DOI: 10.5061/dryad.cfxpnvxhm
title: Materials for rattlesnake chemoreceptor gene expression

description: Predatory species who hunt for their prey rely on a suite of integrated
 characters, including sensory traits which are also used for non-predatory
 behaviors. Linking the evolution of sensory traits to specific selection
 pressures therefore requires a deep understanding of the underlying
 genetics and molecular mechanisms producing these complex phenotypes.
 However, this relationship remains poorly understood for complex sensory
 systems that consist of proteins encoded by large gene families. The
 chemosensory repertoire of rattlesnakes includes hundreds of type-2
 vomeronasal receptors (V2Rs) and olfactory receptors (ORs), representing
 the two largest gene families found in the genome. To investigate the
 biological importance of this chemoreceptor diversity, we assessed gene
 expression in the eastern diamondback rattlesnake (Crotalus adamanteus)
 and identified sex- and age-biased genes. We found V2R expression in the
 vomeronasal epithelium was limited to juvenile snakes, suggesting the
 sensory programming of this tissue may be correlated with early life
 development. In the olfactory epithelium, we found subtle expression
 biases which were more indicative of life history rather than development.
 We also found transcriptional evidence for dosage compensation of
 sex-linked genes and trait integration in the expression of transcription
 factors (TFs). We overlay our molecular characterizations in C.
 adamanteus onto updated OR and V2R phylogenies, providing a
 genetic road map for future research on these receptors. Finally, we
 investigated the deeper macroevolutionary context of the most highly
 expressed V2R gene spanning the rise of tetrapods and estimated the
 strength of positive selection for individual amino acid residues in the
 predicted protein structure. We hypothesize that this gene may have
 evolved as a conserved signaling subunit to ensure consistent G-protein
 coupled receptor (GPCR) functionality, potentially relaxing signaling
 constraints on other V2R paralogs and promoting ligand binding
 specificity.
description: # Materials for rattlesnake chemoreceptor expression
 [https://doi.org/10.5061/dryad.cfxpnvxhm](https://doi.org/10.5061/dryad.cfxpnvxhm) ## Description of the data and file structure Supplemental information to accompany Hogan et al., 2025 "Life history and chromosome organization determine chemoreceptor gene expression in rattlesnakes". Article published in the Journal of Heredity. ### Files and variables #### File: supp04\_selection\_stats.xlsx **Description:** Statistical tests and results for interpreting gene sequences. Tab labels and contents: "TM domain info" = trans-membrane alignment positions for *C. adamanteus* OR and V2R gene alignment and for the multi-species conserved V2R alignment; "FEL raw" = HyPhy FEL selection test results for *C. adamanteus* ORs followed by V2Rs; "FEL Extra vs Intra vs TM" = proportional z-test results comparing protein domain distribution of selected sites detected by FEL for *C. adamanteus* ORs followed by V2Rs; "MEME raw" = HyPhy MEME selection test results for *C. adamanteus* ORs followed by V2Rs; "MEME Extra vs Intra vs TM" = proportional z-test results comparing protein domain distribution of selected sites detected by MEME for *C. adamanteus* ORs followed by V2Rs; "all EBF&gt;100" = lists of Estimated Bayes Factor scores &gt; 100 reported from MEME for *C. adamanteus* ORs, V2Rs, and for the multi-species analysis on a conserved V2R; "Chrm ancestral recon" = percent breakdowns of chromosome ancestral gene tree reconstructions calculated for *C. adamanteus* ORs and V2Rs; "Chrm vs selection" = data (left) and stats results (right) using another z-test to compare the chromosomal distribution of significantly selected sites detected using MEME for *C. adamanteus* ORs followed by V2Rs below;  ##### Variables * Significant z-test results in this spreadsheet will be shown with red text in a yellow cell * Cell colors = colors were added to cells in this spreadsheet to facilitate reading and to distinguish tests performed on ORs and tests performed on V2Rs. We designated green to OR data, and blue to V2R data. For the slides reporting proportional tests, we color coded extracellular tests with light red, and intracellular tests with light blue. * NA = represents amino acid sites which had no known protein domain or function. * nuc = nucleotiode * AA = amino acid * Extracellular = outside of cell * Intracellular = cytoplasmic region inside of cell * TM = trans membrane * alpha = value used to represent non-synonymonous mutations * beta = value used to represent synonymonous mutations * LRT = Log ratio test result * q - and q + = selection test parameter generated by MEME * EBF = estimated Bayes factor * LogL = result of the log L test performed by HyPhy * Interaction site = an amino acid or codon position which has the specific proteomic functionality #### File: supp03\_combined\_trees\_aligned.txt **Description:** Three consecutive Nexus alignments, ordered *C. adamanteus *ORs first, *C. adamanteus * V2Rs second, and multi-species conserved V2R alignment third. #### File: supp02\_Deseq2\_results.xlsx **Description:** Results from our differential gene expression tests using Deseq2 in R. Tab labels and contents: "Olfactory AGE" = Full Deseq2 results of *C. adamanteus* olfactory epithelium RNA aligned to whole genome comparing juvenile and adult rattlesnakes; "Olfactory SEX" = Full Deseq2 results of *C. adamanteus* olfactory epithelium RNA aligned to whole genome comparing male and female rattlesnakes; "Vomeronasal AGE" = Full Deseq2 results of *C. adamanteus* vomeronasal epithelium RNA aligned to whole genome comparing juvenile and adult rattlesnakes; "Vomeronasal SEX" = Full Deseq2  ##### Variables * Gene_cds_id = column containing the gene ID reported in the genome. * baseMean = the mean expression value used by Deseq2 to calculate differential expression. * log2FoldChange = the main differential expression result, reported as positive or negative by Deseq2 based on direction of bias. * lfcSE = standard error reported by Deseq2 * stat = test statistic value used internally by Deseq2 for calculating differential expression. * pvalue = p value reported by Deseq2 prior to adjusting for over dispersion. * padj = adjusted p value reported by Deseq2, which we utilized for statistical cutoffs in our analyses.