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A large fraction of the noxious components of spider venoms are a functionally diverse family of structurally related polypeptides with an inhibitor cystine knot (ICK) motif. The cysteine-rich nature of these toxins makes structural elucidation difficult, and most studies have focused on venom components from the small handful of medically relevant spider species such as the highly aggressive Brazilian wandering spider Phoneutria nigriventer. To alleviate difficulties associated with the study of ICK toxins in spiders, we devised a comprehensive approach to explore the evolutionary patterns that have shaped ICK functional diversification using venom gland transcriptomes and proteomes from phylogenetically distinct lineages of wandering spiders and their close relatives. We identified 626 unique ICK toxins belonging to seven topological elaborations. Phylogenetic tests of episodic diversification revealed distinct regions between cysteine residues that demonstrated differential evidence of positive or negative selection, which may have structural implications towards the specificity and efficacy of these toxins. Increased taxon sampling and whole genome sequencing will provide invaluable insights to further understand the evolutionary processes that have given rise to this diverse class of toxins.<\/jats:p>","DOI":"10.3390\/toxins15020112","type":"journal-article","created":{"date-parts":[[2023,1,30]],"date-time":"2023-01-30T01:33:37Z","timestamp":1675042417000},"page":"112","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Killer Knots: Molecular Evolution of Inhibitor Cystine Knot Toxins in Wandering Spiders (Araneae: Ctenidae)"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1535-6929","authenticated-orcid":false,"given":"Michael S.","family":"Brewer","sequence":"first","affiliation":[{"name":"Department of Biology, East Carolina University, Greenville, NC 27858, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"T. Jeffrey","family":"Cole","sequence":"additional","affiliation":[{"name":"Department of Biology, East Carolina University, Greenville, NC 27858, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.toxicon.2018.09.004","article-title":"Entomo-venomics: The evolution, biology and biochemistry of insect venoms","volume":"154","author":"Walker","year":"2018","journal-title":"Toxicon"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Sunagar, K., and Moran, Y. (2015). The rise and fall of an evolutionary innovation: Contrasting strategies of venom evolution in ancient and young animals. 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