DOI: 10.5061/dryad.m0cfxpp25
title: Phylogenomics of scorpions reveal contemporaneous diversification of scorpion mammalian predators and mammal-active sodium channel toxins

description: Scorpions constitute a charismatic lineage of arthropods and comprise more
 than 2,500 described species. Found throughout various tropical and
 temperate habitats, these predatory arachnids have a long evolutionary
 history, with a fossil record that began in the Silurian. While all
 scorpions are venomous, the asymmetrically diverse family Buthidae harbors
 nearly half the diversity of extant scorpions, and all but one of the 58
 species that are medically significant to humans. However, the lack of a
 densely sampled scorpion phylogeny has hindered broader inferences of the
 diversification dynamics of scorpion toxins. As redress, we assembled a
 phylogenomic dataset of 100 scorpion venom transcriptomes and/or genomes,
 emphasizing the sampling of highly toxic buthid genera. To infer
 divergence times of venom gene families, we applied a phylogenomic node
 dating approach for the species tree in tandem with phylostratigraphic
 bracketing to estimate minimum ages of mammal-specific toxins. Our
 analyses establish a robustly supported phylogeny of scorpions,
 particularly with regard to relationships between medically significant
 taxa. Analysis of venom gene families shows that mammal-specific sodium
 channel toxins have independently evolved in five lineages within
 Buthidae. Temporal windows of mammal-specific toxin origins are correlated
 with the basal diversification of major scorpion mammal predators such as
 carnivores, shrews, bats and rodents. These results suggest an
 evolutionary model of relatively recent diversification of buthid sodium
 channel toxin (NaTx) homologs in response to diversification of scorpion
 predators.
description: <em><strong>Phylogenomics and
 dating</strong></em> Scorpions were
 collected by hand in field theaters across Brazil, Egypt, Israel, and the
 US, commonly with the aid of ultraviolet lighting. Milking and dissection
 of venom glands, RNA extraction, and paired-end transcriptome sequencing
 was performed on the Illumina HiSeq 2500 platform for 42 species. New
 datasets were combined with 45 venom gland RNA-Seq datasets and one genome
 we previously generated. Orthologous loci were drawn from MCL clustering
 of 3564 orthogroups computed from a larger analysis of Chelicerata and
 outgroup taxa (Ballesteros and Sharma 2019). Three matrices were assembled
 with minimal taxon occupancy thresholds: Matrix 1 (at least 115 species),
 Matrix 2 (at least 109 species), and Matrix 3 (at least 103 species).
 Phylogenetic inference of these concatenated matrices was computed with
 IQ-TREE v. 1.6 (Nguyen et al. 2014) implementing the best-fitting amino
 acid substitution model per partition. Species trees were estimated using
 the coalescent method implemented in ASTRAL v.3 (Mirarab and Warnow 2015),
 using the collection of orthologous gene trees as inputs. Analysis of the
 smallest matrix (Matrix 3) was trialed using Phylobayes-mpi v. 1.7
 (Lartillot and Philippe 2004) with four independent chains under the CAT +
 GTR + G<sub>4</sub> model. Divergence time estimation was
 computed on Matrix 2 using the approximate likelihood calculation as
 implemented in Codeml and MCMCtree (both part of the PAML v. 4.8 software
 package (Yang 2007; dos Reis and Yang 2019).
 <em><strong>Toxin
 evolution</strong></em> Cysteine-stabilized
 a-helix and b-sheet fold (CSab), disulphide-directed beta-hairpin (DDH)
 and Inhibitor cystine knot (ICK) homologs from scorpion venom were
 retrieved from the complete dataset used in the scorpion phylogenetic
 analyses following our recent approaches (Santibáñez-López et al. 2018;
 Santibáñez-López et al. 2019b), and from UniProt. Gene trees were
 conducted using IQ-TREE for the entire dataset (1,353 CSab-ICK scorpion
 toxins, with 41 DDH scorpion toxins as outgroups), and for each of the
 four main clades recovered: (a) sodium channel toxins (NaTx); (b)
 potassium channel toxins (KTx); (c) chlorine channel toxins (ClTx); and
 (d) calcins. Comparative analyses between the subclades recovered within
 the NaTx included the search for repetitive motifs in their mature peptide
 using Multiple Em for Motif Elicitation (Bailey et al. 2015). The mature
 peptide of the two main clades within the NaTx (Aah2-like and Cn2-like)
 were separately analyzed using CLANS clustering (Frickey and Lupas 2004).
 Estimation of minimum ages for mammal-specific toxins was derived from
 molecular dating performed herein, using the ages of the most inclusive
 clades of taxa as minimum age estimates for gene age (phylostratigraphic
 bracketing). Divergence times for scorpion predators such Herpestidae
 (Carnivora), Chiroptera, Eulipotyphla and Rodentia were retrieved from a
 recent analysis of mammal diversification times (Upham et al. 2019) and
 compared against the mammal-specific toxin origins.