Phenotypic variation among populations, as seen in the signaling traits of many species, provides an opportunity to test whether similar factors generate repeated phenotypic patterns in different parts of a species’ range. We investigated whether genetic divergence, abiotic gradients, and sympatry with closely related species explain variation in the dewlap colors of Amazon Slender Anoles, Anolis fuscoauratus. To this aim, we characterized dewlap diversity in the field with respect to population genetic structure and evolutionary relationships, assessed whether dewlap phenotypes are associated with climate or landscape variables, and tested for non-random associations in the distributions of A. fuscoauratus phenotypes and sympatric Anolis species. We found that dewlap colors vary among but not within sites in A. fuscoauratus. Regional genetic clusters included multiple phenotypes, while populations with similar dewlaps were often distantly related. Phenotypes did not segregate in environmental space, providing no support for optimized signal transmission at a local scale. Instead, we found a negative association between certain phenotypes and sympatric Anolis species with similar dewlap color attributes, suggesting that interactions with closely related species promoted dewlap divergence among A. fuscoauratus populations. Amazon Slender Anoles emerge as a promising system to address questions about parallel trait evolution and the contribution of signaling traits to speciation.

Brief description of the methods for data collection:

Dewlap color variation data

To characterize geographic dewlap color variation in Anolis fuscoauratus, we used data from our comprehensive herpetofaunal inventories in Amazonia and the Atlantic Forest over the last two decades. To this purpose, we sampled individuals by hand or pitfall traps. We only included in our environmental and species co-occurrence analyses 32 sites for which dewlap color information was available (pictures or field notes) from the 63 sites that were included in genetic analyses.

We also obtained data on the presence of all other anole species sympatric with A. fuscoauratus at a given site based on our field inventory data. To reduce the chance of undetected species, we only included data from surveys that lasted a minimum of one week and involved at least three herpetologists searching for animals both night and day. The final dataset included occurrence data for 11 other anole species at the 32 sites for which A. fuscoauratus dewlap coloration data were available: Anolis auratus, A. chrysolepis, A. dissimilis, A. nasofrontalis, A. ortonii, A. planiceps, A. punctatus, A. scypheus, A. tandai, A. trachyderma, and A. transversalis.

Genetic data

A double-digest restriction site associated DNA library (ddRAD) was generated at the University of Wisconsin Biotechnology Center. Briefly, DNA extractions were digested with the restriction enzymes PstI and MspI, and the resulting fragments were tagged with individual barcodes, PCR-amplified, multiplexed, and sequenced in a single lane on an Illumina HiSeq 2500 platform. The number of paired-end reads ranged from ~1.15 to 8.85 million per individual, with a read length of 100 base pairs. De-multiplexed raw sequence data were deposited in the Sequence Read Archive (BioProject PRJNA492310; BioSample accessions SAMN18340748-18340924).

We used Ipyrad v. 0.7.30 to de-multiplex and assign reads to individuals based on sequence barcodes (allowing no mismatches from individual barcodes), perform de novo read assembly (minimum clustering similarity threshold = 0.95), align reads into loci, and call single nucleotide polymorphisms (SNPs). A minimum Phred quality score (= 33), sequence coverage (= 6x), read length (= 35 bp), and maximum proportion of heterozygous sites per locus (= 0.5) were enforced, while ensuring that variable sites had no more than two alleles (i.e., a diploid genome). Moreover, for inclusion in the final datasets, we ensured that each locus was present in at least 70% of the sampled individuals.

To estimate population genetic structure and admixture in Anolis fuscoauratus, we generated in Ipyrad a final dataset composed of 118,434 SNPs at 16,368 loci (including no outgroups). A single SNP was then extracted from each locus to minimize sampling of linked SNPs. We used VCFtools v. 0.1.16 to filter out SNPs whose minor allele frequency (MAF) was lower than 0.05.

To estimate phylogenetic relationships, we generated in Ipyrad a second dataset composed of 135,952 SNPs at 17,302 RAD loci (now including outgroup taxa and linked SNPs), ensuring that each locus was present in at least 70% of the sampled individuals.

Environmental data

We used 17 variables in environmental analyses: cover of evergreen broadleaf trees, deciduous broadleaf trees, shrubs, herbaceous vegetation, and regularly flooded vegetation, annual cloud cover, elevation, slope, terrain roughness, and terrain ruggedness, all obtained from the EarthEnv database. As climatic variables, we used annual mean temperature, maximum temperature of the warmest month, mean temperature of the warmest quarter, annual precipitation, precipitation of the wettest month, and precipitation of the wettest quarter, obtained from the Chelsa database, as well as the climatic moisture index, a metric of relative wetness, obtained from the ENVIREM database. Values were extracted for each environmental variable from the 32 sites for which A. fuscoauratus dewlap color information was available in QGIS v. 3.4.5.

Please refer to the manuscript's Material and Methods for details on the analyses.

Please send questions to ivanprates [at] gmail [dot] com

This repository contains information associated with the manuscript entitled:

Evolutionary drivers of sexual signal variation in Amazon slender anoles.

By Ivan Prates, Annelise D'Angiolella, Paulo R. Melo-Sampaio, Miguel T. Rodrigues, Kevin de Queiroz, and Rayna C. Bell.

The manuscript is currently (March 2021) in press in Evolution.

The following information is provided:

1. R and Unix shell scripts used to prepare and filter the data and perform all analyses;

2. Filtered and assembled genetic (ddRAD) data;

3. Outputs from phylogenetic, genetic structure, environmental, and co-occurrence analyses;

4. Supplementary tables, figures, and text.

Raw read data were deposited in the Sequence Read Archive and can be retrieved at:

https://www.ncbi.nlm.nih.gov/sra/PRJNA492310

Supplementary files as follows:

Figure S1. Violin plots depicting the ranges of all 17 environmental variables.

Table S1. Locality information for Anolis fuscoauratus and sympatric Anolis species used in the co-occurrence analyses.

Table S2. Specimen and locality information of individuals used in the genetic analyses.

Table S3. Locality information and data used in the environmental analyses.

Table S4. Loadings of variables used in environmental principal component analyses.

Text S1. Protein precipitation protocol used for genomic DNA extraction.

Text S2. Phylogenetic tree including node support values and outgroup taxa.