DOI: 10.6084/m9.figshare.710637.v1
title: Phylogenetic analyses of the vertebrate voltage-gated calcium channel L-type alpha 1 subunit gene family

description: Sequence based phylogenetic analyses of vertebrate voltage-gated calcium channel alpha 1 subunits (CACNA1) of L-type - CACNA1S, CACNA1C, CACNA1D and CACNA1F - using amino acid sequences predicted from the Ensembl (http://www.ensembl.org) genome browser. Species and genome assembly information, database identifiers, location data and annotation notes for all identified sequences are included in the Excel workbook 'Supplementary Table CACNA1L.xlsx'. <strong>File information:</strong> Species included in these analyses, with abbreviations: human (<em>Homo sapiens</em>, Hsa), mouse (<em>Mus musculus</em>, Mmu), grey short-tailed opossum (<em>Monodelphis domestica</em>, Mdo), chicken (<em>Gallus gallus</em>, Gga), Carolina anole lizard (<em>Anolis carolinensis</em>, Aca), zebrafish <em>(Danio rerio</em>, Dre), three-spined stickleback (<em>Gasterosteus aculeatus</em>, Gac), medaka (<em>Oryzias latipes</em>, Ola), green spotted pufferfish (<em>Tetraodon nigroviridis</em>, Tni), transparent sea squirt (<em>Ciona intestinalis</em>, Cin) and fruit fly (<em>Drosophila melanogaster</em>, Dme). Alignment file included in FASTA-format: 'align_CACNA1L.fasta'. This file format can be opened by most sequence analysis applications as well as text editors. The alignment was created using ClustalX 2.0.12 (http://www.clustal.org/clustal2/) with standard settings (Gonnet weight matrix, gap opening penalty 10.0 and gap extension penalty 0.20). For short, incomplete or diverging gene predictions in Ensembl, the nucleotide sequence including the gene prediction (with introns) as well as the flanking sequence was collected and the Genscan gene prediction server (http://genes.mit.edu/GENSCAN.html) was used to identify exons that had not been predicted. Sequences that were still divergent with regard to exon-intron boundaries were curated manually by following consensus for splice donor and acceptor sites as well as sequence homology to other family members. Remaining highly divergent, non-alignable, regions were removed from the final alignment. The alignment was edited using the BioEdit Sequence Alignment Editor (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Phylogenetic tree files are included in Phylip/Newick format with the extension '.phb'. This file format can be opened by freely available phylogenetic tree viewers such as FigTree (http://tree.bio.ed.ac.uk/software/figtree/) and TreeView (http://darwin.zoology.gla.ac.uk/~rpage/treeviewx/). All trees were made using the alignment described above. Corresponding figures for each phylogenetic tree are also included as PDF-files. The neighbor joining (NJ) tree, 'NJ_tree_CACNA1L.phb', was made using standard settings in ClustalX 2.0 (http://www.clustal.org/clustal2/), supported by a non-parametric bootstrap analysis with 1000 replicates. The Phylogenetic Maximum Likelihood (PhyML) tree, 'PhyML_tree_CACNA1L.phb', was made using the PhyML3.0 algorithm (http://www.atgc-montpellier.fr/phyml/‎) with the following settings: amino acid frequencies (equilibrium frequencies), proportion of invariable sites (with optimised p-invar) and gamma shape parameters were estimated from the alignments, the number of substitution rate categories was set to 8, BIONJ was chosen to create the starting tree, both NNI and SPR tree optimization methods were considered and both tree topology and branch length optimization were chosen. The JTT model of amino acid substitution was chosen using ProtTest 3.0 (https://bitbucket.org/diegodl/prottest3/downloads). The tree is supported by a non-parametric bootstrap analysis with 100 replicates. Both trees are rooted with the fruit fly <em>Ca-α1D</em> sequence.