Date: 2023-06-25

Dataset Title: Supplementary Materials and Supporting Data for Srodawa et al. "Evolution of Three Finger Toxin Genes in Neotropical Colubrine Snakes (Colubridae)"

Dataset Creators: K. Srodawa, P.A. Cerda, A.R. Davis Rabosky, and J.M. Crowe-Riddell

Dataset Contact: P.A. Cerda pacerda@umich.edu

Funding: American Museum of Natural History, University of Michigan Ecology and Evolutionary Biology Department, University of Michigan Museum of Zoology, University of Michigan Rackham Graduate School, University of Michigan

Key Points: 
    - Historically understudied rear-fanged snakes possess venoms that are comprised of highly toxic neurotoxin.
    - Parallel evolution of heterodimeric 3FTx

Research Overview: Snake venom research has historically focused on front-fanged species (Viperidae and Elapidae), limiting our knowledge of venom evolution in rear-fanged snakes across their ecologically-diverse phylogeny. Three finger toxins (3FTxs) are a known neurotoxic component in the venoms of some rear-fanged snakes (Colubrinae, Colubridae), but it is unclear how prevalent 3FTxs are both in expression within venom glands and more broadly among colubrine species. Here, we used a transcriptomic approach to characterize the venom expression profiles of four species of colubrine snakes from Neotropics that were dominated by 3FTx expression (in the genera Chironius, Oxybelis, Rhinobothryum, and Spilotes) and reconstructed the gene trees of three finger toxins. We found evidence of the parallel evolution of heterodimeric three finger toxins and evidence of strong positive selection. Our results highlight the importance of exploring the venoms of understudied species in reconstructing the full evolutionary history of toxins across the tree of life.

Methodology: We used a transcriptomic approach to determine the venom gland transcriptome for five species of neotropical rear-fanged snakes. We sequenced extracted RNA on an Illumina NovaSeq 6000 and assembled the transcriptome using Trinity. We then annotated the transcriptome by identifying expressed sequences. We also estimated the abundance of each toxin sequence. We constructed a Bayesian and a RAxML gene tree using a protein alignment of three finger toxins identified in this study and gathered from NCBI GenBank.

Files contained here:
This deposit consists of two compressed folders: a data folder (Data.zip) and a supplementary Materials folder (supplementaryMaterials.zip)

- Data: contains supporting data for related publication (see citation below)
    - fig3.R: R script (Written in version 4.2.2 on a Mac operating system) that visualizes figure 3 and supplemental figure 1. Uses the following packages
        -phytools v1.2-0
        -ape v5.2-6
        -nationalparkcolors v0.1.0 (instructions to install found here: https://github.com/katiejolly/nationalparkcolors)
    - 3FTx_Bayesian.tre: tree file generated from Bayesian phylogenetic reconstruction methods
    - 3FTx_Protein_Alignment.fasta: Amino acid alignment of 3 Finger toxin sequences from species surveyed in associated publication (sequence names ending with 3FTx-#) and sequences gathered from GenBank (sequencing names ending with genBank accession numbers). 
    - 3FTx_RAxML.tre: tree file generated from RAxML phylogenetic reconstruction methods

- supplementaryMaterials: Contains supplemental materials for related publications (see citation below). Naming scheme is as follows: [TableNumber]_[TaxonID][dataFileContent].csv.

    TaxonID is as follows: Chmu3332 = Chironius multiventris A, Chmu3577 = Chironius multiventris B, Oxae3102 = Oxybelis aeneus, Rhbo3018 = Rhinobothryum bovallii, Spsu0562 = Spilotes sulphureus.

    The following files describe the toxin annotation from each individual and contain the following variables: taxonID; seqID=Name gived from assembler; log2FPKM+1 = log scale value for FPKM (Fragments per million fragments); accessionNumber = genBank accession number; blastHit = full name of blast sequence; blastSpecies = species of blast hit; gene = gene name of blast hit; geneFamily = Toxin gene family of blast hit; eValue = support value for blast hit; TPM = Transcripts per million (proxy for normalized gene expression); FPKM = Fragments pre million fragments (proxy for gene expression); transcript_rank = rank value for sequence

        - TableS1_Chmu3332TranscriptAnnotation.csv
        - TableS2_Chmu3577TranscriptAnnotation.csv
        - TableS3_Oxae3102TranscriptAnnotation.csv
        - TableS4_Rhbo3018TranscriptAnnotation.csv
        - TableS5_Spsu0562TranscriptAnnotation.csv
    
    The following files describe the toxin gene family abundance and percentage recovered from each individual and contain the following variables: geneFamily = toxin family; total_TPM = sum of TPM values for each sequence of the respective toxin family; percent_TPM = total_TPM / sum of total_TPM * 100

        - TableS6_Chmu3332ToxinTotals.csv
        - TableS7_Chmu3577ToxinTotals.csv
        - TableS8_Oxae3102ToxinTotals.csv
        - TableS9_Rhbo3018ToxinTotals.csv
        - TableS10_Spsu0562ToxinTotals.csv
    
    Table S11 contains the codon locations where each selection test found signitures of positive or negative selection. 
       - TableS11_selectionTestSummary.xlsx
            FUBAR =  Fast, Unconstrained Bayesian AppRoximation for Inferring Selection
            MEME = Mixed Effects Model of Evolution
            M2 = Codml M2 model of selection
            Nterm start/end = codon sites where the N-terminus region starts and ends
            Loop start and end codons are signified by the following schema: L(Loop)RomanNumeral(I-V)Start/End(s or e)
                Example: LIe (Loop one end) or LVs (Loop five start)

    
    TableS12 contains the species names and genBank accession numbers for 3FTx sequences recovered from NCBI genBank.
        - TableS12_genbankAccessionNumbers.csv

    FigureS1 displays the RAxML tree visualized in Data/fig3.R
        - FigureS1.pdf

Related Publication(s):
Srodawa, K., Cerda, P.A., Davis Rabosky, A.R., Crowe-Riddell, J.M. Evolution of Three Finger Toxin Genes in Neotropical Colubrine Snakes (Colubridae). Forthcoming

Use and Access:
This data set is made available under a Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/.

To Cite Data: 
Srodawa, K., Cerda, P. A., Davis Rabosky, A. R., Crowe-Riddell, J. M. Supplementary Materials and Supporting Data for Srodawa et al. "Evolution of Three Finger Toxin Genes in Neotropical Colubrine Snakes (Colubridae)" [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/01w7-8d39