The deep-rooted origin of disulfide-rich spider venom toxins
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eLife assessment
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.
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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.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Shaikh and Sunagar addresses the question of the origin of spider venom proteins. It has been known for many years that an important component of spider venoms is a diverse group of small proteins known as disulfide-rich peptides (DRPs). However, it has not been clear whether this group of proteins has a common origin or evolved convergently in different lineages. The authors collected sequences of the genes encoding these proteins from publicly available genomes of spiders from a range of families. They aligned the sequences using the structural cysteines as guides and carried out a phylogenetic analysis of the different sequences, ultimately classifying the different proteins into over 50 super-families. One thing that is not clear from the text or from the references …
Author Response
Reviewer #1 (Public Review):
The manuscript by Shaikh and Sunagar addresses the question of the origin of spider venom proteins. It has been known for many years that an important component of spider venoms is a diverse group of small proteins known as disulfide-rich peptides (DRPs). However, it has not been clear whether this group of proteins has a common origin or evolved convergently in different lineages. The authors collected sequences of the genes encoding these proteins from publicly available genomes of spiders from a range of families. They aligned the sequences using the structural cysteines as guides and carried out a phylogenetic analysis of the different sequences, ultimately classifying the different proteins into over 50 super-families. One thing that is not clear from the text or from the references cited (I am not an expert on spider venom) is how many of these superfamilies were known before and how many are novel. There is also no clear indication of what criteria were used to define a subset of sequences as a superfamily. Nonetheless, the authors show that all these superfamilies have a single common ancestor, predating the divergence of araneomorphs and mygalomorphs and that the DRPs underwent independent diversification in each of these two lineages.
We have identified 78 novel superfamilies in this study and 33 were previously identified (Pineda et al. 2020 PNAS). We had previously described information in lines 90, 101 and 106 regarding the description of novel superfamilies from previous studies and the ones described in this study.
Line 90 “Recently, using a similar approach, 33 novel spider toxin superfamilies have been identified from the venom of the Australian funnel-web spider, Hadronyche infensa (9).”
Line 101 “This approach enabled the identification of 33 novel toxin superfamilies along the breadth of Mygalomorphae (Figures S1 and S2).”
Line 106 “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 (Figures S3 and S4).”
Spider toxin superfamilies have been named after gods/deities of death, destruction and the underworld based on nomenclature introduced by Pineda et al. (2014 BMC genomics). We have now included this explanation in the manuscript under the methods and results sections. We have also provided additional details pertaining to this nomenclature in Table S1.
The authors also looked at selective forces acting on the sequences using dN/dS analyses. They reach the conclusion that there are different modes of selection acting on different sequences based on their role - defensive or predatory venoms - building on previous work by the lead author on venom sequence evolution in diverse animals.
All in all, this is an admirable piece of molecular evolution work, providing new data on the evolution of spider venom proteins. There are some confusions in terminology that need to be cleared up, and somewhat more context needs to be given for non-specialists as detailed in the points below:
We thank the reviewer for their constructive and critical suggestions, as well as the kind words of encouragement. Their suggestions have helped us in significantly improving the quality of our work.
Suggestion 1) Common names of the main spider infraorders should be given.
We thank the reviewer for their helpful input. We have now introduced spider infraorders with well-known spiders and their common names under the introduction section. Furthermore, we have also included a schematic representation of the spider phylogeny, and highlighted lineages under investigation as Figure 1.
Suggestion 2) Opisthothelae is not the common ancestor of Mygalomorphae and Araneamorphae, but the clade that encompasses those two clades. This incorrect statement appears in several places. Further on, it is stated that Opisthothelae is the common ancestor of all extant spiders. This is wrong both from a terminological point of view (a clade cannot be ancestral to another clade) and from a factual point of view, since there are extant spiders not included in Opisthothelae.
We thank the reviewer for pointing out this oversight. We have now corrected it to suborder Opisthothelae as the clade encompassing Mygalomorphae and Araneomorphae spiders.
Suggestion 3) Several proteins and proteins families are mentioned without being introduced, e.g. knottin. Please provide short descriptions.
We have now provided a short introduction to terms such as Knottin.
Reviewer #2 (Public Review):
This interesting study looks into the evolution of putative spider venom toxins, specifically disulfide-rich peptides (DRPs). The authors use published sequence data to gain new insights into the evolution of DRPs, which are the major component of most spider venoms. Through a series of sequence comparisons and phylogenetic analyses they identify a substantial number of new spider toxin superfamilies with distinct cysteine scaffolds, and they trace these back to a primitive scaffold that must have been present in the last common ancestor of mygalomorph and araneomorph spiders. Looking at the taxonomic distribution of these putative venom DRPs, they conclude that mygalomorph and araneomorph DRPs have evolved in different ways, with the former being recruited into venom at the level of genera, and the latter at the level of families. In addition, they perform selection analyses on the DRP superfamilies to uncover the surprising result that mygalomorph and araneomorph DRPs have evolved under different selective regimes, with the evolution of the former being characterised by positive selection, and the latter by purifying (negative) selection.
However, I don't think that in the current state of the manuscript these conclusions are robustly supported for several reasons. First, it seems that not all previously published data were included in the phylogenetic analyses that were used to identify new superfamilies of DRPs.
We have, indeed, analysed all spider toxin sequences available to date. We have relied on the signal and propeptide regions for identifying novel superfamilies, which is an accepted convention: Pineda et al. (2014 BMC Genomics); Pineda et al. (2020 PNAS).
Although many additional superfamilies can be identified, we have only retained those sequences for which there were at least 5 representatives for the identification of toxin superfamilies, and 15 representatives for selection analyses to ensure robustness. This filtering step ensured that the generated alignments, phylogenetic trees, and evolutionary assessments were robust and devoid of noise that stems from single-representative groups. Adding in those sequences would have enabled us to identify many more superfamilies, solely based on the signal and propeptide examination, but it wouldn’t have been possible to support them with other lines of evidence that were provided for all other superfamilies in this study, jeopardising the overall quality of the manuscript. Nonetheless, there is strong evidence that the left-out sequences are also related to the ones analysed in this study (Figure S10). In future, when more transcriptomes are sequenced, it would be possible to designate these newer toxin superfamilies with much stronger support.
Second, much of the data were obtained from whole-body transcriptome data, which leaves a degree of uncertainty that these data indeed derive from the venom glands that produce the toxins.
We respectfully disagree with the reviewer that ‘much of the data’ are from the whole-body transcriptomes. Nearly all sequences in our study are sourced from Pineda et al. (2014 BMC Genomics and 2020 PNAS), Sunagar et al (2013 Toxins), Cole and Brewer (2020 bioRxiv) and transcriptome sequence assembly data from established online repositories NCBI (NR and TSA) and ENA. All the above-mentioned studies (KS is a part of many of these) under their methods section clearly state that the transcriptomes were generated using mRNA isolated from venom gland tissue (BioProject accessions: PRJEB14734; PRJEB6062; PRJNA189679, PRJNA587301 and PRJNA189679, where source tissue type is designated as venom gland).
We would like to direct the reviewer’s attention to the following excerpts from reference papers from which data for this study has been sourced:
- Pineda S et al. (2020 PNAS): “Three days later, they were anesthetized, and their venom glands were dissected and placed in TRIzol reagent (Life Technologies). Total RNA from pooled venom glands was extracted following the standard TRIzol protocol.”
- Sunagar et al (2013 Toxins): “Paired venom glands were dissected out and pooled from nine mature females on the fourth day after venom depletion by electrostimulation. Total RNA was extracted using the standard TRIzol Plus method ...”
- Cole and Brewer (2020 bioRxiv): “... the venom glands of each ctenid were dissected out, whole RNA was isolated from the venom glands …”
We would also like to point out that hexatoxins are widely studied and are some of the most well-understood spider venom toxins. Many representatives have been functionally characterised and shown to be potent in affecting prey and predatory species [Sunagar et al (2013 Toxins); Pineda et al. (2014 BMC Genomics and 2020 PNAS); Volker, et al. (2020 PNAS) - KS is a part of most of these studies as well]. However, the current technologies do not permit the high-throughput screening of the enormous diversity of toxins in spiders, which is why not every toxin sequence identified from the venom gland is functionally characterised. Nonetheless, venom researchers will not contest the role of these highly expressed venom gland proteins in envenoming, especially given that they share significant sequence identities with toxins that are functionally well-characterised.
The only exception to the above is non-ctenid araneomorph toxin superfamily sequences, which are retrieved from whole-body transcriptomes (Cole and Brewer; 2020 bioRxiv). The authors of the paper indicated these as putative toxins. As explained above, homologs of these peptides are well-characterised to be venom toxins. Additionally, in our phylogenetic trees (Figures 3, 4, S6 and S9), they are nested within the toxin clades, reaffirming their identity.
Third, the taxonomic representation of mygalomorph and araneomorph diversity in this study is so sparse that it becomes impossible to distinguish whether toxin recruitments have happened at the level of genera, families, or even higher-level taxa.
We respectfully disagree with this suggestion. The taxonomic breadth investigated in this study isn’t sparse. Analysed sequences belong to groups across the breadth of the spider phylogeny. To address this criticism, we are now including a schematic representation of spider phylogeny, where lineages under investigation are highlighted (Figure 1A). Given this broader taxonomic breadth, all of our interpretations are parsimoniously extendable to their common ancestors. For instance, we establish the common origin of all DRPs in the members of these widespread spider families. Therefore, not including sequences from other sister groups will not invalidate this hypothesis, and the most parsimonious explanation will be that the missing members too are likely to have DRPs in their venom (which is also a common understanding of the spider venom research). Whether DRPs dominate the venoms of these missing groups will only come to light upon investigation, but their presence in the venom is highly likely. Moreover, please do note that we have analysed nearly all sequences available in the literature to date.
As for the recruitment of the toxin superfamily at the taxon level, we would like to point out the phylogenies in Figures 2 and 3 that clearly show the differential recruitment events. We would also like to point out lines 120 and 136 state that this may not only be a result of recruitment and could arise from differential rates of diversification (also evident in other analyses presented in Figures 5 and Tables S2 and S3).
Line 120 “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 genus (Figure 2).”
Line 136 “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 be recruited at the level of the spider family, rather than the genus. As a result, and in contrast to mygalomorph DRPs, araneomorph toxin superfamilies were found to be scattered across spider lineages (Figure 3; Figure S6; node support: ML: >90/100; BI: >0.95).”
Adding any number of missing lineages will neither change the fact that araneomorphs ‘appear’ to have recruited these superfamilies at the genera level, nor the family-level recruitment of toxin superfamilies in a large number of examined mygalomorphs.
We have now introduced a new figure (Figure 7) that highlights the different scenarios that explain the observed differences in the evolution of mygalomorph and araneomorph spider toxins. We have also included additional text in the manuscript to explain this better.
Fourth, only a selection of DRP superfamilies was used for natural selection analyses, without the authors explaining how this selection was made. Yet, they attempted to draw general conclusions about toxin evolution in mygalomorphs and araneomorphs, even though most of the striking differences they found were restricted to just two mygalomorph genera, and one family of araneomorphs.
From our experience and previous reports [Sunagar and Moran (2015, PLoS genetics); Sunagar, et al. (2012, MBE); Yang, Z. (2007, MBE)], the unavailability of enough sequences from datasets results in inaccurate estimation of omega values. For instance, if there are only a couple of sequences in a superfamily, both of which are slightly different from one another, then even these minor differences in them would be exaggerated. Hence, we have resorted to performing selection analysis on datasets for which there are at least 15 sequences. No doubt that this conservative approach reduces the number of datasets analysed, but it also ensures that our findings are well-supported. We have now clarified this in our manuscript under the methods section.
However, we did previously include sequences from all toxin superfamilies described to date in our alignment figure (Fig S10) and analysed their signal and propeptide regions. They were only excluded from selection analyses. It can be seen that they too are DRPs, but they belong to distinct superfamilies from the ones being described here.
If these concerns are addressed this study can shed important new light on venom toxin evolution in one of the most diverse venomous taxa on Earth.
We thank the reviewer for their constructive inputs and suggestions which have enabled us to make this manuscript more accessible to a wider audience.
Reviewer #3 (Public Review):
This work aims to elucidate the evolutionary origins of disulfide-rich spider toxin superfamilies and to determine the modes of natural selection and associated ecological pressures acting upon them. The authors provide a compelling line of evidence for a single evolutionary origin and differing factors (e.g., prey capture strategies and methods of anti-predator defense) that have shaped the evolution of these toxins. Additionally, the two major spider infraorders are claimed to have experienced differing selective pressures regarding these toxins.
The results presented here are novel and generally well-presented. The evidence for a single origin of DRP toxins in spiders is exciting and changes the paradigm of spider venom evolution.
The data are well analyzed, but the methods lack enough detail to reproduce the results. More information regarding the parameters passed to each software package, version numbers of all software employed, and models of molecular evolution employed in phylogenetic analyses are among the necessary missing information.
We thank the reviewer for their kind words and constructive and critical suggestions. Their suggestions have contributed towards improving the quality of our work. Upon their suggestion, we have now expanded the methods section to include more details.
The differences in the evolutionary pressures between mygalomorphs and RTA-clade spider DRP toxins are clear, but expanding RTA results to all araneomorphs may be overreaching. Additional araneomorph sequence data is available, despite the claims within this manuscript (e.g., see Jiang et al.. 2013 Toxins; He et al.. 2013 PLoS ONE; and Zobel-Thropp et al.. 2017 PEERJ). These papers include cDNA sequences of spider venom glands and contain representatives of inhibitory cysteine knot toxins, which are DRP toxins. These data would greatly enhance the strengths of the results presented herein.
In response to the expansion of RTA results to araneomorphs, we would like to point out that RTA comprises about 50% of the diversity recorded in Araneomorphae. The araneomorph data analysed in our study covers a range of araneomorph family divergence time Agelenidae (<70 MYA), Pisauridae (<50 MYA) and Theridiidae (~200 MYA, Magalhaes 2020, Biological Reviews 95.1). We report a strong signature of purifying selection influencing the evolution of araneomorph toxin SFs, despite the long evolutionary time separating them (50 - 200 MYA). We firmly believe that further addition of toxin sequence data from other groups will not deviate from the general trend of molecular evolution observed in both these lineages across such large period of time; barring certain certain exceptions (such as SF13 a defensive toxin identified from Hadronyche experiencing purifying selection; Volker, et al. 2020 PNAS).
We had initially excluded non-ctenid datasets from our analyses on account of poor sequence annotation and lack of representative sequence data. However, we have now incorporated Dolomedes mizhoanus (DRP) (Jiang et al. 2013 Toxins) and Latrodectus tredecimguttatus (non-DRP) (He et al. 2013 PLoS ONE) toxin dataset into our analyses, following reviewer’s suggestion. This has led to identification of 5 novel superfamilies, providing additional support to our spider venom evolution hypothesis.
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eLife assessment
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.
-
Reviewer #1 (Public Review):
The manuscript by Shaikh and Sunagar addresses the question of the origin of spider venom proteins. It has been known for many years that an important component of spider venoms is a diverse group of small proteins known as disulfide-rich peptides (DRPs). However, it has not been clear whether this group of proteins has a common origin or evolved convergently in different lineages. The authors collected sequences of the genes encoding these proteins from publicly available genomes of spiders from a range of families. They aligned the sequences using the structural cysteines as guides and carried out a phylogenetic analysis of the different sequences, ultimately classifying the different proteins into over 50 super-families. One thing that is not clear from the text or from the references cited (I am not an …
Reviewer #1 (Public Review):
The manuscript by Shaikh and Sunagar addresses the question of the origin of spider venom proteins. It has been known for many years that an important component of spider venoms is a diverse group of small proteins known as disulfide-rich peptides (DRPs). However, it has not been clear whether this group of proteins has a common origin or evolved convergently in different lineages. The authors collected sequences of the genes encoding these proteins from publicly available genomes of spiders from a range of families. They aligned the sequences using the structural cysteines as guides and carried out a phylogenetic analysis of the different sequences, ultimately classifying the different proteins into over 50 super-families. One thing that is not clear from the text or from the references cited (I am not an expert on spider venom) is how many of these superfamilies were known before and how many are novel. There is also no clear indication of what criteria were used to define a subset of sequences as a superfamily. Nonetheless, the authors show that all these superfamilies have a single common ancestor, predating the divergence of araneomorphs and mygalomorphs and that the DRPs underwent independent diversification in each of these two lineages.
The authors also looked at selective forces acting on the sequences using dN/dS analyses. They reach the conclusion that there are different modes of selection acting on different sequences based on their role - defensive or predatory venoms - building on previous work by the lead author on venom sequence evolution in diverse animals.
All in all, this is an admirable piece of molecular evolution work, providing new data on the evolution of spider venom proteins. There are some confusions in terminology that need to be cleared up, and somewhat more context needs to be given for non-specialists as detailed in the points below:
- Common names of the main spider infraorders should be given.
- Opisthothelae is not the common ancestor of Mygalomorphae and Araneamorphae, but the clade that encompasses those two clades. This incorrect statement appears in several places. Further on, it is stated that Opisthothelae is the common ancestor of all extant spiders. This is wrong both from a terminological point of view (a clade cannot be ancestral to another clade) and from a factual point of view, since there are extant spiders not included in Opisthothelae.
- Several proteins and proteins families are mentioned without being introduced, e.g. knottin. Please provide short descriptions.
-
Reviewer #2 (Public Review):
This interesting study looks into the evolution of putative spider venom toxins, specifically disulfide-rich peptides (DRPs). The authors use published sequence data to gain new insights into the evolution of DRPs, which are the major component of most spider venoms. Through a series of sequence comparisons and phylogenetic analyses they identify a substantial number of new spider toxin superfamilies with distinct cysteine scaffolds, and they trace these back to a primitive scaffold that must have been present in the last common ancestor of mygalomorph and araneomorph spiders. Looking at the taxonomic distribution of these putative venom DRPs, they conclude that mygalomorph and araneomorph DRPs have evolved in different ways, with the former being recruited into venom at the level of genera, and the latter …
Reviewer #2 (Public Review):
This interesting study looks into the evolution of putative spider venom toxins, specifically disulfide-rich peptides (DRPs). The authors use published sequence data to gain new insights into the evolution of DRPs, which are the major component of most spider venoms. Through a series of sequence comparisons and phylogenetic analyses they identify a substantial number of new spider toxin superfamilies with distinct cysteine scaffolds, and they trace these back to a primitive scaffold that must have been present in the last common ancestor of mygalomorph and araneomorph spiders. Looking at the taxonomic distribution of these putative venom DRPs, they conclude that mygalomorph and araneomorph DRPs have evolved in different ways, with the former being recruited into venom at the level of genera, and the latter at the level of families. In addition, they perform selection analyses on the DRP superfamilies to uncover the surprising result that mygalomorph and araneomorph DRPs have evolved under different selective regimes, with the evolution of the former being characterised by positive selection, and the latter by purifying (negative) selection.
However, I don't think that in the current state of the manuscript these conclusions are robustly supported for several reasons. First, it seems that not all previously published data were included in the phylogenetic analyses that were used to identify new superfamilies of DRPs. Second, much of the data were obtained from whole-body transcriptome data, which leaves a degree of uncertainty that these data indeed derive from the venom glands that produce the toxins. Third, the taxonomic representation of mygalomorph and araneomorph diversity in this study is so sparse that it becomes impossible to distinguish whether toxin recruitments have happened at the level of genera, families, or even higher-level taxa. Fourth, only a selection of DRP superfamilies was used for natural selection analyses, without the authors explaining how this selection was made. Yet, they attempted to draw general conclusions about toxin evolution in mygalomorphs and araneomorphs, even though most of the striking differences they found were restricted to just two mygalomorph genera, and one family of araneomorphs.
If these concerns are addressed this study can shed important new light on venom toxin evolution in one of the most diverse venomous taxa on Earth.
-
Reviewer #3 (Public Review):
This work aims to elucidate the evolutionary origins of disulfide-rich spider toxin superfamilies and to determine the modes of natural selection and associated ecological pressures acting upon them. The authors provide a compelling line of evidence for a single evolutionary origin and differing factors (e.g., prey capture strategies and methods of anti-predator defense) that have shaped the evolution of these toxins. Additionally, the two major spider infraorders are claimed to have experienced differing selective pressures regarding these toxins.
The results presented here are novel and generally well-presented. The evidence for a single origin of DRP toxins in spiders is exciting and changes the paradigm of spider venom evolution.
The data are well analyzed, but the methods lack enough detail to reproduce …
Reviewer #3 (Public Review):
This work aims to elucidate the evolutionary origins of disulfide-rich spider toxin superfamilies and to determine the modes of natural selection and associated ecological pressures acting upon them. The authors provide a compelling line of evidence for a single evolutionary origin and differing factors (e.g., prey capture strategies and methods of anti-predator defense) that have shaped the evolution of these toxins. Additionally, the two major spider infraorders are claimed to have experienced differing selective pressures regarding these toxins.
The results presented here are novel and generally well-presented. The evidence for a single origin of DRP toxins in spiders is exciting and changes the paradigm of spider venom evolution.
The data are well analyzed, but the methods lack enough detail to reproduce the results. More information regarding the parameters passed to each software package, version numbers of all software employed, and models of molecular evolution employed in phylogenetic analyses are among the necessary missing information.
The differences in the evolutionary pressures between mygalomorphs and RTA-clade spider DRP toxins are clear, but expanding RTA results to all araneomorphs may be overreaching. Additional araneomorph sequence data is available, despite the claims within this manuscript (e.g., see Jiang et al. 2013 Toxins; He et al. 2013 PLoS ONE; and Zobel-Thropp et al. 2017 PEERJ). These papers include cDNA sequences of spider venom glands and contain representatives of inhibitory cysteine knot toxins, which are DRP toxins. These data would greatly enhance the strengths of the results presented herein.
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