The evolutionary history of chelicerate metallothioneins reveals de novo emergence and metal-binding specialization across the subphylum.

Metallothioneins (MTs) are a diverse family of proteins involved in metal homeostasis and detoxification, enabling organisms to adapt to varying metal availability across ecosystems. While extensively studied in vertebrates and gastropods, MTs remain poorly characterized in many arthropods, particularly chelicerates. Identifying and characterizing chelicerate MTs is therefore key to deciphering how this diverse group of animals -which includes spiders, scorpions, and ticks- copes with metal-related challenges in different environments. In this study, we have identified over 450 MTs from 221 chelicerate species and classified them into three structurally and phylogenetically distinct types: MT1, MT2, and MT3. Bidomain MTs are the most widespread across chelicerate lineages, while short monodomain forms and large variants occur in specific groups. Metal-binding characterization of eight representative MTs have revealed diverse metal selectivity -including Zn-, Cd-, and Cu-thioneins, as well as multipurpose forms- and biding capacity, ranging from 3 to 13 divalent ions per molecule. Evolutionary analyses suggest that MT1s are ancestral, MT2s appeared in Euchelicerates, and MT3s likely emerged in spiders. These findings establish a framework for understanding the MT diversity of chelicerates and reveal functional adaptations of potential evolutionary and ecological relevance, particularly those related to their ability to inhabit ecosystems with widely varying metal availability.