Editor's evaluation: The deep-rooted origin of disulfide-rich spider venom toxins

Abstract

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Spider venoms are a complex concoction of enzymes, polyamines, inorganic salts, and disulfide-rich peptides (DRPs). Although DRPs are widely distributed and abundant, their bevolutionary origin has remained elusive. This knowledge gap stems from the extensive molecular divergence of DRPs and a lack of sequence and structural data from diverse lineages. By evaluating DRPs under a comprehensive phylogenetic, structural and evolutionary framework, we have not only identified 78 novel spider toxin superfamilies but also provided the first evidence for their common origin. We trace the origin of these toxin superfamilies to a primordial knot – which we name ‘Adi Shakti’, after the creator of the Universe according to Hindu mythology – 375 MYA in the common ancestor of Araneomorphae and Mygalomorphae. As the lineages under evaluation constitute nearly 60% of extant spiders, our findings provide fascinating insights into the early evolution and diversification of the spider venom arsenal. Reliance on a single molecular toxin scaffold by nearly all spiders is in complete contrast to most other venomous animals that have recruited into their venoms diverse toxins with independent origins. By comparatively evaluating the molecular evolutionary histories of araneomorph and mygalomorph spider venom toxins, we highlight their contrasting evolutionary diversification rates. Our results also suggest that venom deployment (e.g. prey capture or self-defense) influences evolutionary diversification of DRP toxin superfamilies. Editor's evaluation This is an important survey of disulfide-rich peptides (DRPs), which comprise a large fraction of the most functionally important components of spider venom. While spider DRPs were thought to have evolved independently numerous times throughout the spider tree of life, the authors make a solid case for the idea that they all stem from a single common ancestral protein. The study makes a significant advance towards formalizing the diversity of spider venoms, which will be of interest both to scientists working on protein evolution and to those working on functional venomics. https://doi.org/10.7554/eLife.83761.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest The majority of spiders rely on their venom to defend themselves, to hunt, or both. Armed with this formidable weapon, they have managed to conquer every continent besides Antarctica since they first emerged about 495 million years ago. A closer look at spider venoms hints at an intriguing evolutionary history which has been rarely examined so far. The venom of other animals, such as snakes or scorpions, is usually formed of a wide range of unrelated toxins; in contrast, spiders rely on a single class of proteins, known as disulfide-rich peptides, to create their deadly venom cocktail. This family of molecules is impressively diverse, with each peptide having a distinct structure and mode of action. Its origins, however, have remained elusive. To fill this knowledge gap, Shaikh and Sunagar scanned the sequences of all disulfide-rich peptides generated to date, bringing together a dataset that includes 60% of all modern-day spiders. The analyses allowed the identification of 78 new superfamilies of spider toxins. They also revealed that all existing peptides originate from a single molecule, which Shaikh and Sunagar named after the powerful Hindu goddess Adi Shakti. This ancestral toxin was present 375 million years ago in the last common ancestor of modern-day spiders. The work also highlighted that disulfide-rich peptides evolved under different pressures in various groups of spiders; this may be because some species primarily use their venom for hunting, and others for defence. While the ‘hunters’ may need to constantly acquire toxins with new roles and structures to keep their edge over their prey, those that rely on venom to protect themselves may instead benefit from relying on tried-and-tested toxins useful against a range of infrequent predators. Finally, the analyses revealed that the disulphide-rich peptides of Mygalomorphae tarantulas, which form one of the three major groups of spiders, are much more diverse than the related toxins in other spiders. The underlying reason for this difference is still unclear. Several life-saving drugs currently on the market are based on toxins first identified in the venoms of snakes, cone sails or lizards. Similar discoveries could be unlocked by better understanding the range of deadly molecules used by spiders, and how these came to be. Introduction With their killer instinct and deadly toxins, spiders have been at the centre of many myths and folktales from times immemorial. They are an archetypal arthropod group with mid-Cambrian or early Ordovician origin, nearly 495 million years ago (MYA; Lozano-Fernandez et al., 2016). Because of their unique ability to secrete silk and venom, spiders have successfully colonised diverse ecological niches. They are amongst the most successful predators on the planet, with over 50,000 species and 129 families described to date (King, 2004; WSC, 2022). The majority of spiders are equipped with chelicerae harbouring venom glands, with Symphytognathidae, Uloboridae, and certain Mesothelae species being the only exceptions (King, 2004; Mullen and Vetter, 2019). Spider venoms are a concoction of enzymes, polyamines, nucleic acids, inorganic salts and disulfide-rich peptides (DRPs) (Senji Laxme et al., 2019b; King and Hardy, 2013). They are predominantly rich in DRPs that are characterised by a diversity of structural motifs, including Kunitz (Yuan et al., 2008), disulfide-directed β-hairpin (Wang et al., 2000), disulfide-stabilised antiparallel β-hairpin stack (DABS; Pineda et al., 2020) and inhibitor cystine knot (ICK) – also known as knottins (Pallaghy et al., 1994; Undheim et al., 2016). Despite the fact that DRPs constitute three-quarters of spider venom, our evolutionary understanding of their origin and diversification has remained elusive. This knowledge gap stems from a lack of sequence and structural data for DRPs from diverse spider lineages and the prevalence of significant sequence divergence in these toxins. Here, we examined DRP sequences from the spiders of the Mygalomorphae infraorder (includes funnel-web spiders and tarantulas), family Theridiidae (includes the red-back spiders), and the Retrolateral Tibial Apophysis (RTA) clade from Araneomorphae, which constitutes over 58% of spider genera (2527 genera) described to date (Figure 1A). A molecular phylogenetic framework implemented in this study resulted in the identification of 78 novel toxin superfamilies and suggests a deep-rooted origin of venom DRPs in spiders. Our findings also highlight the role of distinct prey capture strategies of Araneomorphae and Mygalomorphae in shaping the recruitment and diversification of venom DRPs. Furthermore, by comparatively evaluating spider venom toxins employed for anti-predatory defense and prey capture, we also unravel the impact of the purpose of venom deployment on the evolution of spider venoms. Thus, sequence, phylogenetic, structural and evolutionary assessments in this study have provided insights into the fascinating origin and early diversification of this predominant spider venom component. Figure 1 Download asset Open asset Schematic representation of Araneae phylogeny and their venom superfamilies. Panel A here shows a cladogram of Araneae with lineages under investigation indicated in red. In panel B, representative signal peptide alignments of toxin superfamilies are shown with sequence conservation of >90% highlighted in blue. Results Novel spider toxin superfamilies Superfamilies (SF) of venom toxins in spiders have been classified based on their signal peptide and propeptide sequences (Pineda et al., 2014). This premise was first used to describe the Shiva superfamily of toxins from Atracidae spiders (Pineda et al., 2014). Recently, using a similar approach, 33 novel spider toxin superfamilies have been identified from the venom of the Australian funnel-web spider, Hadronyche infensa (Pineda et al., 2020). Since gene phylogenies have not been extensively utilised while classifying spider venom toxins, our understanding of their origin and diversification has been severely limited. In this study, we relied on the strong conservation of signal peptide and propeptide regions in identifying several novel spider venom toxin superfamilies, following the same strategy as [Figure 1B; Pineda et al., 2020; Pineda et al., 2014]. Blast searches were used to identify the homology between largely divergent toxin superfamilies. Toxin sequences were found to share strong sequence conservation within a superfamily. Cysteine residues, which are involved in the formation of disulphide bonds and, thereby, are extremely vital in determining protein structure and function, were used as guides to manually refine sequence alignments. This approach enabled the identification of 33 novel toxin superfamilies along the breadth of Mygalomorphae (Figure 2—figure supplements 2 and 3). Among these, 31 superfamilies belonged to the DRP class, whereas the other two were enzymatic non-DRP toxins, including the first report of Neprilysin (SF103) and CAP (CRiSP/Allergen/PR-1; SF104) from Atracidae spiders (Figure 4—source data 1). Moreover, analyses of Araneomorphae toxin sequences using the strategy above resulted in the identification of 45 novel toxin superfamilies from Araneomorphae, all of which but one (SF109) belonged to the DRP class of toxins (Figure 3—figure supplements 2 and 3). Overall, among all novel spider toxin superfamilies identified in this study, the majority (n=75) were DRPs, reinstating the dominance of this toxin type in spider venoms. Based on the arrangement of cysteine residues involved in the formation of disulphide bonds, these DRPs could be further segregated into ICK-like (n=28), DABS (n=13) and novel disulphide patterned non-ICK (n=34) superfamilies (Pineda et al., 2020). We named these novel spider toxin superfamilies after deities of death, destruction, and the underworld (Supplementary file 1), following a nomenclature system introduced by Pineda et al., 2014. The identification of novel toxin superfamilies was further supported by phylogenetic and principal component analyses. Reconstruction of evolutionary histories using Bayesian inference (BI) and maximum-likelihood (ML) approaches retrieved monophyletic groups of toxin superfamilies (Figures 2 and 3; node support: ML:>90/100; BI:>0.95; refer to figure supplement provided for complete phylogeny with branch lengths). Interestingly, the plesiotypic DRP scaffold seems to have undergone lineage-specific diversification in Mygalomorphae, where the selective diversification of the scaffold has led to the origination of novel toxin superfamilies corresponding to each Linnaean genus (Figure 2). In our Bayesian and maximum-likelihood phylogenetic tree reconstructions, these toxin scaffolds were found to form distinct clades, further supporting this claim (Figure 2—figure supplement 1; node support: ML: ML:>90/100; BI:>0.95). Figure 2 with 4 supplements see all Download asset Open asset The Bayesian phylogeny of mygalomorph spider venom toxin superfamilies. This figure represents the Bayesian phylogeny of Mygalomorphae spider toxin superfamilies, where branches with high (BPP >0.95) and low (BPP <0.95) node supports are shown in thick black and thin grey lines, respectively. Coloured spheres alongside tree tips represent the spider genera, while the coloured outer circle indicates the spider family in which the respective toxin superfamily has been identified (Atracidae [red], Barychelidae [orange], and Theraphosidae [blue]). Figure 2—source data 1 This zip archive contains sequence alignment used to perform phylogenetic analyses and the Bayesian phylogeny of Mygalomorphae DRP toxin superfamilies. https://cdn.elifesciences.org/articles/83761/elife-83761-fig2-data1-v2.zip Download elife-83761-fig2-data1-v2.zip Figure 3 with 4 supplements see all Download asset Open asset The Bayesian phylogeny of araneomorph spider venom toxin superfamilies. This figure represents the Bayesian phylogeny of Araneomorphae spider toxin superfamilies, where branches with high (BPP >0.95) and low (BPP <0.95) node supports are shown in thick black and thin grey lines, respectively. Coloured spheres, alongside tree tips, represent the spider genera, while the coloured outer circle indicates the spider family (Agelenidae [red], Ctenidae [green], multiple araneomorph families [purple]: Ctenidae, Gnaphosidae, Homalonychidae, Lycosidae, Oxyopidae, Pisauridae, Psechridae, Salticidae, Thomisidae, Xenoctenidae) in which the respective toxin superfamily has been identified. Figure 3—source data 1 This zip archive contains sequence alignment used to perform phylogenetic analyses and the Bayesian phylogeny of Araneomorphae DRP toxin superfamilies. https://cdn.elifesciences.org/articles/83761/elife-83761-fig3-data1-v2.zip Download elife-83761-fig3-data1-v2.zip A similar pattern was also observed in the case of Araneomorphae, where certain toxin SFs (n=12) were found to have diversified within individual genera, corresponding to the Linnaean taxonomy (Figure 3). However, we also documented a large number of DRP toxins (n=32) that were found to have diversified in a family-specific manner, wherein, a toxin scaffold seems to have a more ancient recruitment, corresponding to the Linnean family, rather than the Linnaean genus. As a result, and in contrast to mygalomorph DRPs, araneomorph toxin superfamilies were found to be scattered across spider lineages (Figure 3; Figure 3—figure supplement 1; node support: ML:>90/100; BI:>0.95). Moreover, Principal component analysis (PCA) of toxin sequences further provided evidence for the monophyly of mygalomorph and araneomorph SFs, where each toxin superfamily formed a distinct group in PCA plots (Figure 2—figure supplement 4; Figure 3—figure supplement 4). Furthermore, sequence alignments of DRPs clearly highlighted the homology among DRP toxin superfamilies (Figure 4; Figure 4—figure supplement 1; node support: ML:>90/100; BI:>0.95). Six cysteine residues were found to be nearly universally conserved across 101 DRP toxin SFs (Figure 4B; Figure 4—figure supplement 2). Our findings enabled us to trace the origin of spider venom DRPs in Opisthothelae, the clade that encompasses Araneomorphae and Mygalomorphae (Magalhaes et al., 2020). Thus, we highlight for the first time that all DRP toxins in spiders may have had a common molecular origin, nearly 375 MYA. It should be noted, however, that functional analyses have been performed only on a handful of mygalomorph toxins, with even fewer studies focusing on araneomorph toxin superfamilies, and that it would be inaccurate to speculate on the functions of these toxins based on homology. Figure 4 with 2 supplements see all Download asset Open asset The Bayesian phylogeny and cysteine framework representation of spider venom DRPs. This figure depicts the Bayesian phylogeny and alignment of representative sequences of Araneae DRP toxin superfamilies, where branches with high (BPP >0.95) and low (BPP <0.95) node supports are shown in thick black and thin grey lines, respectively. The coloured outer circle in panel A indicates the infraorder of spiders (Mygalomorphae and Araneomorphae shown in dark and light brown, respectively) in which the respective DRP superfamily was identified. In panel B, cysteine framework conserved across toxin SFs is highlighted in blue. Figure 4—source data 1 This zip archive contains sequence alignment used to perform phylogenetic analyses and the Bayesian phylogeny of Araneae DRP toxin superfamilies. A list of accession IDs for sequence data utilised in this study has also been provided. https://cdn.elifesciences.org/articles/83761/elife-83761-fig4-data1-v2.zip Download elife-83761-fig4-data1-v2.zip Molecular evolution of spider venom DRP toxins To evaluate the nature and strength of the selection that has shaped spider venom DRPs, we employed site-specific models that detect selection across sites. Our findings suggest that the majority of Mygalomorphae toxin superfamilies (12/19 SFs) have evolved under the influence of positive selection (ω ranging between 1.1 and 2.9; positively selected sites [PS]: 0–26), while the remaining few have experienced negative or purifying selection (ω ranging between 0.7 and 0.8; PS: 0–13; Figure 5, Figure 5—source data 1). In stark contrast, nearly all of the Araneomorph toxin superfamilies that we investigated here were found to have evolved under a strong influence of negative selection (ω ranging between 0.2 and 1.0; PS: 0–10; Figure 5, Figure 5—source data 1). We further assessed whether these changes documented across sites have a significant effect on the biochemical and structural properties of amino acids using TreeSAAP (Figure 5—source data 1). Outcomes of these analyses revealed the accumulation of replacement changes in Mygalomorphae toxin superfamilies that result in radical shifts in amino acid properties, potentially influencing their structure and function (Figure 5—source data 1). Figure 5 with 1 supplement see all Download asset Open asset Molecular evolution of spider toxin superfamilies. This figure shows the distribution of ω values (Y-axis) for araneomorph and mygalomorph spider venom toxin superfamilies (X-axis). The horizontal dotted black line represents neutral evolution (ω=1), with ω values above and below it indicating positive (ω>1) and negative (ω<1) selection, respectively. Figure 5—source data 1 Molecular evolution of toxin superfamilies. (a): Positively selected sites detected by the Bayes Empirical Bayes approach implemented in M8; (b): Fast Unconstrained Bayesian AppRoximation (c): Sites detected as experiencing episodic diversifying selection (0.05 significance) by the Mixed Effects Model Evolution (MEME). Sites detected at 0.99 and 0.95 significance are indicated in the parenthesis; (d): number of sites under pervasive diversifying selection at the posterior probability ≥0.9 (FUBAR); (e): Number of sites under pervasive purifying selection at the posterior probability ≥0.9 (FUBAR); ω: mean dN/dS. Biochemical properties evaluated: Equilibrium Const. – ionisation, COOH (pK); Hydropathy (h); Long-range n.b. energy (El); Polarity (p); Total n.b. energy (Et). Structural properties: α−helical tendencies (Pα); β-structure tendencies (Pβ); Average # surrounding residues (Ns); Bulkiness (Bl); Chromatographic index (RF); Coil tendencies (Pc); Compressibility (K°); Helical contact energy (Ca); Mean r.m.s. fluctuation displacement. (F); Molecular volume (Mv); Molecular weight (Mw); Partial specific volume (V°); Polar requirement (Pr); Power to be – C-term. α-helix (αc); Power to be – middle, α-helix (αm); Power to be – N-term., α-helix (αn); Refractive index (µ); Solvent accessible reduct. ratio (Ra); Thermodynamics transfer hydrophobicity (Ht). https://cdn.elifesciences.org/articles/83761/elife-83761-fig5-data1-v2.zip Download elife-83761-fig5-data1-v2.zip To comparatively evaluate the nature of selection that shapes venom components deployed either for prey capture or antipredator defence, we employed maximum-likelihood and Bayesian approaches. In these analyses, we identified toxin superfamilies SF74, SF77, SF79, SF89, SF90, SF92, and SF99 as predatory toxins (i.e. toxins deployed for prey capture – refer to the discussion section for the principle considered for this classification), whereas SF13 (i.e. Ares SF) was classified as a defensive spider venom toxin superfamily (i.e. toxins deployed for antipredator defence) as described previously (Herzig et al., 2020). Assessment of molecular evolutionary regimes identified a significant influence of positive selection on venom toxins that are employed for prey capture (ω ranging between 1.2 and 2.9; PS: 0–11, Figure 5—source data 1, Figure 5—figure supplement 1), relative to those that are chiefly or exclusively used for antipredatory defence (ω=0.8; PS: 3; Figure 5—source data 1, Figure 5—figure supplement 1). Discussion The deep evolutionary origin and diversification of the primordial knot Prior attempts to explore the phylogenetic and evolutionary histories of spider venom DRPs have hypothesised independent origin and lineage-specific diversification of DRP venom toxins (Rodríguez de la Vega, 2005). In contrast, recent literature, primarily focusing on Hadronyche infensa, suggests that the diverse disulfide-rich venom arsenal of this Australian funnel-web spider is a derivative of an ancestral ICK motif that underwent several rounds of duplication and diversification (Pineda et al., 2020). Often restricted to a specific spider lineage, or given the inconsistent ways of classifying spider venom toxins, previous attempts have failed to provide a broader perspective on the evolution of these peptides (Ferrat and Darbon, 2005; Chen et al., 2008). Given their very long evolutionary histories, genes encoding DRP toxins have undergone significant diversification, making it difficult to precisely trace their phylogenies. Together with the lack of structural and functional data for these toxins, all of the aforementioned factors have impeded our understanding of the origin and evolution of this predominant spider venom component. To address this knowledge gap, we employed sequence comparisons, phylogenetic inferences and evolutionary analyses, which enabled the identification of 76 novel spider venom toxin superfamilies (45 from Araneomorphae [44 DRP and 1 non-DRP] and 33 from Mygalomorphae spiders [31 DRP and 2 non-DRP]). Our findings strongly suggest a deep-rooted origin of DRP spider venom toxin superfamilies (Figure 4A), possibly from a single ancestral DRP or knottin scaffold, which we name ‘Adi Shakti’, after the original creator of the universe according to Hindu mythology. We propose that all of the extant spider disulfide-rich peptide toxin superfamilies (n=101) in Mygalomorphae and Araneomorphae, which include those that were previously reported (n=26), as well as the ones identified in the present study (n=75), have originated from this ‘primordial knot’, further undergoing lineage-specific gene duplication and diversification (Figures 2—4). The origin and diversification of these superfamilies can be explained by a mechanism that is similar to the combinatorial peptide strategy, wherein certain venomous animals, such as cone snails, generate a remarkable diversity in their mature toxin peptides while preserving the signal and propeptide regions (Escoubas and Rash, 2004; Zhu et al., 2000; Olivera et al., 1995). Rapid events of diversification, preceded by repeated rounds of gene duplication, form the basis of the combinatorial peptide library strategy (Sollod et al., 2005). These hyper-mutational events have been previously shown to be restricted to the mature peptide regions of toxins (Conticello et al., 2001). In contrast, the signal and propeptide regions, which are vital for the precise secretion and folding of proteins, respectively, evolve under the strong influence of negative selection pressures (Duda and Palumbi, 1999) - a molecular evolutionary trend also reported in venom coding genes of snakes (Brust et al., 2013). Spider venom coding genes appear to have followed a similar strategy. However, unlike the cone snail venom coding genes that have a recent evolutionary origin (<35–50 MYA; Olivera, 1997; Duda and Kohn, 2005), spider venom toxins have likely originated from an ancestral scaffold in Opisthothelae, the clade that encompasses Mygalomorphae and Araneomorphae spiders (over 99% of all spider genera described to date), nearly 375 MYA (Magalhaes et al., 2020). Given their significant sequence divergence since their deep-rooted evolutionary origin, the entire protein-coding gene, including the signal and propeptide regions, has accumulated significant differences. Consistent with this hypothesis, the majority of positively selected sites (~96%) identified in spider venom DRP toxins (all sites in Araneomorphae, and all but two sites in Mygalomorphae) were restricted to the mature peptide region, whereas the signal and propeptide regions harboured a minor proportion of these sites (1% and 3%, respectively; Figure 5—source data 1). It has been theorised that the plesiotypic (or ancestral) DRP scaffold comprised of eight cysteines that formed four disulfide bonds (Pineda et al., 2020; Cole and Brewer, 2021). However, the evolutionary history of DRP scaffold in spider venom has been riddled with events of duplication and diversification, which may have resulted in multiple gain and loss of structural and functional residues, including cysteines. As a result, we find that DRP toxins in extant spiders are comprised of distinct scaffolds with a range of cysteine pairs (4–12 cysteines forming 2–6 disulfide bonds). For example, an individual spider from the Haplopelma genus may contain SF90, SF91, SF99 and SF100 toxin superfamilies in its venom with 6, 8, 12, and 10 cysteines, respectively. This makes it very difficult to trace the nature and cysteine skeletal structure of the plesiotypic scaffold – something that could be answered in future with the help of comparative genomics and synteny analyses. Our extensive phylogenetic and evolutionary analyses provide insight into the common origin of DRP toxins in spiders, dating back to the common ancestor of Mygalomorphae and Araneomorphae. However, we refrain from speculating on the exact nature of this plesiotypic scaffold. Contrasting weaponisation strategies: recruitment versus innovation Venom is an intrinsically ecological trait that has underpinned the evolutionary success of many animals (Suranse et al., 2022). The ability of venomous organisms to incapacitate prey and predators emanates from toxins that exhibit an array of biochemical activities and target divergent pathways. Many venomous lineages deploy a wide range of toxins from phylogenetically unrelated superfamilies. Venomous snakes, for example, have ‘recruited’ a myriad of toxins, including snake venom metalloproteinases, snake venom serine proteases, three-finger toxins, phospholipase A2s, L-amino acid oxidases, Kunitz-type serine protease inhibitors, kallikreins, lectins, DNases, and hyaluronidases (Casewell et al., 2013; Faisal et al., 2021; Dutta et al., 2017; Senji Laxme et al., 2019a; Casewell et al., 2020; Figure 6). Similarly, spider venoms typically possess many forms of enzymes (e.g. phospholipases, proteases and chitinases), polyamines, salts, and disulphide-rich toxins (King and Hardy, 2013; Figure 6). However, spider venom DRPs with diverse ion channel targeting activities, such as sodium, potassium, calcium, and chloride ion channels, predominate in the venoms of nearly all spiders, constituting three-quarters of the venom (Figure 6). Phylogenetic and evolutionary assessments in this study trace the evolutionary origin of DRPs in Opisthothelae, the suborder that includes the majority of spiders described to date. This strategy, wherein, a molecular scaffold with a single deep-rooted evolutionary origin constitutes the major content of the venom, is unique to spiders. Venoms of most other animals are, instead, composed of unrelated toxin types, derived from distinct scaffolds/gene superfamilies in varying proportions. Thus, instead of recruiting distinct toxins with diverse functions into their venoms like the majority of venomous animals, spiders seem to have diversified a single molecular template to generate a commensurate functional diversity in their venoms. These findings not only shed light on the fascinating evolutionary history of spider venoms but also highlight an unrealized potential of molecular scaffolds in underpinning the dramatic structural and functional diversification of the venom arsenal. Figure 6 Download asset Open asset Distinct toxin scaffold recruitment strategies in spiders and snakes. This figure depicts distinct toxin scaffold recruitment strategies in (A) spiders and (B) advanced snakes. The Araneae phylogeny highlights the domination of disulfide-rich peptide toxins in spiders [Atracidae: Atrax sp.; Theraphosidae: Poecilotheria formosa; Theridiidae: Latrodectus mactans; Ctenidae: Phoneutria nigriventer: e.g., Palagi et al., 2013; Oldrati et al., 2017; Diniz et al., 2018], whereas venoms of advanced snakes are constituted by diverse phylogenetically unrelated toxin superfamilies (Viperidae: Daboia russelii, Elapidae: Naja naja, Colubridae: Spilotes sulphureus: e.g., Senji Laxme et al., 2021a; Senji Laxme et al., 2021b; Modahl et al., 2018). Doughnut charts, portraying the major molecular scaffolds in venom are also shown disulfide-rich peptides (yellow), snake venom metalloproteinases (SVMP, red), phospholipase A2 (PLA2, green), three-finger toxins (3 FTx, blue) and other minor components (black). Structures of the major scaffolds are also shown, with helices coloured in green, β-strands in blue and disulfide bonds in orange. Distinct recruitment and diversification of spider venom superfamilies in Mygalomorphae and Araneomorphae In addition to suggesting the common evolutionary origin of DRP toxins, Bayesian and maximum-likelihood phylogenies provided intriguing insights into the early diversification of DRPs in spiders. Mygalomor