Category: Anole Genome Research Page 1 of 2

Anole Genomes Webinar

Tomorrow (30 June 2020) I will be presenting a webinar on our ongoing work assembling Anolis genomes. The webinar is hosted by Dovetail Genomics who provided the core technology we used to generate high quality genome sequences. The talk is at 11AM EST. If you want to watch live and have the chance to ask questions, you can register here. If you can’t make it but still want to hear what we are up to, Dovetail will post the video on their website alongside other speakers in the series.

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.

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?

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 by staustinreview.com 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

Gorman_Dominica_1965_Anolis_oculatus

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.

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 (http://www.birderslounge.com/2008/07/green-anole-amore/), A. marmoratus (from willy.ramaekers flickr: http://www.flickr.com/photos/27048739@N02/), Polychrus marmoratus (from Pierson Hills flickr: http://www.flickr.com/photos/nclarkii/), chickens (from http://ww.animalpicturesarchive.com/)

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).

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

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