to Cuba, the Bahamas, and the Cayman islands. Following introductions, A. sagrei can reach high population densities and undergo rapid range expansion. In a recently published contribution, we provide the first record of this aggressive invasive lizard in Brazil.

In 2017, we recorded specimens of A. sagrei within the limits of an International Airport in the metropolitan area of the city of Rio de Janeiro, southeastern Brazil. The observation of juveniles and mating couples suggests that the species is established locally.

The origin, geographic extent, and potential for spread of A. sagrei in Rio de Janeiro and Brazil are currently unclear. It is also unclear whether this species will be able to colonize natural habitats, such as the surrounding Atlantic Rainforest.

The establishment of brown anole populations elsewhere has led to shifts in substrate use by native anoles and promoted major shifts in the structure of local insect assemblages. As such, this species has the potential of affecting local ecological communities in Brazil. However, the effects of A. sagrei on the local fauna – including native lizards that we sampled in the area – are difficult to predict.

This is the second case of an established exotic anole species in Brazil. Populations of the Cuban green anole, Anolis porcatus, were recently detected in several sites in the Baixada Santista coastal region, state of São Paulo.

To know more:

Oliveira J.C.F., Castro T.M., Drago M.C., Vrcibradic D., Prates I. (2018). A second Caribbean anole lizard species introduced to Brazil. Herpetology Notes, 11: 761-764.

PDF available here (at the bottom of the webpage).

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