Category Archives: Anole Genome Research

The Genetics of Anolis Lizard Tail Regeneration: (Re)generating Major Internet Buzz

Anolis carolinensis duo with regenerated tails. Photo credit: Joel Robertson.

Anolis carolinensis duo with regenerated tails. Photo credit: Joel Robertson.

Readers of this blog are well aware of autotomy in lizards – self-amputation of the tail – that usually occurs as a result of sub-lethal predation. Readers of this blog are also familiar with the fascinating ability of many lizards to regenerate new tails post-autotomy. Lizards are the closest relatives to humans that can regenerate a fully functional appendage in the adult stage, and understanding the molecular basis of this process can shed light on the latent regenerative capacities in mammals. A new paper published this week in PLOS ONE (Hutchins et al. 2014) provides the first insights into the genetic mechanisms of lizard tail regeneration, using Anolis carolinensis as a model. Via the high-throughput sequencing of RNA from regenerating green anole tails, and the mapping of these sequences to the A. carolinensis genome, the authors describe the genes that are expressed during the regeneration process, shedding light on potential targets for future human therapies.

Disclaimer: I am not an author on the paper, although I do work in the Kusumi Lab with the authors.

While the ability to regenerate a fully functional appendage in the adult phase is likely a deeply homologous trait across animals, it is not uniformly conserved across vertebrates. Fish, as in the zebrafish model (Gemberling et al. 2013), and amphibians, as in the salamander models (Knapp et al. 2013) can regenerate both limbs and tails, suggesting that while the ancestral vertebrate was equipped with this ability, it seems mammals have during their evolution somehow lost it. Evolutionary hypotheses explaining exactly why some taxa lose the ability to regenerate adult appendages are far and wide, ranging from the stochastic to ecologically-specific fitness trade-offs (reviewed in Bely and Nyberg 2010).

But what are the proximate (i.e. genetic) reasons as to why lizards remain strong regenerators while mammals are left holding the short end of the regeneration stick? Continue reading The Genetics of Anolis Lizard Tail Regeneration: (Re)generating Major Internet Buzz

Exploring the Anolis Y Chromosome

Sex chromosomes have historically been identified by inspecting chromosome spreads under a light microscope and looking for a morphologically distinct or heteromorphic pair of chromosomes – typically and X and Y or a Z and W. However, heteromorphic sex chromosomes are absent in many animal groups, particularly fish, amphibians, and lizards, making it difficult to determine whether a species with genetic sex determination has an XY or ZW system. As a consequence, the study of sex chromosome evolution in clades in which cryptic or homomorphic sex chromosomes are prevalent has been hampered by a lack of identified sex chromosomes in these groups. New methods are needed to find the sex chromosomes in these species and increase our understanding of homomorphic sex chromosome biology, the evolution of sex determining systems, and patterns of sex chromosome evolution overall.

David Zarkower and I have a paper in press at Molecular Ecology Resources that uses high-throughput DNA sequencing to identify sex-specific genetic markers as a means to reveal sex chromosome systems in species that lack heteromorphic sex chromosomes. We are using a newly developed DNA sequencing technique called restriction site associated DNA sequencing or RAD-seq. RAD-seq sequences the DNA flanking very specific DNA sequences (restriction enzyme recognition sites) scattered throughout the genome, generating tens of thousands of genetic markers. RAD-seq is a powerful technique for exploring genetic variation in ‘nonmodel’ species because it does not require a fully sequenced genome, requires relatively modest sequencing capacity, and can detect even minor genetic differences among individuals. We are using RAD-seq to 1) identify sex-specific molecular markers (i.e., bits of DNA found in individuals from one sex but not the other), and 2) using these markers to determine whether a species has XY or ZW sex chromosomes. Species with male-specific markers will have an XY system while species with female-specific will have a ZW system.

We are interested in using RAD-seq to screen various vertebrate species for sex chromosomes, but first wanted to validate the technique using a species with a known sex-determining mechanism. We chose the green anole (Anolis carolinensis) because its X and Y chromosomes are small and homomorphic. Therefore A. carolinensis sex chromosomes should provide a rigorous test of this technique and success with Anolis suggests there may be broad utility using this technique in other groups with homomorphic sex chromosomes.

We performed RAD-seq on seven male and ten female A. carolinensis and recovered one male-specific molecular marker. We confirmed that the marker was male-specific using PCR and also found that this genetic marker is conserved in some additional Anolis species, confirming homology among the Y chromosomes of these species (Anolis sex chromosome homology has been discussed previously on Anole Annals 1, 2). These results highlight the potential utility of RAD-seq as a tool to discover the sex chromosome systems of large numbers of species in a rapid, cost-effective manner.

PCR validation of the male-specific RAD-seq marker in Anolis carolinensis.

PCR validation of the male-specific RAD-seq marker in Anolis carolinensis.

In addition to learning about Anolis sex chromosomes the male-specific molecular marker we identified can be used to sex individuals of many Anolis species using a simple PCR-based assay, particularly species in the A. carolinensis group and in the Norops clade. This enables identification of an individual’s sex prior to the onset of secondary sexual characteristics, for example in embryos, thereby aiding developmental studies of sexually dimorphic phenotypes. The importance of sexual dimorphism to Anolis ecology and evolution has been examined previously (1, 2, 3, 4), but there is certainly much more to learn, particularly about how sexually dimorphic traits develop and evolve. The ability to sex Anolis embryos is an important step to advance this research.

Phylogenetic relationships among sampled species illustrating the sex-specific amplification of the gene rtdr1y in selected anole species. The autosomal gene kank1 was used as an internal positive control in all reactions. Bands labelled with ‘NS’ are nonspecific PCR products.

Phylogenetic relationships among sampled anoles illustrating the sex-specific amplification of the gene rtdr1y in selected anole species. The autosomal gene kank1 was used as an internal positive control in all PCR reactions. Bands labelled with ‘NS’ are nonspecific PCR products.

Reconstructing the History of Anole Sex Chromosomes


George Gorman in Dominica

In the 1960s and 70’s evolutionary cytogenetics experienced a remarkable burst of interest and scholarship. Thanks largely to the efforts of George Gorman (at right) and others working at the Museum of Comparative Zoology, anoles played a central role in this research (some historical detail has previously been posted on AA). Among their findings was the occurrence of heteromorphic sex chromosomes, sex chromosomes that are visibly distinguishable from each other under a microscope, in several Anolis species but not others. Furthermore, Gorman and colleagues discovered that those Anolis species with heteromorphic sex chromosomes all had male heterogamety, with some having an XX/XY system while others had an XXXX/XXY system. Chromosomes from nearly 100 Anolis species were examined during this period and about 1/3 of those species had heteromorphic sex chromosomes. Interest in chromosome evolution waned in the 1980’s as DNA sequence data became increasing accessible, but there has been a recent resurgence thanks, in part, to sex chromosomes.

Continue reading Reconstructing the History of Anole Sex Chromosomes

ASU Green Anole Genome Reannotation Now Available on Ensembl

Green anole (Anolis carolinensis). Photo courtesy of Karla Moeller.

Green anole (Anolis carolinensis). Photo courtesy of Karla Moeller.

Ensembl Release 71 includes many updates for Anolis carolinensis, including the addition of the Arizona State University (ASU) Anole Genome Project annotation recently published in BMC Genomics (Eckalbar et al., 2013). This release includes an updated Ensembl gene set and aligned RNA-Seq data from a number of tissues, including embryo, lung, liver, heart, dewlap, skeletal muscle, adrenal gland, ovary, and brain, which have been added to the track viewer. These RNA-Seq data from individual tissues and from the ASU reannotation or the “Anole Genome Project” can be viewed just below the Ensembl gene tracks, as in this example.

Anoles (and Alligators) Give a New View on the Evolution of Vertebrate Eevelopment

One of the key features of vertebrates is the backbone, which is formed in development by a clock-like segmentation process called somitogenesis. Most of what we know about the genes that control somitogenesis comes from studies of just 4 vertebrate species–the mouse, the chicken, the African clawed frog (Xenopus laevis), and the zebrafish. Until now, we haven’t had a good window into the evolution of somitogenesis from the perspective of a non-avian reptile. The green anole (Anolis carolinensis) is now providing this perspective as a 5th model system for molecular developmental studies.

In a recently published paper (Eckalbar et al., Developmental Biology, 2012), we have shown that green anole embryos share molecular features of somitogenesis with the mouse and the chicken, which are also amniotes. Surprisingly, the green anole also retains expression patterns that match those of the non-amniote species, Xenopus and zebrafish, and that have been lost in the mouse and chick. The American alligator (Alligator mississippiensis), which together with birds are classified in a group called the Archosauria, are intermediate in somitogenesis features between anoles and chicken. These findings reshape our view of what was happening in the backbone development of the amniote ancestor, the first vertebrate whose eggs were fully adapted for life on land.

For those in the anole research community, RNA-Seq transcriptome data sets (Illumina HiSeq2000; 28 and 38 somite-pair stages) have been released together with this paper. Transcriptome data links can be found at the AnolisGenome portal and also directly from the NIH Gene Expression Omnibus. We aim to get more transcriptome sequence to the Anolis research community in 2012.

Anoles on Genbank

With the recent sequencing of the Anolis carolinensis genome and Thom’s recent post on resources for other anole species I got to wondering how many DNA sequences are available for anoles?  In an effort to answer this question, I searched for DNA sequence data from Anolis and other genera now considered part of Anolis (Norops, Chamaeleolis, Chamaelinorops, and Phenacosaurus) on the NCBI’s popular GenBank database.  I found that Genbank‘s nucleotide database contains over 29,ooo unique anole sequences. Not surprisingly, the most sequence (25,973) are from A. carolinensis.  Remaining sequences are divided among 216 anole species. The top species after carolinensis are: krugi (433), distichus (378), sagrei (351) and cristatellus (328).  Is anyone else surprised by these totals?  I would have guessed sagrei would be second.  I think A. distichus will at least double in the next few years, partly because I’m doing lots of sequencing from this species myself.

Only 29 species are represented by more than 10 sequences and half of the 216 species represented in GenBank are represented by a single (usually mitochondrial) sequence. The availability of this data highlights our prospects for asking evolutionary and ecological questions across the rest of anoline diversity, but also highlights the huge amount of work ahead if we are interested in making broad genus-wide comparisons. Admittedly, Genebank lags behind current research as most of us only post sequences at the time of publication (we have hundreds of sequences to be added in the next few years).

What Do You Get When You Combine Three Lizards and a Chicken?

Anolis carolinensis (, A. marmoratus (from willy.ramaekers flickr:, Polychrus marmoratus (from Pierson Hills flickr:, chickens (from

New primers for sequencing nuclear loci from Anolis!

Availability of genomic loci for sequencing has long been a major stumbling block to evolutionary inference in non-model taxa.  In anoles, for example, several decades of work relied almost exclusively on mitochondrial DNA.  As part of the Anole genome sequencing initiative, my lab group collaborated with the Broad Institute to identify conserved primers that can be used to amplify nuclear loci from across Anolis.  We ultimately tested 200+ primer pairs, most of which were identified by comparing the genome of Anolis carolinensis to genomic data from two related lizards (Anolis marmoratus and Polychrus marmoratus) and the chicken (others came from recent work in the Jackman lab). Continue reading What Do You Get When You Combine Three Lizards and a Chicken?

Media Coverage of the Anole Genome Paper

Photo by David E. Scott/Savannah River Ecology Laboratory, Aiken, S.C.

We’ll try to keep this post updated with links to coverage of the anole genome paper (please use the comments to tell us about new articles as they appear!):

Commentaries: Science 2.0Why Evolution is TrueNatureNational Geographic, Dust TracksmyFDL (are you a septic of evolution?)

Press Release and Summaries: Broad Institute Press ReleaseBloomberg, Harvard GazetteRedorbit, International Business Times (and some amusing chatter about this article), TruthDive, io9, R&D Daily, GenomeWeb Daily

Anole Genome Paper Published Today!

Image copyright Andrew M. Shedlock.

The anole genome paper is out in Nature today (although links on Nature’s own page only take you to a list of authors at the present time, I’m assuming this glitch will be fixed shortly).  Nature also published a brief commentary highlighting some of the most interesting discoveries from this work.  For more coverage of work related to the genome, check out this post and stay tuned to Anole Annals – we’ll have a bunch more genome posts over the next few days.

How the Green Anole Was Selected To Be The First Reptile Genome Sequenced

As the publication of the anole genome approaches, one might ask: “Just how was Anolis carolinensis selected to be the first non-avian reptile to have its genome sequenced?” Turns out that it’s a long and convoluted story, and this is one man’s first-hand account.

To set the stage, we have to go back to the early days of genome sequencing, all the way back to 2005. This was a time when to sequence a genome was a really big, time-consuming, extremely expensive affair (the human genome had cost ca. $2 billion; by 2005, the price had dropped to ca. $20 million per genome). Such a big deal, in fact, that there was an NIH committee that decided which species would be sequenced, and assigned them to one of the three genome sequencing centers (Baylor University, Washington University in Saint Louis and the Broad Institute in Cambridge) that had been created as part of the human genome sequencing initiative. The first few species selected were chosen exclusively with regard to their potential relevance to human health. They were the laboratory model systems, the workhorses of biomedical research, such as the mouse, chimp, Xenopus, chicken, Drosophila and C. elegans.

By 2005, a couple of mammals had been sequenced and representatives of all classes of vertebrates except one: reptiles. Continue reading How the Green Anole Was Selected To Be The First Reptile Genome Sequenced

Anolis Transposable Elements and the Evolution of Amniote Genomes

Interested in transposable elements in the Anolis genome? You should be!

As DNA sequences that can move about the genome, transposable elements – or TEs – are also called “jumping genes”. These are some of the most important components of genomes, accounting for much of the variation in genome size and structure across vertebrates. The activity of TEs add to the genetic variation of populations in neutral, deleterious, and sometimes adaptive ways. In the human genome, TEs can insert into genes and cause numerous genetic diseases such as muscular dystrophy (Cannilan and Batzer 2006).

We published a review in last month’s issue of Mobile Genetic Elements (Tollis & Boissinot 2011) describing the diversity and abundance of TEs found so far in the Anolis genome, and how they impact our understanding of genome evolution in reptiles and mammals. The Anolis genome contains an extraordinary diversity of TEs, including DNA transposons (“cut and paste” elements) and long terminal repeat (LTR) and non-LTR retrotransposons (“copy and paste” elements). Even though there are many different kinds of TEs in Anolis, within most TE families there are low copy numbers relative to the human genome, suggesting that purifying selection keeps tight control. Continue reading Anolis Transposable Elements and the Evolution of Amniote Genomes

The Origins of Anolis carolinensis

Fig. 1: Figures illustrating Cuban origins of A. carolinensis from our 2005 paper in Molecular Ecology. Green shading indicates the range and phylogenetic position of A. carolinensis, blue shading indicates Cuban populations related to A. carolinensis. The arrows indicate possible dispersals from Cuba, some of which are supported by phylogenies (including the dispersal from Cuba to the continental United States indicated by the bold arrow).

With all this discussion of the green anole’s genome, it seems like a good time to remind everyone of how Anolis carolinesis came to be the model organism that it is today.  The simple answer, of course, is that A. carolinensis is the only species of anole endemic to the continental United States.  As such, its always been the anole species most accessible to the broadest range of researchers.  The deeper answer – and the focus of this post – concerns how A. carolinesis happened to become the continental United States’s only native anole in the first place.

Continue reading The Origins of Anolis carolinensis

Anole Annals: Your One Stop Anole Genome Information Source

For information on why the anole genome is useful for evolutionary studies, go here.

For information on how the genome is already being used in research, try here, here, here, here and here.

For the history of discovery and study of anoles, go here.

For the evolutionary history of the green anole, check this one out.

For a great story, don’t miss this one.

For great pictures of anoles and their dewlaps, try here, here, and here  (among others).

For many other topics in anole ecology, behavior, and diversity, try looking up terms in the blog’s search window.

What’s The Anole Genome Good For?

One of these species has had its genome sequenced, and the other has independently evolved to look very similar and live in the same environment. The anole genome will make anoles an even more powerful group in which to study evolutionary convergence. Photos by Melissa Losos (left) and Pete Humphrey (right).

When the genome of Anolis carolinensis is finally published, most attention will focus on how this genome, the first reptile to be sequenced (not including birds), differs from other vertebrate genomes, and what these differences may tell us about genome evolution. No doubt this will be interesting, but the real value of this genome–in my unbiased opinion–resides in the questions we finally will be able to address about the evolutionary process, particularly in one model system of evolutionary study, Anolis lizards. Chris Schneider published a perceptive article, “Exploiting genomic resources in studies of speciation and adaptive radiation of lizards in the genus Anolis,” on this topic three years ago, and I will briefly expand on his points here.

An anole genome will be useful for evolutionary studies in two ways. First, a long-standing question in evolutionary biology concerns the genetic basis of convergent evolution (i.e., when two or more evolutionary lineages independently evolve similar features). Do convergent phenotypes arise by convergent evolution of the same genetic changes, or do different lineages utilize different mutations to produce the same phenotype? In other words, does convergence at the phenotypic level result from convergent change at the genetic level, or can different genetic changes produce the same phenotypic response? In the last few years, molecular evolutionary biologists have produced a wealth of studies investigating whether convergent changes in coat color in rodents, eye and spine loss in fish, bristle loss in fruit flies and many other changes are the result of changes in the same gene, even some times by the very same genetic mutation. Underlying these questions are more fundamental questions about constraints and the predictability of evolution (these topics have been reviewed a number of times in the last couple of years, most recently in a paper by me, in a paper which refers to other recent reviews).

The anole ecomorphs, habitat specialists behaviorally and morphologically adapted to use different parts of the environment. The same set of ecomorphs (with several exceptions) have evolved independently on each island in the Greater Antilles. Figure from "Lizards in an Evolutionary Tree," based on earlier figures in Ernest Williams' work.

Anolis lizards are, of course, the poster child for evolutionary studies of convergent evolution. Indeed, convergence has run rampant in this clade. AA has prattled on endlessly about the famous anole ecomorphs, a set of habitat specialist types that have evolved repeatedly on each island in the Greater Antilles to occupy different habitat niches. This convergence is usually studied in terms of limb length, tail length, and toepad dimensions: arboreal species have big toepads, twig species short legs, grass species long tails, and so on, with these traits independently evolving many times. But the ecomorphs are convergent in many other traits that have received less attention: head and pelvis dimensions, sexual dimorphism in both size and shape, territorial and foraging behavior, to name a few, and the more closely we look, the more convergent traits we find. And, further, anole convergence is not entirely an ecomorph phenomenon; some traits vary within an ecomorph class, but are convergent among species in different ecomorph classes, for example, thermal physiology and dewlap color.

In other words, there’s more convergence in Anolis than you can shake a stick at, and the availability of the anole genome sequence will provide the tools to investigate its underlying genetic basis. Continue reading What’s The Anole Genome Good For?

Not Your Typical Genome: Homogeneous Anole Genome Lacks Isochores Common in Other Amniotes

Figures from Fujita et al. illustrating relative homogeneity of GC content across the anole genome (left) and shifts in GC3 along branches in the vertebrate tree, with black branches indicating descreases of GC3 and gray branches indicating increases of GC3 (right).

Genomes are rarely homogeneous aggregations of Gs, As, Ts, and Cs.  Indeed, variation in  basepair frequency can have important implications for how genomes, and the organisms they generate, evolve.  Regions with relatively homogenous GC content that extend for more than 300 kb known as isochores are prominent features of previously sequenced amniote genomes.  Isochores are associated with a range of important variables, including gene density, intron length, DNA replication timing, and gene expression.  GC-rich isochores also tend to experience high rates of recombination, resulting in elevated effective population sizes and increased efficiency of purifying selection relative to drift.

Continue reading Not Your Typical Genome: Homogeneous Anole Genome Lacks Isochores Common in Other Amniotes

Anole Genome Research: New Primers for All!

Table from Portik et al.'s Conservation Genetics paper reporting new primer pairs for amplification of nuclear loci (left side) and a phylogeny generated using some of these loci from Stanley et al.s' 2011 MPE paper on cordylids (right panel).

A new study by Portik et al. used the anole genome to develop more than 100 new primer pairs for the amplification of nuclear-encoded DNA from squamates, some of which have already proven useful for inferring relationships within and among species.  Portik et al.’s carefully thought out strategy for marker development – which focused on rapidly evolving protein-coding loci – ensures that their loci will be particularly useful for phylogenetic analyses.  First, Portik et al.  focused on intronless protein-coding genes, with the goal of limiting length variation and simplifying alignment.  Second, recognizing  low variability relative to non-coding regions as a potential limitation of protein-coding loci, Portik et al. focused exclusively on developing markers from loci that are  more variable than the first third of RAG-1 (one of the most useful and widely-used of the nuclear genes used  previous phylogenetic studies of squamates).  This strategy yielded 104 genes and led to development of primers for 170 gene fragments ranging from 407-2,492 bp.  Portik et al. conducted limited PCR testing on 70 of these loci and found varying degrees of success across five squamate families, including Scincidae, Varanidae, Agamidae, Cordylidae, and Gekkonidae.  More importantly, some of the loci have already proven useful for phylogenetic studies of skinks (Portik et al. 2010 , Portik et al. 2011), cordylids (Stanley et al. 2011) and iguanids (anole genome paper, which is currently in press at Nature).

While high throughput sequencing technology will eventually render PCR primers and Sanger sequencing nothing more than curiosities from a previous generation, this time is  at least a few years away.  In the meantime, Portik et al. have given the herpetological community some very useful new tools to play with.