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We then used BEDTools intersect iteratively to identify sites callable in all samples in a given population Quinlan and Hall Given this information, we calculated average nucleotide diversity separately for the autosomes and putatively X-linked scaffolds as the arithmetic mean of per site diversity across all callable sites. Specifically, we first mapped samples individually using default parameters and then did a second pass of mapping with default parameters except for including information about identified splice junctions for all samples using the "—sjdbFileChrStartEnd" command Li ; Li, Genome Project Data Processing Subgroup et al.

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We mapped trimmed re to the A. We measured RNA-seq statistics using the stats function of bamtools2. S2Supplementary Material online. In this study, we conducted analyses to infer additional X-linked loci in the green anole, quantify patterns of sex-biased gene expression across the genome, and characterize the extent of dosage compensation on the anole X chromosome. In addition, we present evidence for dosage compensation on the anole X chromosome. Comparative analysis: To identify putative X-linked genes, we conducted bidirectional best hit alignment between the chicken and anole genomes.


In therian mammals, gene dosage is upregulated on the X chromosome in both males and females, and females experience silencing of one X chromosome Payer and Lee although gene-by-gene escape from inactivation can occur, Carrel and Willard ; in the platypus, a monotreme mammal with a chromosomal sex determination system that is derived independently of therian mammals, the X chromosome exhibits partial inactivation Deakin et al.

Diversity analyses: For independent verification of the putative X-linked transcripts, we analyzed patterns of genetic diversity across the genome. However, it is unknown whether the limited of taxa that have been studied to date biases perceived trends about dosage compensation. Lastly, one script replaced internal and terminal stop codons with gaps. For example, population 1 consisted of biological replicate 1 for each of the four individuals, population 2 consisted of biological replicate 2 for each for the four individuals, and so on.

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Dosage compensation is a process whereby genes on the sex chromosomes achieve equal gene expression. Finally, we show that X-linked genes have a higher ratio of non-synonymous to synonymous substitution rates than autosomal genes, a pattern consistent with fast-X.

Gene sequences that did not meet these requirements were discarded from the analysis. We then aligned genes from each anole scaffold back to the galGal4 genome.

Materials and methods

We called genes as differentially expressed if the q -value—a P value with a Benjamini—Hochberg correction for multiple testing Trapnell et al. X-linked genes were present at each threshold and here present data using an FPKM threshold of 2 supplementary fig.

For each alignment step, we used a cutoff score of to include genes for further analysis. Total RNA was isolated from each sample separately, all samples were barcoded and multiplexed with paired-end sequencing libraries generated using manufacturer protocols and sequenced on an Illumina HiSeq Hutchins et al. S1Supplementary Material online.

After sequencing the samples were de-multiplexed and analyzed separately. Therefore, to identify putative X-linked genes in the green anole, we used a comparative genomics approach.

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We also found that our were robust when we analyzed expression after ing to where re mapped in the chicken genome galGal4; supplementary table S3Supplementary Material online. Next, we obtained a list of transcript IDs of genes located on the chicken chromosome 15 from Ensembl BioMart Ensembl v79 Kinsella et al. No pseudoautosomal genes have yet been described and pseudoautosomal regions in A. The identification of many genomic regions that are haploid in males but diploid in females suggest that the A. Y-linked degeneration is expected to result in changes in X-linked transcript levels, a form of dosage compensation to achieve similar X-linked expression in males and females Wilson Sayres and Makova X chromosome expression, however, has not yet been comprehensively characterized in the green anole.

In the absence of dosage compensation, however, the expression of sex-linked genes is expected to reflect their overall copy in each sex i.

Chicken genomic regions are shown on the top chromosome, while anole scaffolds that may be X-linked and the ly described X-linked LGb are shown on the bottom. We compared genome-wide levels of transcription between males and females, and between the X chromosome and the autosomes in the green anole, Anolis carolinensi s. Briefly, regenerating tail samples were collected 25 days post autotomy and sliced into five sections along the proximal—distal axis.

This gene-by-gene pattern has been observed in birds Dean et al. The green anole, Anolis carolinensisis a squamate model used to investigate reproductive physiology and behavior Lovern et al. The latter process is expected to generate a higher ratio of non-synonymous to synonymous substitutions on the X chromosome than the autosomes—a phenomenon called the fast-X effect Vicoso and Charlesworth ; Meisel and Connallon Other factors may result in a similar ature: genetic drift on the Z chromosome relative to the autosomes has also been shown to result in a "fast-X" effect Mank et al.

In addition to dosage compensation, natural selection is expected to behave differently on the sex chromosomes vs. We estimated confidence intervals for autosomal diversity, X-linked diversity, and the ratio of X to autosomal diversity by sampling bootstrap replicates with replacement. This primarily allowed us to control for differences in sequencing coverage and quality among replicates, but also provided five hermaphrodite dating Tempe AZ and technically independent estimates of diversity among these individuals. Additionally, we also conducted an analysis of genes with 50 or more re in males and genes with 50 or more re in females.

We calculated genetic diversity within each set of biological replicates. Differential expression: We conducted the differential expression analysis using Cuffdiff in Cufflinks2. When dividing the X chromosome into regions based on linkage groups, we discovered that genes in the first reported X-linked region, anole linkage group b LGbexhibit complete dosage compensation, although the rest of the X-linked genes exhibit incomplete dosage compensation.

Our data further suggest that the mechanism of this dosage compensation is upregulation of the X chromosome in males. Based on these analyses, we propose that the green anole X chromosome contains at least genes. Shawn M. Rupp, Timothy H. Webster, Kimberly C. Olney, Elizabeth D. Hutchins, Kenro Kusumi, Melissa A. In species with highly heteromorphic sex chromosomes, the degradation of one of the sex chromosomes will result in unequal gene expression between the sexes e. This resulted in five biological replicates from each of the four individuals for a total of twenty samples.

Genes are color-coded by scaffold.

Location of proposed X-linked sequences in the chicken genome. If most new mutations are recessive, then natural selection will be more efficient on the X chromosome, both removing harmful variants and also increasing the frequency of beneficial X-linked variants. Given that the minimum expression threshold can skew when examining patterns of dosage compensation, we examined the relative expression with five different FPKM thresholds 0, 1, 2, 3, and 4similar to Smith et al.

In total, these genes spanned eight scaffolds fig. Substitution rate analysis: We downloaded pairwise alignments between the AnoCar2. This pattern is explained in part by the time since gametologous Y-linked alleles were pseudogenized or lost Wilson Sayres and Makova Given the variation among the few systems that have been studied, it is necessary to examine dosage compensation in evolutionarily independent chromosomal sex determination systems.

Lines connect orthologous genes between the two genomes. If genes on the green anole X chromosome have evolved dosage compensation between males and females, relative expression values between the sexes will not be sufficient to identify X-linked transcripts. In the presence of dosage compensation, genes on the sex chromosomes in males and females will be expressed at approximately similar levels, nearing a sex ratio of 1.

Among these scaffolds, each contains at least hermaphrodite dating Tempe AZ gene ly reported to be X-linked Rovatsos et al.

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Genomic analyses are crucial for a comprehensive picture of anole sex chromosome differentiation, dosage compensation, and atures of natural selection. For these analyses, we focused exclusively on biallelic SNPs, to which we applied a series of filters, removing sites at which the sequencing depth was less than 2, Fisher strand bias was greater than 30, the mean mapping quality was less than 30, or the site quality was less than We then only examined sites that were callable in all samples within a given population on scaffolds containing at least callable sites per population.

If the anole X and Y chromosomes are as differentiated as preliminary data suggests, and if it follows the trend of other male-heterogaemtic systems, then we expect to observe dosage compensation on the anole X chromosome. We found that the same trends for autosomal vs. As an additional line of evidence to verify these X-linked scaffolds, we conducted an analysis of genetic diversity on the autosomes and the putative X-linked scaffolds.

In addition, genes on the X chromosome exhibited higher ratios of nonsynonymous to synonymous substitution than autosomal genes, consistent with the fast-X effect.

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Diversity is expected to be reduced on X chromosomes relative to autosomes due to a smaller effective population size and more efficient natural selection Ellegren Relative expression could only be computed for genes with detectable expression values for both males and females.

We present evidence for dosage compensation between the sexes, and between the sex chromosomes and the autosomes. Twenty-three additional genes were later identified on LGb, bringing the total of known X-linked genes to In a subsequent analysis, quantitative PCR in male and female samples identified an additional 38 genes that are consistent with male-heterogamety Rovatsos et al.

We conducted analysis both including and excluding alignments with internal stop codons to avoid inflating ratios of non-synonymous to synonymous substitution rates by including pseudogenes, and here we present the analysis with genes with internal stop codons excluded. Each list was run as a target set with a list of gene IDs for the whole genome as the background set, and we specified that only hermaphrodite dating Tempe AZ with a multiple testing corrected P value of 0.

In contrast, XY males only possess one copy of the X chromosome, so recessive X-linked alleles are directly exposed to natural selection. We included genes annotated from both the Ensembl v79 and a ly published annotation using transcriptomes Eckalbar et al. By default, Cuffdiff normalizes read count estimates between samples, so models for inter-sample normalization of raw read counts were not applied to read estimates returned by Cuffdiff Dillies et al.