DOI: 10.5061/dryad.jh9w0vtf8
title: Convergent_nAChR venom resistance Mammals 2022

description: Convergent evolution is central to the study of adaptation and has been
 used to understand both the limits of evolution and the diverse patterns
 and processes which result in adaptive change. Resistance to snake venom
 α-neurotoxins (αNTXs) is a case of widespread convergence having evolved
 several times in snakes, lizards, and mammals. Despite extreme toxicity of
 αNTXs, substitutions in its target, the nicotinic acetylcholine receptor
 (nAChR), prevent αNTX binding and render species resistant. Recently, the
 published meerkat (Herpestidae) genome revealed that meerkats have the
 same substitutions in nAChR as the venom-resistant Egyptian mongoose
 (Herpestidae), suggesting that venom-resistant nAChRs may be ancestral to
 herpestids. Like the mongoose, many other species of feliform carnivores
 prey on venomous snakes, though their venom resistance has never been
 explored. To evaluate the prevalence and ancestry of αNTX resistance in
 mammals, we generate a dataset of mammalian nAChR utilizing museum
 specimens and public datasets. We find five instances of convergent
 evolution within feliform carnivores, and an additional eight instances
 across all mammals sampled. Tests of selection show that these
 substitutions are evolving under positive selection. Repeated convergence
 suggests that this adaptation played an important role in the evolution of
 mammalian physiology and potentially venom evolution.
description: # Convergent_nAChR Venom Resistance Mammals 2022 Dataset DOI:
 [10.5061/dryad.jh9w0vtf8](https://doi.org/10.5061/dryad.jh9w0vtf8) Author:
 Dr. Danielle H. Drabeck Affiliation: University of Minnesota Date of data
 collection: 2022 Date of publication: 2025 ## 1. OVERVIEW This dataset
 accompanies the manuscript “Convergent evolution of α-neurotoxin
 resistance in mammalian nicotinic acetylcholine receptors (nAChR).” It
 contains sequence alignments, phylogenetic trees, and codeml control files
 used to test for positive selection on the nAChR α-subunit across mammals.
 The dataset enables replication of analyses showing repeated, independent
 amino acid substitutions conferring snake venom α-neurotoxin resistance in
 multiple mammalian lineages. ## 2. PURPOSE OF THE DATASET The purpose of
 this dataset is to: \- Document convergence in the nAChR α-subunit gene
 among mammals. \- Evaluate molecular evidence for α-neurotoxin resistance
 via convergent amino acid substitutions. \- Provide data used in
 codon-based models (PAML codeml) for positive selection and ancestral
 state reconstruction. \- Facilitate reuse by researchers studying
 coevolution, convergence, or toxin resistance. ## 3. DESCRIPTION OF FILES
 Mammal_Full.phy – Full codon alignment of mammalian nAChR α-subunit
 sequences used in PAML analyses. Dropped_Alignment.phy – Reduced alignment
 excluding low-quality sequences. FINAL_MamTree.tre – Mammalian species
 tree used for all selection analyses. DROPOUT_Tree.tre – Pruned version of
 the tree matching Dropped_Alignment.phy. GeneTree_SiteModel_M8.tre – Gene
 tree estimated under the M8 site model. M8_Ancestors.fasta – Reconstructed
 ancestral sequences under the M8 model. codeml_BS_Alt.ctl /
 codeml_BS_Alt_A.ctl – PAML control files for branch-site model alternative
 hypotheses. codeml_BS_Null.ctl / codeml_BSNull_A.ctl – PAML control files
 for branch-site null models. Sitecodeml.ctl / DropoutSitecodeml.ctl – PAML
 control files for site-model analyses. README.txt – Metadata summary
 included in the dataset. ## 4. METHODS SUMMARY Sequence acquisition: nAChR
 α-subunit coding sequences were obtained from public databases (NCBI
 GenBank, Ensembl) and museum-derived genomic extracts. Alignment:
 Sequences were aligned at the codon level using MACSE v2 to preserve
 reading-frame integrity. Phylogenetic analysis: Species trees were curated
 from TimeTree and reconciled with taxonomic references. Selection tests:
 Positive selection was tested using codeml (PAML v4.9) under site and
 branch-site models (M7 vs. M8, Null vs. Alt). Likelihood ratio tests were
 used to detect sites or branches under selection. Ancestral
 reconstruction: Posterior probabilities of ancestral states were estimated
 under the M8 model to infer substitution histories across mammalian
 lineages. ## 5. KEY FINDINGS SUPPORTED BY THESE DATA \- Independent amino
 acid substitutions at α-neurotoxin binding sites evolved multiple times in
 mammals. \- Venom resistance occurs in several feliform carnivore lineages
 and at least eight other mammalian groups. \- Branch-site models support
 positive selection acting on binding-site residues. ## 6. FILE USE AND
 REPRODUCIBILITY PAML control files (*.ctl) can be used directly with the
 provided alignments and trees. File paths may need adjustment for local
 directories. Analyses were run using PAML v4.9 on Linux with parameters
 described within each control file. ## 7. FUNDING AND ACKNOWLEDGMENTS This
 dataset was supported by the National Institute of General Medical
 Sciences (K12GM119955) and the University of Minnesota. Museum specimens
 and genomic resources were provided by collaborating institutions listed
 in the associated publication. ## 8. DATA LICENSE AND CITATION All data
 are released under the Public Domain (CC0 1.0 Universal) license. Cite as:
 Drabeck, D.H. (2025). Convergent evolution of nAChR venom resistance in
 mammals. Dryad, Dataset.
 [https://doi.org/10.5061/dryad.jh9w0vtf8](https://doi.org/10.5061/dryad.jh9w0vtf8) ## Files and variables File: GeneTree_SiteModel_M8.tre Description: Gene tree inferred under the PAML site model M8 (beta & ω&gt;1), used to identify codon sites evolving under positive selection across mammals. File: M8_Ancestors.fasta Description: FASTA file containing reconstructed ancestral sequences for the nAChR α-subunit, estimated under the M8 model to infer the evolutionary history of toxin-binding site substitutions. File: Mammal_Full.phy Description: Full codon alignment of mammalian nAChR α-subunit sequences, including all taxa analyzed for selection and ancestral reconstruction in PAML. File: DROPOUT_Tree.tre Description: Pruned phylogenetic tree corresponding to the reduced “Dropped_Alignment.phy,” containing only taxa retained after quality filtering. File: DropoutSitecodeml.ctl Description: PAML control file specifying parameters for site-model analyses using the reduced “Dropped_Alignment.phy” dataset and “DROPOUT_Tree.tre.” File: Dropped_Alignment.phy Description: Reduced codon alignment excluding sequences with missing data or low alignment quality, used for validation and sensitivity analyses. File: codeml_BS_Alt.ctl Description: Branch-site model control file (alternative hypothesis) allowing ω&gt;1 on specified lineages to test for episodic positive selection. File: codeml_BSNull_A.ctl Description: Branch-site model control file (null hypothesis, variant A) fixing ω=1 on foreground branches to compare against the alternative model. File: codeml_BS_Null.ctl Description: Standard branch-site model control file for null hypothesis tests (ω fixed at 1), paired with “codeml_BS_Alt.ctl” for likelihood ratio testing. File: codeml_BS_Alt_A.ctl Description: Alternate branch-site control file using different model initialization parameters to ensure convergence of likelihood optimization. File: Sitecodeml.ctl Description: PAML control file for site-model analyses (M7 vs. M8) using the full “Mammal_Full.phy” alignment and “FINAL_MamTree.tre.” File: FINAL_MamTree.tre Description: Curated mammalian species tree topology used as the reference phylogeny for all codeml analyses and ancestral reconstructions. File: README.txt Description: Metadata file summarizing dataset contents, methods, and usage details for reproducibility and citation. Variables: Each alignment file contains codon sequences corresponding to the α-subunit of the nicotinic acetylcholine receptor (nAChR). Taxon names: standardized mammalian species identifiers. Codon positions: aligned nucleotide triplets representing protein-coding regions. Branch labels (in .tre files): indicate species or foreground lineages tested for positive selection. ω (omega): ratio of nonsynonymous to synonymous substitution rates estimated by PAML. Posterior probabilities: used to identify sites under positive selection in the M8 model. ## Code/software All analyses were performed using freely available, open-source software. The primary tool used was PAML (Phylogenetic Analysis by Maximum Likelihood), version 4.9, for codon-based models of molecular evolution. The included control files (*.ctl) are formatted for use with the “codeml” program within PAML. Alignments were prepared using MACSE v2.0 (Ranwez et al., 2018), which maintains reading-frame integrity for protein-coding sequences. Tree visualizations and format conversions were performed using FigTree v1.4.4 and IQ-TREE v2.2 for initial phylogenetic inference. Workflow summary: 1\. Codon alignments were generated in MACSE (v2.0) from nAChR α-subunit nucleotide sequences. 2\. Phylogenetic trees were pruned and formatted in FigTree and IQ-TREE to match alignment taxon sets. 3\. Selection analyses were performed in PAML v4.9 using the control files provided here: \- Site models (M7 vs. M8) using “Sitecodeml.ctl” and “DropoutSitecodeml.ctl.” \- Branch-site models (Null vs. Alt) using “codeml_BS_Null.ctl,” “codeml_BS_Alt.ctl,” and their variant A counterparts. 4\. Ancestral reconstructions were generated under the M8 model, producing “M8_Ancestors.fasta” and “GeneTree_SiteModel_M8.tre.” No proprietary software was used. All scripts and control files can be run directly with PAML v4.9 or higher on any Unix/Linux system. Results can be viewed or verified with standard text editors or tree-viewing programs (e.g., FigTree, Dendroscope, or ETE3 in Python). ## Access information All data included in this dataset are either newly generated or derived from publicly available genomic resources. Source data: \- Nucleotide and protein sequences for the nicotinic acetylcholine receptor α-subunit (CHRNA1) were obtained from the National Center for Biotechnology Information (NCBI; [https://www.ncbi.nlm.nih.gov](https://www.ncbi.nlm.nih.gov)) and the Ensembl Genome Browser ([https://www.ensembl.org](https://www.ensembl.org)). \- Publicly available genome assemblies and annotations were accessed under their respective open-access licenses (GenBank and Ensembl release terms). \- Museum-derived DNA sequences were generated as part of this study and are deposited here for unrestricted public use under a CC0 1.0 Universal (Public Domain Dedication) license. No additional access restrictions apply. All files necessary to reproduce analyses are hosted on Dryad and do not require special software beyond those listed in the Code/software section.