Convergent evolution of gene expression in two high-toothed stickleback populations

<div><p>Changes in developmental gene regulatory networks enable evolved changes in morphology. These changes can be in <i>cis</i> regulatory elements that act in an allele-specific manner, or changes to the overall <i>trans</i> regulatory environment that interacts with <i>cis</i> regulatory sequences. Here we address several questions about the evolution of gene expression accompanying a convergently evolved constructive morphological trait, increases in tooth number in two independently derived freshwater populations of threespine stickleback fish (<i>Gasterosteus aculeatus</i>). Are convergently evolved <i>cis</i> and/or <i>trans</i> changes in gene expression associated with convergently evolved morphological evolution? Do <i>cis</i> or <i>trans</i> regulatory changes contribute more to gene expression changes accompanying an evolved morphological gain trait? Transcriptome data from dental tissue of ancestral low-toothed and two independently derived high-toothed stickleback populations revealed significantly shared gene expression changes that have convergently evolved in the two high-toothed populations. Comparing <i>cis</i> and <i>trans</i> regulatory changes using phased gene expression data from F1 hybrids, we found that <i>trans</i> regulatory changes were predominant and more likely to be shared among both high-toothed populations. In contrast, while <i>cis</i> regulatory changes have evolved in both high-toothed populations, overall these changes were distinct and not shared among high-toothed populations. Together these data suggest that a convergently evolved trait can occur through genetically distinct regulatory changes that converge on similar <i>trans</i> regulatory environments.</p></div

<i>Trans</i> changes are more likely to be shared across populations.

<p>(A) Genes with significantly different evolved expression in both freshwater populations relative to marine fish, showing significantly correlated changes in gene expression in PAXB_FW and CERC_FW dental tissue. (B) Freshwater dental tissue had a significant but small number of shared <i>cis</i>-regulatory changes. (C) Freshwater dental tissue showed significantly correlated changes in <i>trans</i> expression changes. A-C show genes with significant expression changes between populations and quantifiable (i.e. genes with transcripts containing a polymorphic SNP covered by at least 20 reads) <i>cis</i>-regulatory changes in both populations. Density (color) was estimated with a Gaussian kernal density estimator. (D-F) Bar graphs show the number of genes with shared or divergent expression patterns from the above panels. (G-I) Similar to (A-C), but showing only genes in the BiteCode gene set.</p

Evolved changes in <i>cis</i>-regulation.

<p>(A) Cartoon showing the two different regulatory changes detectable by our F1 hybrid system. Both genes 1 and 2 show an evolved increase of expression in freshwater fish, but the freshwater allele of gene 1 but not gene 2 is expressed more highly in F1 hybrids. Therefore, gene 1 has evolved its increased gene expression through <i>cis</i>-regulatory changes, while gene 2 was modulated by <i>trans</i> regulatory changes. (B) Density plot showing the measured <i>cis</i>-regulatory changes. Neither population displayed a significant allelic bias, as measured by a Wilcoxon signed-rank test. (C-D) Gene expression changes in both parental and hybrid dental tissue–genes are color-coded based on the role of <i>cis</i> and/or <i>trans</i> change in PAXB_FW (C) or CERC_FW (D) dental tissue. Dashed line indicates the first principal component axis.</p

<i>Trans</i> changes predominate evolved dental gene expression changes.

<p>(A-B) Proportion of differentially expressed genes displaying opposing and concordant <i>cis</i> and <i>trans</i> changes in PAXB_FW (A) or CERC_FW (B) dental tissue. Genes whose expression differences were mostly explained by <i>cis</i> changes tended to be more concordant (<i>P</i> = 5.0e-17, 0.002 for PAXB_FW and CERC_FW, respectively) than those mostly explained by <i>trans</i> changes. (C) Density of the relative percentage of gene expression differences which are explained by <i>cis</i> changes in PAXB_FW and CERC_FW dental tissue. (D) Cumulative percentage of percentage of gene expression due to <i>cis</i> changes. Genes in CERC_FW samples display a higher percentage <i>cis</i> change than genes in PAXB_FW samples (<i>P</i> = 1.25e-22, Mann-Whitney U test).</p

ScafKeyForNewFasta.csv

Key for scaffold order in fasta file

FileS7 ensGene_revised.gtf

Revised .gtf file of Ensembl gene predictions. Coordinates of gene predictions were converted to the revised assembly coordinates. All Ensembl-predicted genes were included, except ENSGACT00000019430, which spans two scaffolds (11 and 79) that are not adjacent in the revised genome assembly. File is zipped

FileS4 NewScaffoldOrder.csv

Revised scaffold order for each chromosome (consensus of FTC and BEPA). Revised coordinates (based on this study) and original assembly coordinates are presented. Orientations are defined relative to original genome assembly. The orientation of some scaffolds was not detected in this study. These scaffolds are labeled as having 'unknown' orientation; their orientation was not altered relative to their orientation in the original genome assembly. Chromosome 'M' is the mitochondrial genome sequence, which was not analyzed in this study but is replicated in the revised genome assembly

FileS6 revisedAssemblyMasked.fa.zip

Repeat masked fasta file containing revised genome assembly based on consensus scaffold order and orientation as described in File S4 in the Glazer et al. manuscript. Repeat masked fasta file is based off the repeat masked version of the original genome assembly, which was masked with RepeatMasker. File is zipped

Supplemental methods, results, figures, and tables from Jaw size variation is associated with a novel craniofacial function for galanin receptor 2 in an adaptive radiation of pupfishes

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel function for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a postulated Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions

convertCoordinate.R

This R function converts between the 'old' and 'new' stickleback assembly coordinate systems. The 'old' coordinate system is the assembly described in the Jones et al 2012 stickleback genome paper. It requires access to the FileS4 NewScaffoldOrder.csv file. It has 4 inputs: chr, pos, direction, and scafFile. It returns a list of [chromosome, position]. See README or comments in convertCoordinate.R for further details