Ancient origin and dynamic evolution of bivalent spider toxins.

Bivalent peptide toxins comprising 2 cysteine-rich domains have evolved from single-domain precursors on multiple occasions in animal venoms, resulting in enhanced molecular target selectivity and avidity. Although bivalent toxins are emerging as prevalent in animal venoms, the genomic and evolutionary processes driving the transitions between single- and multi-domain architectures remain poorly understood. Here, we investigated the evolution of bivalent inhibitor cystine knot (ICK) toxins in spider venom. We first generated a genome assembly of the tree-dwelling funnel-web spider Hadronyche cerberea, revealing a massive expansion of ICK toxin-encoding genes, including the bivalent π-hexatoxin-Hc1a. All ICK toxin genes share a conserved 3-exon structure, flanked by transposable elements (TEs) that may have facilitated gene expansion. This gene structure is shared by the Hc1a subfamily, where the entire mature bivalent toxin is encoded by the third exon. Leveraging de novo transcriptome assemblies from 86 spider species along with venom proteomic data, we show that bivalency in the Hc1a subfamily is of ancient origin and evolved via intra-exonic duplication not involving introns. This was followed by domain expansion and recurrent domain losses mediated by point mutations, deletions, and unequal crossing-over facilitated by high interdomain sequence similarity. In contrast, the bivalent toxin DkTx from Cyriopagopus schmidti is confined to a small group of tarantulas, where it appears to have evolved once, with subsequent domain losses potentially linked to TE activity. Our findings reveal that singular events of domain duplication can give rise to complex, asymmetrical evolutionary trajectories shaped by gene instability and selective retention of functional domains.