Venom Mass Fingerprints for Species Delimitation, Reloaded: A Test Case with Italian Scorpions (Euscorpius)

 

Venom Mass Fingerprints for Species Delimitation, Reloaded: A Test Case with Italian Scorpions (Euscorpius)

Abstract

Scorpion venoms are complex mixtures of bioactive compounds including proteins, peptides and enzymes. Although venoms exhibit substantial intraspecific variation, some compounds appear to be species-specific, highlighting their potential as chemotaxonomic markers. Venom mass fingerprints (MFPs) derived from Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) have been proposed as a means of delimiting species. However, their utility for species delimitation has rarely been evaluated against independent sources of taxonomic evidence. In the present study, the performance of MALDI-TOF MS-based venom peptide barcoding (mass ranges of m/z 800‒4500 and m/z 3000‒10,000) was assessed in 52 populations of eight Italian species of the scorpion genus Euscorpius Thorell, 1876, representing different levels of evolutionary divergence, by comparing venom MFPs with mitochondrial Cytochrome c Oxidase Subunit I (COI) DNA barcodes. Non-metric multidimensional scaling (NMDS) based on Jaccard distances of 52 venom MFPs (one per population) recovered seven distinct, statistically supported species clusters that were mostly congruent with analyses based on COI. Hierarchical clustering analyses using the intersection-based distances of the same MFPs consistently recovered the two major Euscorpius species complexes and revealed geographically structured subclades congruent with COI data. Venom profiles successfully discriminated among both closely and distantly related species in similarity-based analyses, recovering patterns broadly consistent with established phylogenetic hypotheses. Phylogenetic reconstruction with Maximum Likelihood, based solely on venom characters, obtained less resolution at deeper evolutionary levels, with lower nodal support and some incongruence with the topology obtained from the DNA sequences. The results demonstrate that venom MFPs constitute a useful source of data for species delimitation, particularly for resolving cryptic species complexes and morphologically conserved taxa, complementing morphological and DNA sequence data in integrative taxonomy. Broader taxon sampling, together with improved signal detection and character selection (filtering ion signals), may increase the utility of venom MFPs for phylogenetic analysis by increasing the number of phylogenetically informative characters.

Blasco-Aróstegui, J., Roppo, M., Prendini, L., & Predel, R. (2026). Venom Mass Fingerprints for Species Delimitation, Reloaded: A Test Case with Italian Scorpions (Euscorpius). Toxicon: X, 100265. https://doi.org/10.1016/j.toxcx.2026.100265