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https://doi.org/10.1038/s41467-021-21093-8 OPEN Phylogenetic analyses suggest centipede venom arsenals were repeatedly stocked by horizontal gene transfer ✉ ✉ Eivind A. B. Undheim1,2,3 & Ronald A. Jenner 4

Venoms have evolved over a hundred times in animals. Venom toxins are thought to evolve mostly by recruitment of endogenous proteins with physiological functions. Here we report 1234567890():,; phylogenetic analyses of venom proteome-annotated venom gland transcriptome data, assisted by genomic analyses, to show that centipede venoms have recruited at least five gene families from bacterial and fungal donors, involving at least eight horizontal gene transfer events. These results establish centipedes as currently the only known animals with venoms used in predation and defence that contain multiple gene families derived from horizontal gene transfer. The results also provide the first evidence for the implication of horizontal gene transfer in the evolutionary origin of venom in an animal lineage. Three of the bacterial gene families encode virulence factors, suggesting that horizontal gene transfer can provide a fast track channel for the evolution of novelty by the exaptation of bacterial weapons into animal venoms.

1 Centre for Biodiversity Dynamics, Department of Biology, NTNU, Trondheim, Norway. 2 Centre for Ecological and Evolutionary Synthesis, Department of Bioscience, University of Oslo, Blindern, Oslo, Norway. 3 Centre for Advanced Imaging, University of Queensland, St Lucia, QLD, Australia. 4 Department of ✉ Life Sciences, Natural History Museum, London, UK. email: [email protected]; [email protected]

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orizontal gene transfer (HGT) between kingdoms and Results and discussion domains of life has contributed to the evolution of a Overall support for HGT. Several methods are available for H 25 diversity of novel adaptive traits in animals, including the identifying HGT . A combination of phylogenetic analyses of ability of bdelloid rotifers to withstand desiccation, the ability of candidate HGT gene families including both potential donor and springtails to feed on decaying organic matter, and the ability of host sequences, and confirming their presence in host genomes is plant-parasitic nematodes to degrade plant cell walls1–7. HGT has considered to be the most robust method. We used this approach also contributed to the evolution of venom, one of the most to identify putative HGTs from non-metazoan sources into convergently evolved animal adaptations. Venoms are complex, centipede venoms. Table 1 summarizes the support for all typically proteinaceous, secretions that are used primarily for inferred HGTs that have contributed to centipede venom predation and defence by a wide phylogenetic range of animals. arsenals. The robustly supported phylogenetic nesting of clades of However, although animal venoms have evolved at least a hun- centipede sequences within paraphyletic backbones of non- dred times independently8, the contribution of HGT to the metazoan donor sequences supports HGT for five of the seven evolution of venom arsenals has so far been shown to be minor. gene families: β-pore-forming toxin (β-PFTx), centipede pepti- HGT is a well-supported hypothesis for only three gene dylarginine deiminase (centiPAD), protein with a domain of families present in arthropod and cnidarian venoms. Phylogenetic unknown function (DUF3472), pesticidal crystal protein domain- analyses, in some cases supported by genomic information, containing protein-like protein (PCPDP-like), and uncharacter- strongly suggest that bacteria were the source of type D phos- ized protein family 5 (unchar05). The phylogenetic nesting of pholipases found in the venoms of sicariid spiders, scorpions, and centipede geotoxin 2 (GEOTX02) within fungal sequences is less ticks9, and of pore-forming toxins expressed in the venom glands well supported, while the centipede sequences for uncharacterized of ticks as well as gland cells in the digestive system of cnidarians, protein family 16 (unchar16) group in a clade that is sister to a although it is debated whether these should be considered part of clade of oomycete sequences. Furthermore, by confirming that the venom system or not10. Similarly, glycoside family 19 chit- five of the genes map to protein-coding genes with introns in the inases found in the venom of chalcidoid parasitoid wasps were genome of the geophilomorph centipede Strigamia maritima, probably transferred from parasitic fungi11. Other potential cases which is the only published centipede genome26, we show that of HGT contributing to insect venoms currently lack phylogenetic they are bona fide centipede genes rather than the result of support12–14, while the direction of HGT of neurotoxic α- contamination or symbionts. Importantly, a recent study27 that latrotoxins present in the venom of theridiid spiders and bacteria examined the presence of contamination in the genome of S. remains uncertain15. Although HGT is currently not considered maritima confirms that none of our HGT candidates map to the to be a major mechanism of venom evolution, venoms are only genomic scaffold for which there are signs of contamination nevertheless a promising research area given the existence of (scaffold JH431684; C. M. Francois, pers. comm.). many tens of thousands of mostly unstudied venomous animal We bolster our conclusions about HGT with three ancillary species. Many venoms also contain a substantial number of criteria. First, all seven putative HGT gene families are present in proteins with few or no known metazoan homologues16–21, and both centipede venom gland transcriptomes and milked venom these may include HGT candidates. proteomes, which argues against them being accidental contamina- One venomous lineage that contains a large diversity of tion. Second, each putative HGT gene is consistently expressed in unassignable venom proteins22,23 is centipedes (Chilopoda). the venom glands of multiple species collected from disparate Centipedes are one of the oldest terrestrial venomous lineages, geographic locations and habitats, which would not be expected if with a fossil record going back 418 million years24. Living species the sequences derived from local contaminants. Third, putative belong to five orders: Scutigeromorpha (long-legged house cen- HGT sequences from different centipede species that are con- tipedes), Lithobiomorpha (stone centipedes), Geophilomorpha taminants would be expected to group with related non-centipede (long-bodied earth centipedes), Scolopendromorpha (the most sequences in different places in gene trees, rather than cluster familiar centipedes, including large tropical species), and Cra- together in a single clade. The strong clustering of the centipede terostigmomorpha (two species from Tasmania and New Zeal- sequences into well-supported clades in our gene trees, and the lack and). All of these have complex venoms that are used for of the haphazard interleaving of putative donor and centipede predation and defence. While most of the protein families con- sequences in any of our trees strongly suggest that the putative HGT tained in centipede venoms were recruited from gene families that genes are bona fide centipede sequences. Fulfilment of these are widespread in animals, others have few or no metazoan ancillary criteria in addition to the phylogenetic nesting of the homologues. This pattern suggests that the evolutionary origins of centipede sequences within paraphyletic groups of donor sequences, several centipede venom toxins could lie outside the animal and the presence of five of the seven genes in the genome of S. kingdom. maritima, further decreases the probability that our results are due We show that multiple HGTs have stocked centipede venom to contamination or symbionts. Below we will discuss the full arsenals throughout their evolution. Phylogenetic analyses of support for our conclusions for each of the genes, and the possibility venom gland transcriptome and venom proteome data assisted by that the genes that could not be checked against the S. maritima genomic analyses identified seven gene families encoding cen- genome (centiPAD and PCPDP-like) could be due to symbionts. tipede venom proteins and peptides that were horizontally transferred between bacteria, fungi, oomycetes, and centipedes. Our analyses reveal between 10 and 12 HGT events. At least eight Bacterial pore-forming toxins transferred twice into cen- HGTs involved five gene families that transferred from bacteria tipedes. Centipede β-PFTxs were recruited into the ancestral and fungi into centipede venoms, whereas the direction of two or centipede venom proteome, with subsequent losses from crater- three HGTs between centipedes and fungi and oomycetes remain ostigmomorph and geophilomorph venoms23. This gene family uncertain. Three of the protein families in bacterial donor taxa are belongs to the bacterial aerolysin-like β-pore-forming toxin virulence factors involved in pathogenicity, suggesting that cen- superfamily, which Moran et al.10 showed was transferred at least tipedes have repurposed bacterial weapons as venom components six times from bacteria to eukaryotes, including animals. We did involved in predation and/or defence. Our findings suggest that not specifically design our phylogenetic dataset to provide a HGT can be an important factor shaping the evolution of animal precise estimate of when and where all non-centipede HGTs venoms. occurred, but our findings agree with and extend their results.

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Table 1 Summary of gene families horizontally transferred into centipede venoms.

Gene HGT source Number of Phylogenetic location of HGT Phylogenetic location of Mapped to Strigamia maritima HGT recruitment into venom genomeb eventsa β-PFTx Bacteria 2 (1) Arthropoda or Chilopoda; within Chilopoda SMAR004242, SMAR004243, Lithobiomorpha SMAR012417 centiPAD Bacteria 2 Within Scutigeromorpha; within Within Scutigeromorpha; n/a Lithobiomorpha within Lithobiomorpha DUF3472 Bacteria 1 or 2 (1) In the stem of Pleurostigmophora Within Scolopendromorpha SMAR002991, SMAR002992, or Amalpighiatac; or in Epimorpha SMAR002993, SMAR008653 and within Lithobiomorpha GEOTX02 Fungid 1 or 2 Geophilomorpha Geophilomorpha (group 1: SMAR012843, SMAR003678, SMAR004759); (group 2: SMAR012429, SMAR005429); group 3: SMAR014279; (group 4: SMAR009615, SMAR004692, SMAR001285, SMAR007268, SMAR006394, SMAR009617, SMAR010233) PCPDP-like Bacteria 1 Lithobiomorpha Lithobiomorpha n/a unchar05 Fungi 2 Geophilomorpha, within Geophilomorpha SMAR002275, SMAR004333, Lithobiomorpha SMAR005016, SMAR002277, SMAR015613 unchar16 Oomycetesd 1 Unknown Craterostigmomorpha SMAR001399, SMAR001400 n/a The absence of these genes from the genome of S. maritima is uninformative because the HGT events happened elsewhere in the tree. aThe number in parentheses shows the number of times the gene was recruited into the venom proteome if that differs from the number of HGT events23. bThe identity of all paralogous loci is given. All are protein-coding loci with introns. Different paralog groups are indicated in parentheses. cDue to uncertainty about centipede phylogeny52 we cannot distinguish between a single HGT into Pleurostigmophora (non-scutigeromorph centipedes), followed by a loss in Craterostigmomorpha, or a HGT into Amalpighiata (Lithobiomorpha + Epimorpha). Both these hypotheses suggest a loss in henicopid lithobiomorphs. dThe direction of transfer is ambiguous.

Although the structure of the gene tree is complex (Fig. 1; see venom, cnidarian β-PFTxs, which were horizontally transferred Supplementary Fig. 1 for full tree), it shows that centipede β- independently from those found in the venoms of centipedes and PFTxs transferred twice from bacteria, once into the stem lineage arachnids, are secreted into the pharynx and gut and aid digestion of centipedes or arthropods (upper clade with 94% bootstrap by disintegrating prey tissues, although their paralytic activity support in Fig. 1), and once into the lithobiomorph lineage may also assist in prey immobilisation10,30,31. There is no (located in the lower clade). This inference is supported by tree experimental data for the role of β-PFTxs in centipedes, but they topology tests, which strongly reject monophyly of centipede β- are believed to be at least in part responsible for the cytolytic PFTxs (see Supplementary Data 1). The structure of the tree, activities of centipede venoms by the formation of transmem- especially the complex interleaving pattern of bacterial, fungal, brane pores32. The great diversity of β-PFTx transcripts expressed plant, and animal sequences in the lower clade of Fig. 1, suggests a in centipede venom proteomes, and the abundance of their complex history of multiple HGTs from bacteria to eukaryotes as expression22,23,33,34, suggest that β-PFTx likely plays important well as losses of β-PFTx. For instance, an early transfer of β-PFTx roles in prey immobilisation and processing. into the arthropod stem lineage implies that it was lost in non- centipede myriapods and pancrustaceans, according to the cur- rent consensus on arthropod phylogeny28. However, the pro- Bacterial exotoxins probable source of PCPDP-like proteins. nounced phylogenetic disjunction of the non-centipede animal We previously detected proteins with a pesticidal crystal protein sequences, and the lack of species from phyla with a strong domain (InterPro accession IPR036716) in the venom of representation in our custom (see “Methods”) and public Lithobius forficatus23. Homologous sequences are also present in sequence databases, such as arthropods, molluscs and nematodes, transcriptomes of other centipedes from both lithobiomorph suggest that multiple HGTs have occurred from bacteria to ani- families (Lithobiidae: L. forficatus, E. cavernicolus; Henicopidae: mals. This interpretation is supported by tree topology tests that A. giribeti, P. validus). All centipede PCPDP-like sequences reject animal monophyly (see Supplementary Data 1). cluster together in a strongly supported clade that is embedded in The β-PFTxs of S. maritima map to three protein-coding a paraphyletic backbone of bacterial PCPDP sequences (Fig. 2; see paralogous genomic loci with introns (see Table 1). The Supplementary Fig. 2 for full tree). The tree also shows that phylogenetic distribution of these paralogs in three sub-clades PCPDP-like proteins were independently transferred into beetles, of centipede sequences in the upper clade of Fig. 1 shows that the a cnidarian and a tardigrade. This is supported by topological tree duplications that produced them happened early in the evolution tests that strongly reject metazoan monophyly (see Supporting of centipedes. However, β-PFTx and the other three protein Data 1). The clade of centipede sequences includes species col- families that were recruited into the ancestral centipede venom lected from the UK, Europe, North America, New Zealand, and are absent from the venom proteome of S. maritima, which shows Australia, and contains no interleaved bacterial sequences. This that streamlining of venom arsenals occurs alongside the strongly suggests that the PCPDP-like sequences are bona fide recruitment and diversification of new components23. centipede sequences rather than bacterial contaminants. The β-PFTxs produced by bacteria are virulence factors that Although on current evidence we cannot categorically reject the contribute to pathogenicity by the lysing of host cells29. possibility that PCPDP-like protein is produced by symbionts, Interestingly, although they are not expressed in their tentacle further evidence against this conclusion is that the centipede

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Parasitiformes (6) 89 Parasitiformes (Ixodes scapularis XP 002433803.1) 89 Actinopterygii (Erpetoichthys calabaricus; 2) Scolopendromorpha (42) 67 Scolopendromorpha (37) 91 Lithobiomorpha (9) Scolopendromorpha (3) 79 Firmicutes (3) Scolopendromorpha (2) 79 Proteobacteria (2) 65 Lithobiomorpha (Anopsobius giribeti; 2) Geophilomorpha (Strigamia maritima male c23080 g1 i1) 55 Scutigeromorpha (58) Scutigeromorpha (6) Parasitiformes (Ixodes scapularis; 12) 94 Branchiopoda (Triops newberryi TR16753 c0 g1 i1) 89 Geophilomorpha (3) 73 Scolopendromorpha (Cryptops hortensis trunk TR26263) 70 Lithobiomorpha (2) 66 Scutigeromorpha (Scutigera coleoptrata VG c1116731 g1 i1) 75 Lithobiomorpha (5) 55 Geophilomorpha (2) 90 Lithobiomorpha (4) Lithobiomorpha (Anopsobius giribeti DN24276 c0 g9 i4) 75 Solifugae (Eremobates sp. TR22997 c0 g2 i2) 75 Opiliones (9) Amblypygi (2) 71 Ricinulei (2) Xiphosura (3) Opiliones (4) Amblypygi (2) Scolopendromorpha (S. morsitans VG c12633 g1 i1) 75 Ricinulei (2) Proteobacteria (Enterovibrio caribbeanicus WP 009601263.1) Proteobacteria (Cystobacter fuscus WP 095986135.1) Ciliophora (5) Proteobacteria (Pseudomonas putida WP 114942594.1) Firmicutes (6) Proteobacteria (Enterovibrio pacificus WP 068905705.1) 92 Anthozoa (Orbicella faveolata XP 020619946.1) Lithobiomorpha (2) Proteobacteria (Acinetobacter guillouiae WP 096733959.1) Brachiopoda (Lingula anatina; 3) 78 Actinopterygii (33) Actinopterygii (32) 70 77 85 Echinodermata (Apostichopus japonicus PIK56663.1) 62 64 Echinodermata (Apostichopus japonicus; 4) 82 Rotifera (Adineta vaga; 2) Acidobacteria (PYX92692.1) Basidiomycota (11) Cyanobacteria (Planktothrix rubescens WP 026785854.1) Basidiomycota (7) 80 Streptophyta(102) Firmicutes (4) Proteobacteria (2) Echinodermata (Acanthaster planci XP 022092298.1) 54 Ascomycota (10) 70 Basidiomycota (3) Anthozoa (2) 63 Bacteroidetes (4) 60 Bacteroidetes (Prevotella copri WP 118155072.1) Bacteroidetes (Joostella marina WP 008615739.1) 83 71 Actinopterygii (Erpetoichthys calabaricus; 3) 59 Platyhelminthes (Macrostomum lignano PAA63075.1) Proteobacteria (3) Bacteroidetes (Dyadobacter jiangsuensis WP 106593456.1) Proteobacteria (Paraburkholderia caryophylli WP 085228623.1) Actinobacteria (4) Actinobacteria (Actinomadura chibensis WP 083980922.1) 73 Actinobacteria (2) Cyanobacteria (Pseudanabaena sp. PCC 6802 WP 019501888.1) 91 Proteobacteria (Methylosinus sp.; 3) Proteobacteria (Sphingomonas sp. 286220 OYY79389.1) Proteobacteria (Kosakonia arachidis WP 090120428.1) 57 Bacteroidetes (Polaribacter butkevichii WP 105048130.1) Insecta (2) Insecta (Bombyx mandarina XP 028038074.1) Insecta (Ostrinia furnacalis XP 028165630.1) Actinobacteria (2) Proteobacteria (Acinetobacter sp. CIP 110321 WP 016162910.1) Firmicutes (2) 63 Mucoromycota (Diversispora epigaea; 8) 56 Bacteroidetes (Alistipes sp. ZOR0009 WP 047450273.1) Thermotogae (Oceanotoga teriensis WP 109604816.1) Actinobacteria (Nocardia abscessus WP 043701198.1) Chloroflexi (Ktedonobacterales bacterium SCAWSG2 WP 129887468.1) 54 Euryarchaeota (Theionarchaea archaeon DG70 KYK36131.1) Euryarchaeota (2) Proteobacteria (Lysobacter enzymogenes WP 078997730.1) Mollusca (3) Platyhelminthes (Schistosoma mansoni XP 018652858.1) Mollusca (Pomacea canaliculata XP 025087227.1) Proteobacteria (2) 91 Bacteroidetes (Fibrella aestuarina BUZ 2 WP 015334044.1) 87 Actinobacteria (18) Bacteroidetes (2) Basidiomycota (Sphaerobolus stellatus SS14 KIJ34445.1) Proteobacteria (4) Proteobacteria (4) 1.0 Proteobacteria (Sphingopyxis sp. Root1497 WP 082544856.1) sequences are very distinct from their nearest bacterial relatives The role of PCPDP-like proteins in centipede venom remains (see below), which is reflected by the relatively long branch unknown, but our results suggest they evolved from bacterial leading to the centipede clade. Lastly, a morphological study of insecticidal pore-forming toxins. The most intensely studied the venom system of L. forficatus found no evidence for bacterial bacterial PCPDPs are pore-forming insecticidal endotoxins symbionts in the venom producing and secreting tissues35. known as Cry toxins or δ-endotoxins, which are used widely in

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Fig. 1 A maximum likelihood tree of β-PFTx sequences shows two clades of centipede β-PFTx sequences nested within a paraphyletic backbone of bacterial sequences. The tree shows that the centipede β-PFTxs originated from at least two bacterial HGTs, one along the centipede or arthropod stem lineage (represented by the clade at the top of the tree with 94% bootstrap support), and one within the lithobiomorph lineage (represented by the cladeof two lithobiomorph sequences lower down the tree). Centipede sequences are coloured blue (present in transcriptomes) and red (present in transcriptomes and venom proteomes). Highlighted sequences are Bacteria (pink), Euryarchaeota (brown), Protozoa (purple), Fungi (yellow), and Streptophyta (cyan). Metazoan sequences are not highlighted. Collapsed clades have the number of included sequences indicated in parentheses. For the uncollapsed tree see Supplementary Fig. 1. The tree was reconstructed using the WAG + R7 model and is displayed as midpoint rooted. Bootstrap support values are shown for each clade, and clades with support <50% are collapsed into polytomies. Clades without bootstrap values have >95% support. Non-centipede images are sourced from Phylopic (www.phylopic.org; credit for the Opiliones image is with Gareth Monger: https://creativecommons.org/licenses/by/3.0/).

GM crops36–39. They are produced by Bacillus species in the result of two HGTs from different bacterial phyla. T. longicornis B. cereus group40,41, especially B. thuringiensis, the entomopatho- centiPAD derives from Gammaproteobacteria, while L. forficatus genic bacterium from which they were first described, and which centiPAD derives from Bacteroidetes (Fig. 3; see Supplementary feeds on the insects killed by the toxin42. Cry toxins consist of Fig. 3 for full tree). The centiPAD sequences are deeply nested three conserved domains: an N-terminal domain of α-helices that within a large tree of bacterial sequences, confirming that human is thought to be responsible for insertion into the cell membrane and bacterial PADs are evolutionarily unrelated45,46. Interest- and pore formation, plus a middle and a C-terminal domain ingly, the nesting of four fungal branches and a sequence derived comprising β-sheets that are involved in receptor interactions, from the black garden ant Lasius niger within the paraphyletic and which may confer host-specific toxicity37,43,44. Cry toxins are backbone of bacterial sequences suggest that PAD was transferred not secreted, but released as parasporal crystalline bodies through multiple times from bacteria to other eukaryotes as well. lysis of the spore-forming bacterial cell. The Cry toxin genes are We cannot categorically reject the possibility that centiPADs located on plasmids, and plasmid transfer may explain why three- are produced by bacterial symbionts, which, if true, would be the domain Cry proteins or genes have been found in several bacterial second example of an animal venom component being produced species outside the B. cereus group37,41. by bacteria47. However, the balance of evidence suggests that In addition to three-domain Cry proteins our tree also contains centiPADs are a bona fide centipede gene family. CentiPAD is a sequences from a broad range of bacterial phyla that only contain prominent component of the venom proteome of T. longicornis22, a single Cry toxin domain, which in all cases is the pore-forming which is incompatible with it being due to accidental bacterial N-terminal domain. The centipede and other eukaryotic PCPDP- contamination. The sequences of T. longicornis can be up to 78% like sequences likewise only contain this N-terminal domain. A similar to the most closely related bacterial PAD sequences, but hint of how centipedes may have repurposed an insecticidal they share unique features that separate them from all bacterial bacterial toxin into a venom protein is suggested by the most sequences grouped in the same clade. Compared to related PAD closely related bacterial sequences. All bacterial sequences that sequences derived from the gammaproteobacterial genera group together with the centipede sequences in the clade at the Pseudomonas, Cedecea, Aeromonas, Serratia, Stenotrophomonas, top of Fig. 2 also only contain the pore-forming N-terminal and Acinetobacter, as well as the betaproteobacterial genera domain, and like the centipede sequences include a signal peptide Achromobacter, Paucibacter, and Undibacterium, the centiPAD region. This suggests that the bacterial proteins are exotoxins that sequences uniquely have a Met593 and a single amino acid are secreted from cells, like the centipede PCPDP-like proteins. deletion at position 606 (see alignment in Supplementary Data 2). Unlike the centipede sequences, the bacterial sequences in this These distinctive differences further support the conclusion that clade also contain C-terminal cell wall-binding repeats (InterPro the T. longicornis centiPADs are bona fide centipede sequences. accession IPR018337), and/or a ricin B lectin domain (InterPro The Lithobius centiPAD sequences group together in a strongly accession IPR000772). Cell wall-binding and ricin domains could supported clade without interleaving bacterial sequences. This help bind such putative exotoxins to bacterial or eukaryotic host clade groups sequences from specimens collected in the UK, cells, enabling the N-terminal perforating domain’s cytolytic continental Europe, and North America23,48,49. This strongly action. The centipede PCPDP-like sequences may derive from suggests that they are bona fide centipede sequences, a conclusion such putative bacterial exotoxins, followed by loss of these target- in line with the lack of evidence for microorganisms in the venom binding domains. Alternatively, the centipede proteins may derive system of L. forficatus35. The European sequences (represented by from a bacterial endotoxin, either a non-secreted single-Cry- UK sequences; an identical German sequence was excluded) form toxin-domain protein, or a true three-domain Cry toxin, by a sister clade to the American sequences. Because the latter were adding a signal peptide. The low sequence similarity of the not determined to species by the original collectors48, it is unclear bacterial and centipede sequences makes it impossible to if they are L. forficatus, which was imported from Europe to distinguish these possibilities. However, it is unlikely that only North America some time before the end of the 19th century50. the N-terminal domain was transferred from bacteria and joined CentiPAD is absent from the transcriptomes of other lithobio- to a native centipede sequence because BLAST searches of the C- morph species: Eupolybothrus cavernicolus, Paralamyctes validus, terminal region of the PCPDP-like sequences against centipede and Anopsobius giribeti51,52. With the exception of E. cavernico- transcriptomes and the genome of S. maritima produce no hits. lus, no venom glands were included in these transcriptomes, so these could be false negatives. However, the mean GC content of the UK centiPAD sequences is on the edge of the first quartile of Two bacterial HGTs of centiPADS. We previously detected the all non-HGT venom protein sequences (0.385 vs. 0.384) from all enzyme peptidylarginine deiminase (PAD) in the venoms of two centipede species analysed in our previous study23 (see distantly related centipede species, Thereuopoda longicornis Supplementary Data 3), which suggests that the HGT probably (order Scutigeromorpha), and Lithobius forficatus (order Litho- occurred relatively recently. biomorpha)22,23. Our phylogenetic analysis shows that these ArecenttransferisalsolikelyfortheT. longicornis centiPADs. sequences are positioned in different parts of the tree, separated The mean GC content of the three T. longicornis centiPAD by many strongly supported nodes. Hence, centiPADs are the sequences (0.588) is extremely skewed in the other direction and

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Proteobacteria (Pseudomonas sp. Irchel 3E13 WP_095156798.1) Firmicutes (2) Firmicutes (4) Firmicutes (5) 78 Proteobacteria (Bacillus anthracis WP_098926211.1) 5365 Proteobacteria (Bacillus toyonensis WP_098806848.1) 92 78 Lithobiomorpha (79) 57 Proteobacteria (Bacillus toyonensis WP_098806846.1) Firmicutes (3) Firmicutes (14) Firmicutes (13) Firmicutes (3) Firmicutes (3) Firmicutes (3) Amoebozoa (Heterostelium album XP_020434409.1) Firmicutes (42) 63 Proteobacteria (7) 69 Firmicutes (2) 66 Amoebozoa (Tieghemostelium lacteum KYQ94056.1) 60 Firmicutes(9) Firmicutes(18) Firmicutes (453) 68 Amoebozoa (Heterostelium album XP_020432798.1) Firmicutes (55) Euryarchaeota (2) Proteobacteria (Bacillus sp. WP_100406991.1) 71 Proteobacteria (2) Actinobacteria (2) 92 Chloroflexi (Ktedonobacterales bacterium WP_129890729.1) 93 Proteobacteria (Corallococcus sp. WP_120623710.1) 75 Nitrospirae (2) Bacteroidetes (11) Proteobacteria (5) Proteobacteria (11) Ascomycota (7) Proteobacteria (Burkholderia sp. WP_082720952.1) Actinobacteria (5) 77 Proteobacteria (2) Tardigrada (Hypsibius dujardini OQV11790.1) 90 Proteobacteria (Lysinibacillus odysseyi WP_036155538.1) Proteobacteria (Thalassospira sp. WP_071239846.1) 64 87 Anthozoa (Exaiptasia pallida KXJ27691.1) 80 Proteobacteria (Brevibacillus reuszeri WP_084765780.1) 90 95 Firmicutes (Cohnella sp. K2E09-144 WP_119147942.1) 85 Proteobacteria (Nitrosomonas oligotropha WP_107801843.1) Bacteroidetes (Tenacibaculum sp. WP_093871939.1) 79 94 Proteobacteria (Desulfobacteraceae OQY47759.1) 82 Bacteroidetes (2) 50 82 85 Proteobacteria (Xanthomonadaceae WP_129831580.1) Bacteroidetes (Lewinellaceae WP_099008298.1) 71 Rhodophyta (Chondrus crispus XP_005716346.1) 78 Proteobacteria (Myxococcus virescens SDE85457.1) 87 93 76 52 Ascomycota (2) 78 Ascomycota (4) 69 Ascomycota (4) Bacteroidetes (Pseudarcicella hirudinis WP_092018062.1) Proteobacteria (Thalassobium sp. PHS66030.1) 95 Proteobacteria (Helicobacter pylori WP_097717626.1) Amoebozoa (Tieghemostelium lacteum KYQ96860.1) 95 Actinobacteria (Nocardia kruczakiae WP_084461239.1) Proteobacteria (Xenorhabdus nematophila WP_013141600.1) Chlorophyta (2) 82 Amoebozoa (6) 61 Insecta (Coleoptera; 8) Proteobacteria (14) 80 Proteobacteria (5) 83 Proteobacteria (5) Proteobacteria (3) Proteobacteria (Minicystis rosea WP_146733695.1) Proteobacteria (11) Actinobacteria (Streptomyces caatingaensis WP_078871461.1) Ascomycota (39) 69 Streptophyta (3) 66 Amoebozoa (37) 62 Amoebozoa (Dictyostelium discoideum XP_646744.1) 93 Amoebozoa (9) Amoebozoa (17) Amoebozoa (Dictyostelium purpureum XP_003285408.1) Amoebozoa (9) Amoebozoa (2) Amoebozoa (8) Amoebozoa (Acytostelium subglobosum XP_012750528.1) Amoebozoa (Acytostelium subglobosum XP_012749234.1) Amoebozoa (3) Amoebozoa (9) Amoebozoa (9) Amoebozoa (7) Amoebozoa (2) 1.0

Fig. 2 A maximum likelihood tree of PCPDP-like sequences shows a clade of centipede sequences nested within a paraphyletic backbone of bacterial sequences. It shows that the centipede sequences originated from a bacterial HGT into the lithobiomorph lineage. Centipede sequences are coloured red. Highlighted sequences are Bacteria (pink), Viridiplantae (cyan), Protozoa (purple), Euryarchaeota (brown), and Fungi (yellow). Metazoan sequences are not highlighted. Collapsed clades have the number of included sequences indicated in parentheses. For the uncollapsed tree see Supplementary Fig. 2. The tree was reconstructed using the VT + G4 model and is displayed as midpoint rooted. Bootstrap support values are shown for each clade, and clades with support <50% are collapsed into polytomies. Clades without bootstrap values have >95% support. Non-centipede images are sourced from Phylopic (www.phylopic.org).

6 NATURE COMMUNICATIONS | (2021) 12:818 | https://doi.org/10.1038/s41467-021-21093-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21093-8 ARTICLE

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:3BBDJPDWLQHBGHLPLQDVHBIDPLO\BSURWHLQBB0\FREDFWHULXPBVSB'/B   :3BBDJPDWLQHBGHLPLQDVHBIDPLO\BSURWHLQBB5KRGRFRFFXVBWXNLVDPXHQVLVB   :3BBDJPDWLQHBGHLPLQDVHBIDPLO\BSURWHLQBB5KRGRFRFFXVBVSB-$B  ((1BK\SRWKHWLFDOBSURWHLQB5+2(5BBB5KRGRFRFFXVBHU\WKURSROLVB6.B  :3BBDJPDWLQHBGHLPLQDVHBIDPLO\BSURWHLQBB5KRGRFRFFXVBPDDQVKDQHQVLVB :3BBDJPDWLQHBGHLPLQDVHBIDPLO\BSURWHLQBB5KRGRFRFFXVBVSB81&0)7VXB Proteobacteria (Serratia marcescens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Fig. 3 A maximum likelihood tree of PAD sequences shows two clades of centiPAD sequences nested within a paraphyletic backbone of bacterial sequences. The tree represents one clade nested within a larger tree (red highlight in inset) made up entirely of bacterial sequences. The tree shows that centiPADs originated from two bacterial HGTs, one within the lithobiomorph lineage, and one within the scutigeromorph lineage. Centipede sequences are in black (present in transcriptomes) and red (present in transcriptomes and venom proteomes). Highlighted sequences are Bacteria (pink) and Fungi (yellow). Metazoan sequences are not highlighted. Collapsed clades have the number of included sequences indicated in parentheses. For the uncollapsed tree see Supplementary Fig. 3. The tree was reconstructed using the WAG + G4 model and is displayed as midpoint rooted. Bootstrap support values are shown for each clade, and clades with support <50% are collapsed into polytomies. Clades without bootstrap values have >95% support. Collembolan image was sourced from Phylopic (www.phylopic.org; credit for the Collembola image is with Birgit Lang: https://creativecommons.org/licenses/by/3.0/). falls outside the 99th percentile (0.557) of all non-HGT centipede for the pathogenic bacterium Porphyromonas gingivalis. Porphyr- venom protein sequences. This skew and the sequence similarity of omonas PAD (PPAD) is a major virulence factor that causes the centipede and bacterial sequences indicate that this HGT may inflammatory gum disease, and is a risk factor for rheumatoid have happened relatively recently. The absence of centiPAD arthritis45,46,54,55. How PPAD contributes to pathogenicity is an sequences from the transcriptomes of other scutigeromorphs active area of research, and it may include defusing the host’s (Scutigerina weberi, Sphendononema guilgingii,andScutigera immune system and the formation of protective biofilms55,56.Itis coleoptrata)23,52 provides further support for a relatively recent unknown what role centiPADs play in centipede venom but HGT.SinceonlythetranscriptomeofS. coleoptrata contains venom modulating the activity of other venom components through gland tissue the other two may be false negatives. We consider this posttranslational modification is one possibility. The centiPAD unlikely, however, because they represent different scutigeromorph sequences from both species have conserved the five catalytic families, while S. coleoptrata and T. longicornis belong to the family residues responsible for PPAD’s enzymatic activity (Asp1372, Scutigeridae. The unique presence of centiPAD in T. longicornis His2321, Asp2323, Asn2928, Cys4010 in the PAD alignment therefore suggests that this gene was transferred after its lineage split in Supplementary Data 5), but they have changed two residues that off from that of S. coleoptrata, which is estimated to have happened determine substrate specificity of bacterial PADs46. by about 200 million years ago53. Bacterial PAD converts peptidylarginine into citrulline residues, One or two bacterial HGTs of DUF3472-domain proteins. and the effects of this process have been most intensely investigated Proteins with a domain of unknown function DUF3472 (InterPro

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Bacteroidetes (4) Bacteroidetes (5) Streptophyta Selaginella moellendorffii XP_0029714289.1 Bacteroidetes (2) Bacteroidetes (3) Bacteroidetes (3) Proteobacteria (15) Proteobacteria (8) Proteobacteria Massilia sp. WP_107873156.1 73 Proteobacteria (4) Basidiomycota (15) Proteobacteria (5) Bacteroidetes (13) Bacteroidetes (23) Bacteroidetes (7) 79 Bacteroidetes (7) 64 Bacteroidetes (2) 90 Zoopagomycota Conidiobolus coronatus KXN70138.1 Bacteroidetes Elizabethkingia meningoseptica WP_016199623.1 Bacteroidetes Elizabethkingia sp. WP__107807162.1 86 Bacteroidetes (3) 78 Bacteroidetes (3) 80 Bacteroidetes (15) 50 Bacteroidetes (2) 66 Bacteroidetes (40) 83 Bacteroidetes (78) Bacteroidetes (99) Copepoda (2) Hydrozoa Hydra vulgaris XP_002157558.3 93 Bacteroidetes (3) 53 Bacteroidetes (3) Bacteroidetes (8) Bacteroidetes Arsenicibacter rosenii WP_071502151.1 Bacteroidetes (12) Bacteroidetes (7) Bacteroidetes (77) Bacteroidetes Arachidicoccus sp. WP__119987790.1 81 85 Ciliophora (12) Collembola (4) Bacterium Sp. AIA99582.1 Bacteroidetes Chryseobacterium bernardetii WP_123870227.1 Bacteroidetes (19) Bacteroidetes (67) Zoopagomycota (3) Mucoromycota (14) Bacteroidetes Sp. KXK19710.1 89 Scolopendromorpha (Scolopendra; 4) Geophilomorpha (Himantarium/Strigamia; 7) 94 Scolopendromorpha (Scolopendra; 4) Geophilomorpha (Strigamia maritima; 2) 91 Scolopendromorpha (Cryptops; 9) 87 Geophilomorpha (Strigamia maritima; 7) Scolopendromorpha (Ethmostigmus/Scolopendra; 5) Scolopendromorpha (Ethmostigmus/Cormocephalus/Cryptops; 14) Lithobiomorpha (Eupolybothrus/Lithobius; 7) 86 Bivalvia (2) Bacterium Sp. RYX84168.1 71 Verrucomicrobia Sp. RME90600.1 88 59 88 Verrucomicrobia (2) 94 Branchiopoda (2) 87 Acidobacteria Sulfopaludibacter sp. SPE28318.1 74 Proteobacteria Insolitispirillum peregrinum WP_076399794.1 Planctomycetes (2) Planctomycetes (17) Verrucomicrobia (7) Proteobacteria Bdellovibrionales PIP89043.1 Copepoda (9) 62 Platyhelminthes Macrostomum lignano PAA79273.1 Actinobacteria (3) Anthozoa (2) 51 Bacteroidetes (9) 62 Firmicutes (4) 7071 Proteobacteria Chromobacterium sp. WP_107798958.1 86 83 70 Gastropoda Pomacea canaliculata PVD23267.1 71 Bacteroidetes/Proteobacteria (7) 81 Calditrichaeota Sp. RPI03071.1 Bacterium Sp. WP_120394777.1 Armatimonadetes Fimbriimonas ginsengisoli AIE84776.1 Proteobacteria (10) Kiritimatielliota/Planctomycetes/Bacteroidetes/ Lentisphaerae/Verrucomicrobia/Firmicutes/ 1.0 Armatimonadetes/Acidobacteria(76) accession IPR021862) are found in the venom of several species of IPR031712). Our phylogenetic analysis places the centipede scolopendromorph centipedes, as well as in geophilomorph and sequences into two clades separated by bacterial and metazoan lithobiomorph venom gland and non-venom gland sequences (Fig. 4; see Supplementary Fig. 4 for full tree). This transcriptomes23,33,34,57,58. In addition, many of the sequences suggests that DUF3472-domain proteins may have transferred have an N-terminal DUF5077 domain (InterPro accession twice from bacteria to centipedes, once into the lineage leading to

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Fig. 4 A maximum likelihood tree of sequences with DUF3472-domains shows two clades of centipede sequences nested within a paraphyletic backbone of bacterial sequences. This suggests that the centipede sequences may have originated from two bacterial HGTs, one into the epimorphan lineage, and one within the lithobiomorph lineage. However, tree topology tests cannot reject centipede monophyly (see Supplementary Data 1). Centipede sequences are coloured blue (present in transcriptomes) and red (present in transcriptomes and venom proteomes). Highlighted sequences are Bacteria (pink), Protozoa (purple), Streptophyta (cyan), and Fungi (yellow). Metazoan sequences are not highlighted. Collapsed clades have the number of included sequences indicated in parentheses. For the uncollapsed tree see Supplementary Fig. 4. The tree was reconstructed using the WAG + R10 model and is displayed as midpoint rooted. Bootstrap support values are shown for each clade, and clades with support <50% are collapsed into polytomies. Clades without bootstrap values have >95% support. Copepod image was sourced from Phylopic (www.phylopic.org; credit for the Collembola image is with Birgit Lang: https://creativecommons.org/licenses/by/3.0/).

Epimorpha (geophilomorphs and scolopendromorphs), and once twice into centipedes. This may also be true for the springtails, into lithobiid lithobiomorphs. Topological tree tests cannot sta- where unchar05 homologues are found in at least two different tistically reject centipede monophyly, but do reject metazoan families, and whose genomes contain hundreds of genes of HGT monophyly (see Supplementary Data 1). This shows that origin5,6. Moreover, the tree also contains a well-supported clade DUF3472-domain proteins have been transferred from bacteria of mite sequences that includes species that have also previously to animals multiple times, like β-PFTxs and PCPDP-like proteins. been shown to have received horizontally transferred fungal DUF3472-domain proteins from S. maritima map to four genes3. protein-coding genomic loci with introns (see Table 1), and the Although a tree topology test cannot reject metazoan tree suggests that these and the multiple copies found in scolo- monophyly in our tree (see Supplementary Data 1), we pendromorphs are the result of several rounds of gene consider the alternative hypothesis of a single early HGT of duplication. unchar05 into animals followed by rampant losses to be less convincing. To explain the large phylogenetic disjunction of the sequences on various levels—within centipedes, within Multiple HGTs between fungi, oomycetes and centipedes. insects, and within animals—would require an immense Centipedes not only express four gene families in their venoms amount of gene loss throughout the animal kingdom to leave that were horizontally transferred from bacteria, but also three just this handful of metazoan homologues, several of which gene families that find their nearest homologues in fungi and represent taxa already known to be recipients of horizontally oomycetes (water molds). GEOTX02 is a peptide present in the transferred genes. venom of the geophilomorph S. maritima, and similar sequences The third gene family that has probably undergone eukaryotic with a corresponding cysteine framework are restricted to a few HGT is Unchar16. It encodes cysteine-rich proteins found in the species of ascomycete fungi. The sequences exhibit two distinct venom gland and non-venom gland transcriptomes of pleuros- cysteine patterns, with 8 or 10 cysteine residues in the mature tigmophoran (non-scutigeromorph) centipedes, as well as in the domain of the peptide, with the latter being restricted to the top venom of the craterostigmomorph Craterostigmus tasmanianus. clade in the tree with 85% bootstrap support (Fig. 5a; see Sup- Unchar16 maps to two protein-coding paralogous loci with plementary Fig. 5 for full tree). The centipede sequences map to introns in the genome of S. maritima (see Table 1). Our searches four paralogue groups of genes with introns in the genome of identified small secretory proteins from plant-parasitic oomycetes S. maritima, with the clade with 74% bootstrap support repre- as homologues based upon sequence similarity and corresponding senting paralogue groups 1–3 and the collapsed clade of eleven cysteine patterns. Unchar16 has undergone marked sequence S. maritima sequences representing paralogue group 4 (see evolution in centipedes, and all centipede sequences group in a Table 1). The tree suggests that the centipede sequences with the well-supported clade when the tree is rooted with oomycetes two different cysteine patterns may have resulted from two (Fig. 5c; see Supplementary Fig. 7 for full tree). However, two HGTs, although a tree topology test cannot reject centipede different HGT scenarios may explain the data depending on how monophyly (see Supplementary Data 1), and the direction of the tree is rooted. these horizontal transfers remains uncertain. The ascomycetes Oomycetes originated at about the same time as centipedes, about included in the tree belong to two orders (Dothideomycetes and 430 million years ago59, so a HGT between early oomycete and Sordariomycetes) and include species known to infect animals centipede lineages is possible if unchar16 was transferred from and plants. The transfers therefore possibly involved an oomycetes into the stem lineage of pleurostigmophoran centipedes. arthropod-infecting ascomycete as either a donor or recipient of However, the early evolutionary history of oomycetes and the GEOTX02. taxonomic distribution of oomycete unchar16 homologues casts Unchar05 is another venom protein family that has been doubt on this scenario. Early diverging oomycete lineages are horizontally transferred between centipedes and fungi. Unchar05 exclusively marine, with the exception of the genus Haptoglossa60,61. is present in the venom of S. maritima but is also found in a trunk Moreover, the oomycete homologues of unchar16 that we identified transcriptome of the lithobiomorph Paralamyctes validus. The belong to the predominantly terrestrial oomycete order Peronospor- two unchar05 transcripts identified in the venom proteome of ales, which is a lineage that evolved much later, in the early S. maritima map to a protein-coding genomic locus with introns Mesozoic about 225-190 million years ago59. This suggests that (SMAR002275), which is one of five paralogous loci (see Table 1), unchar16 may have horizontally transferred much more recently four of which are expressed as transcripts in the venom gland of from centipedes into the peronosporalean lineage of oomycetes—the S. maritima. Our phylogenetic analysis (Fig. 5b; see Supplemen- reverse transfer would require independent HGTs into all four tary Fig. 6 for full tree) shows that unchar05 was transferred into pleurostigmophoran centipede lineages. HGT is known to have centipedes from fungal donors. The centipede sequences group in contributed to the evolution of oomycete secretomes62,63,butwhich a clade with sequences from two species of springtails, Folsomia centipede lineage functioned as a donor of unchar16 in this scenario candida and Orchesella cincta, but neither the centipede nor the remains unclear given the lack of resolution in the tree. On the springtail sequences are monophyletic. This taxonomic interleav- balance of available evidence, we prefer this second scenario, but ing of sequences and the phylogenetic disjunction between the hope that future research will shed further light on this tantalizing centipede species suggest that unchar05 horizontally transferred riddle.

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Fig. 5 Maximum likelihood trees showing eukaryotic HGTs between a Geophilomorpha (Strigamia maritima c37290 g1 i1) 85 Ascomycota (Ophiocordyceps spp.; 2) fungi, oomycetes, and centipedes. a Tree of GEOTX02 homologues 68 Ascomycota (Pseudogymnoascus spp.; 4) showing that the centipede sequences are distributed across two clades, Ascomycota (Glonium stellatum OCL13495.1) 71 Ascomycota (Venturia spp.; 3) and interleaved with ascomycete sequences. The direction and number of Geophilomorpha (Strigamia maritima; 2) HGTs (one or two) is uncertain. The tree was reconstructed using the Geophilomorpha (Strigamia maritima c15356 g1 i1) VT + I + G4 model and is midpoint rooted. For the uncollapsed tree see 85 65 Geophilomorpha (Strigamia maritima c26251 g1 i1) 74 Geophilomorpha (Strigamia maritima c4996 g1 i2) Supplementary Fig. 5. b Tree of unchar05 homologues showing the four Geophilomorpha (Strigamia maritima; 3) 0.5 centipede sequences grouping in a clade with two collembolan sequences, Geophilomorpha (Strigamia maritima; 11) nested within a paraphyletic backbone of fungal sequences. The tree shows

b Basidiomycota (6) that the centipede sequences likely originated from two fungal HGTs, one Chytridiomycota (3) into the geophilomorph lineage, and one within the lithobiomorph lineage. 90 Ascomycota (2) + Parasitiformes (5) The tree was reconstructed using the WAG R5 model and is midpoint Ascomycota (3) rooted. For the uncollapsed tree see Supplementary Fig. 6. c Tree of Ascomycota (Hypoxylon sp. OTB05368.1) unchar16 homologues showing a clade of centipede sequences that is the Basidiomycota (16) Basidiomycota (Fomitopsis pinicola EPS94083.1) sister group to a clade of oomycete sequences. The direction of HGT is 56 Ascomycota (2) unclear. The tree was reconstructed using the VT + R3 model and is rooted 75 Ascomycota (Fusarium longipes RGP72136.1) with the oomycete sequences. For the uncollapsed tree see Supplementary Basidiomycota (2) Insecta (Folsomia candida XP_021950037.1) Fig. 7. For each tree, bootstrap support values are shown for each clade and 85 Lithobiomorpha (Paralamyctes validus; 2) clades with support <50% are collapsed into polytomies. Clades without 63 Geophilomorpha (Strigamia maritima; 2) Insecta(Orchesella cincta ODM87843.1) bootstrap values have >95% support. Centipede sequences are coloured 78 81 Ascomycota (27) black (present in transcriptomes) and red (present in transcriptomes and Ascomycota (7) 71 venom proteomes). Highlighted sequences are Fungi (yellow), Rhodophyta Basidiomycota (Calocera viscosa KZO91091.1) 67 88 Rhodophyta (2) (reddish brown), and Streptophyta (cyan). Metazoan sequences are not 83 Basidiomycota (Dendrothele bispora THV00782.1) highlighted. Collapsed clades have the number of included sequences Ascomycota (Sphaerosporella brunnea KAA8907998.1) 81 Ascomycota (Aspergillus wentii OJJ38332.1) indicated in parentheses. Non-centipede images are sourced from Phylopic Basidiomycota (2) (www.phylopic.org; credit for the Collembola images is with Birgit Lang: Basidiomycota (17) https://creativecommons.org/licenses/by/3.0/). Basidiomycota (Morchella conica RPB15122.1) 79 Ascomycota (Pluteus cervinus TFK60643.1) Ascomycota (5) Streptophyta (Rhodobryum sp. TR14763 c1 g1 i1) 85 0.5 Porifera (2) centipedes are the first known animals with venoms used for predation and defence that contain multiple gene families derived c Oomycetes (7) from HGT. It is likely that HGT contributions to venom Lithobiomorpha (4) Craterostigmomorpha (C. tasmanianus (15) evolution are a much more widespread phenomenon. More than Craterostigmomorpha (C. tasmanianus VG TR27226 c0 g1 i1) a hundred animal lineages have evolved venoms8, and recent 70 Craterostigmomorpha (C. tasmanianus VG TR27226 c0 g1 i5) 74 Craterostigmomorpha (C. tasmanianus VG TR27226 c0 g1 i6) proteotranscriptomic studies of venoms from a wide range of taxa Craterostigmomorpha (C. tasmanianus VG TR27226 c0 g1 i2) have identified substantial numbers of protein families with few Craterostigmomorpha (C. tasmanianus VG TR27226 c0 g1 i3) 16–21 Craterostigmomorpha (C. tasmanianus VG TR27226 c0 g1 i4) or no known metazoan homologues (e.g. ). Such gene Craterostigmomorpha (C. tasmanianus trunk TR37768 c0 g1 i2) families are especially promising for identifying new HGT 75 Lithobiomorpha (4) 80 85 54 Scolopendromorpha (S. morsitans c14987 g1 i1) candidates, but this requires a targeted approach, like the one 61 Scolopendromorpha (2) adopted here, that goes beyond the standard BLAST-based Scolopendromorpha (S. morsitans c9604 g1 i1) annotation pipelines commonly used in venom profiling studies. Scolopendromorpha (6) Scolopendromorpha (4) Our findings expand the insights generated by previous 86 Lithobiomorpha (8) research into how HGT can increase the adaptive versatility of Craterostigmomorpha (C. tasmanianus (2) 1,2 81 Lithobiomorpha (2) organisms . Our results suggest that HGT can allow a venomous 91 91 Craterostigmomorpha (C. tasmanianus trunk TR30700 c1 g1 i2) lineage to reap the immediate adaptive benefits of genes evolved Craterostigmomorpha (C. tasmanianus trunk TR30700 c1 g1 i1) Lithobiomorpha (4) in unrelated lineages if the gene products are preapted to a venom  Geophilomorpha(5) function. For instance, the incorporation of a cytolytic bacterial pore-forming toxin, such as β-PFTx, into the ancestral centipede venom may have conferred an immediate functional benefit, for HGT is a potentially major mechanism of venom evolution. example in prey immobilisation. In this scenario, the pore- Our results suggest that HGT has been a key factor in the forming activity of the bacterial protein is a preaptation that expansion and diversification of centipede venoms in all five would have allowed the protein to take on this function in the orders throughout their evolutionary history (Fig. 6). Because centipede venom without first having to evolve modifications to genes were horizontally transferred from bacteria and fungi both gain a venom function. This parallels, for example, the use of deeply and repeatedly in the phylogeny of centipedes, we expect detoxifying enzymes by herbivorous arthropods that were that the vast majority of centipede species produce venoms that horizontally transferred from, and similarly used, by bacterial include multiple horizontally transferred components. Because and fungal donors64. The selective benefit of the horizontal proteotranscriptomic venom profiles are currently available for transfer of β-PFTx into the earliest centipede venom could have only a small number of the more than 3,100 described species of been substantial because it is just one of two putative toxins that centipedes, new insights into the full impact of HGT on centipede could have been involved in prey immobilization. The other three venom evolution are likely to emerge from future studies. protein families that we reconstructed as present in the ancestral Our findings increase the number of animal venom protein centipede venom are metalloprotease family M12A, glycoside families with well-supported HGT origins from three to at least hydrolase family 18, and centipede CAP1 (cysteine-rich secretory eight, which increases the number of known HGT events stocking proteins, antigen 5 and pathogenesis-related protein family 1), venom arsenals from five or six to at least thirteen. We show that which is the second putative venom toxin23. The recruitment of

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Pleurostigmophora Lithobius (7551), centipedes (7540); centiPAD: L. forficatus (7552), T. longicornis Phylactometria (353555), centipedes: 7540; DUF3472: S. maritima (126957), E. rubripes (62613), Epimorpha Himantarium gabrielis (241672), Sco. morsitans (943129), Sco. subspinipes (55038), C. westwoodi (1096223), Cryptops hortensis (1268897), L. forficatus (7552), cen- tipedes (7540); GEOTX02: S. maritima (1256957), centipedes (7540); unchar05: S. maritima (126957), centipedes (7540); unchar16: Craterostigmus tasmanianus Tl Sc Lf Ec Hen Ct Geo Scolo (60162), Sco. morsitans (943129), L. forficatus (7552), S. maritima (126957), cen- centiPAD centiPAD unchar05 Craterostigmomorpha GEOTX02? tipedes (7540); SLPTX02: centipedes (7540), L. forficatus (7552), Scu. coleoptrata unchar05 -PFTx (29022), C. tasmanianus (60162), C. hortensis (1268897), H. gabrielis (241672), Lithiobius (7551), Sco. Subspinipes (55038), E. rubripes (62613). Results are sum- PCPDP- Lithobiomorpha like marized in Supplementary Data 7. The PCPDP-like gene family didn’t generate any BLAST hits. However, because the sequences contain an insecticidal delta- Scutigeromorpha endotoxin domain known only from bacteria we included this gene family in our analyses as well. SLPTX02 was dropped from further consideration because the broad phylogenetic distribution of homologues suggests an ancient origin of this protein family. DUF3472

Chilopoda -PFTx Construction of phylogenetic datasets. All analyses were performed on amino acid translations of the transcriptome sequences. We used HMMER v3.2.1 Fig. 6 Phylogenetic distribution of centipede venom gene families (http://hmmer.org) with default settings to generate Hidden Markov Models for horizontally transferred from bacteria and fungi. ‘?’ indicates uncertainty each of the seven venom protein families with possible non-metazoan origins, and ’ in the direction of transfer. Taxon abbreviations are as follows. Tl: retained all hits above HMMER s default inclusion threshold (per-sequence E-value of 0.01). Geneious version 11.1.5 (https://www.geneious.com) was used to con- fi Thereuopoda longicornis; Sc: Scutigera coleoptrata; Lf: Lithobius for catus; Ec: struct alignments for training HMMER profiles, using the local paired iterative Eupolybothrus cavernicolus; Hen: Henicopidae; Ct: Craterostigmus alignment method (L-INS-i) in MAFFT v7.45066 (see Supplementary Data 5 and 6 tasmanianus; Geo: Geophilomorpha; Scolo: Scolopendromorpha. for the alignments and profiles). We included in these alignments all the full-length centipede sequences that we generated for these gene families in our previous studies22,23. For β-PFTxs and the PCPDP-like gene family, we additionally inclu- β fi ded a selection of outgroup taxa, and the PCPDP-like alignment was limited to the -PFTx into the ancestral centipede venom represents the rst N-terminal Cry toxin domain. We used the HMMER profiles to search against a known example of HGT contributing to the evolutionary origin local fasta version of the nr database (downloaded from the NCBI FTP Server of venom in a lineage. Horizontal transfer could therefore have ftp://ftp.ncbi.nlm.nih.gov/ on 21 May 2019) for possible homologues of the cen- been a crucial step in setting centipedes on the selective trajectory tipede sequences. We also used these profiles to search a previously published67 custom database of 155 de novo assembled and translated transcriptomes obtained that eventually led to the complex venoms of modern species. from the NCBI Sequence Read Archive (SRA), representing 134 animal species, The fact that the centipede venom homologues of the three with 121 arthropod species including eight millipede whole body and eight cen- horizontally transferred bacterial virulence factors for which there tipede whole body or trunk transcriptomes, as well as seven species of fungi, plants, are functional data have retained the structural domains involved and choanoflagellates (see Supplemental Table S2 in Dash et al.67). This database in pore-formation (β-PFTx and PCPDP-like proteins), or was supplemented with assembled transcriptomes for the centipedes Paralamyctes validus, Anopsobius giribeti, Scutigerina weberi, and Sphendononema guildingii52. conserved the catalytic sites involved in enzymatic action Complementing these transcriptome-based sequence data we used the HMMER (centiPAD), is consistent with a continuity of function and profiles to search for homologues in 25 metazoan Ensembl genomes adaptive value from donor to recipient taxa. Moreover, the gene (http://ensemblgenomes.org) representing these major lineages: Cnidaria: Thelo- duplications that have subsequently occurred in the genome- hanellus kitauei, Nematostella vectensis; Placozoa: Trichoplax adhaerens; Cteno- fi phora: Mnemiopsis leidyi; Deuterostomia: Strongylocentrotus purpuratus; Rotifera: con rmed gene families underlines a commonly observed feature Adineta vaga; Brachiopoda: Lingula anatina; Mollusca: Octopus bimaculoides, of the route to the functional consolidation and diversification of Crassostrea gigas, Lottia gigantea; Annelida: Capitella teleta, Helobdella robusta; horizontally transferred genes2. Our results therefore show that Nematoda: Pristionchus pacificus, Caenorhabditis elegans; Arthropoda, Arachnida: HGT can provide a fast track channel for the evolution of novelty Ixodes scapularis, Sarcoptes scabiei, Tetranychus urticae, Stegodyphus mimosarum; Arthropoda, Pancrustacea: Daphnia pulex, Lepeophtheirus salmonis, Folsomia by the exaptation of bacterial weapons for new functions in candida, Nasonia vitripennis, Apis mellifera, Megaselia scalaris, Anopheles gambiae. animal venoms. Once a comprehensive list of homologues was generated, we removed identical sequences using CD-HIT v4.668, and examined and filtered false positives using CLC Main WorkBench v7 (Qiagen, Aarhus, Denmark) and Geneious v11.1.5 Methods (https://www.geneious.com). In the case of β-PFTx, we also filtered the non- Initial identification of HGT candidates. We used the transcriptomic and pro- chilopod sequences with CD-HIT to only include sequences with <95% sequence teomic data from Undheim et al.22 and Jenner et al.23 to identify HGT candidates identity due to a large number of identified unique homologues (2164 sequences). expressed in centipede venom glands and venoms. Manual inspection of BLAST To create datasets of manageable size for PAD, PCPDP, and DUF3472, while results generated for these studies for more than 90 venom protein families yielded retaining a broad net for capturing putative donor taxa and sampling metazoan sixteen protein families with either non-metazoan hits, and/or few or no metazoan homologues, the identified homologues were first sorted to Kingdom and then hits (β-PFTx, centiPAD, CHILOTX01, DUF3472, GEOTX02, LTHTX01, filtered with CD-HIT to include only sequences with <90% (bacteria, fungi, LTHTX03, PCPDP-like, SCTX01, SCTX02, SLPTX02, SLPTX04, SLPTX06, protists, and viruses) or 70% sequence identity (non-myriapod animals, Archaea, SLPTX30, unchar05, and unchar16.). We performed a protein BLAST search of and plants). Due to the large number of PAD homologues still retained by this these HGT candidate sequences against a local version of the NCBI non-redundant approach (6716 sequences), we then removed all sequences with a pairwise distance (nr) database (downloaded from the NCBI FTP Server ftp://ftp.ncbi.nlm.nih.gov/ to any chilopod sequence >0.5. on 5 June 2019) with BLAST version 2.4.0, and an E-value cut off of e-3. Significant The remaining sequences were aligned using the local paired iterative alignment hits against non-metazoan sequences were found for β-PFTx, centiPAD, DUF3472, method (L-INS-i) in MAFFT v7.304b66. For the alignment of GEOTX02, we first GEOTX02, PCPDP-like, unchar05, unchar16, and SLPTX02. These BLAST results aligned the structurally important conserved cysteines69, and then used the were submitted to the Alienness web server (http://alienness.sophia.inra.fr/cgi/ MAFFT regional alignment ruby script to align the pre-, inter-, and post-cysteine index.cgi), which is a tool designed to detect HGT candidates65. Alienness calcu- regions by local paired iterative alignment method as above. All alignments are lates an Alien Index for each query sequence based on the E-values of the best included in Supplementary Data 4. We used InterProScan70 as implemented in BLAST hits to putative candidate donors (non-metazoan) and recipient (metazoan) Geneious v11.1.5 (https://www.geneious.com) to generate protein domain taxa. The following taxa and taxon codes were excluded from the Alien Index annotations for all alignments (see Supplementary Data 8). The evolutionary calculations as self-hits for the different protein families that generated positive history of each protein family was then reconstructed using a molecular Alien Indices: β-PFTx: Cormocephalus westwoodi (1096223), Ethmostigmus phylogenetic approach. The most appropriate evolutionary model was determined rubripes (62613), Lithobius forficatus (7552), Scutigera coleoptrata (29022), Scolo- using ModelFinder71, before using IQ-TREE v1.5.572 to reconstruct molecular pendra alternans (1329349), Sco. morsitans (943129), Sco. subspinipes (55038), phylogenies by maximum likelihood, and estimating branch support values by Thereuopoda longicornis (353555), Ixodes scapularis (6945), Limulus polyphemus ultrafast bootstrap using 10,000 replicates73. Because taxonomic outgroups could (6850), Strigamia maritima (126957), Cryptops hortensis (1268897), Acuclavella not be designated we used midpoint rooting to root the trees. Trees were visualised merickeli (703423), Damon variegatus (317683), Cryptocellus becki (1642531), in Archaeopteryx v0.992174.

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R.A.J. is (2016). grateful to Luca Venturini and Nathan Kenny for providing bioinformatic assistance, and to 53. Giribet, G. & Edgecombe, G. E. Stable phylogenetic patterns in Greg Edgecombe, Matt Clark, Pete Olson and Ana Riesgo for discussions. We thank scutigeromorph centipedes (Myriapoda: Chilopoda: Scutigeromorpha): dating Gonzalo Giribet and Ligia Benavides for supplying assembled transcriptomes for the diversification of an ancient lineage of terrestrial arthropods. Invertebr. Paralamyctes validus, Anopsobius giribeti, Scutigerina weberi,andSphendononema guil- Syst. 27, 485–501 (2013). dingii. We would like to acknowledge the use of resources provided by UNINETT Sigma2 - 54. Bereta, G. et al. Structure, function, and inhibition of a genomic/clinical the National Infrastructure for High Performance Computing and Data Storage in Norway. variant of Porphyromonas gingivalis peptidylarginine deiminase. Protein Sci. 28, 478–486 (2019). Author contributions 55. Stobernack, T. et al. 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