Molecular Ecology A process of convergent amplification and tissue-specific expression dominate the evolution of toxin and toxin-like genes in sea anemones Journal: Molecular Ecology Manuscript ID MEC-18-1374.R1 Manuscript Type:ForOriginal Review Article Only Date Submitted by the 09-Mar-2019 Author: Complete List of Authors: Surm, Joachim; Queensland University of Technology Faculty of Health, ; Institute of Health and Biomedical Innovation, Smith, Hayden; Queensland University of Technology Faculty of Science and Engineering; Queensland University of Technology Institute for Future Environments Madio, Bruno; University of Queensland Institute for Molecular Bioscience Undheim, Eivind; University of Queensland Centre for Advanced Imaging King, Glenn F; University of Queensland Institute for Molecular Bioscience Hamilton, Brett; University of Queensland Centre for Advanced Imaging; University of Queensland Centre for Microscopy and Microanalysis van der Burg, Chloé; Queensland University of Technology Faculty of Health; Institute of Health and Biomedical Innovation Pavasovic, Ana; Queensland University of Technology, School of Biomedical Sciences Prentis, Peter Venom, Cnidaria, RNA-seq, phylogenetics, mass spectrometry imaging, Keywords: selective pressure Page 1 of 50 Molecular Ecology 1 Article 2 A process of convergent amplification and tissue-specific expression 3 dominate the evolution of toxin and toxin-like genes in sea 4 anemones 5 6 Joachim M. Surm1,2*, Hayden L. Smith3,4, Bruno Madio5, Eivind A. B. Undheim6, Glenn F. King5, Brett 7 R. Hamilton6,7, Chloé A. vanFor der Burg Review1,2, Ana Pavasovic1 ,Only and Peter J. Prentis3,4 8 9 1School of Biomedical Sciences, Faculty of Health, Queensland University of Technology 10 2Institute of Health and Biomedical Innovation, Queensland University of Technology 11 3School of Earth, Environmental and Biological Sciences, Science and Engineering Faculty, 12 Queensland University of Technology 13 4Institute for Future Environments, Queensland University of Technology 14 5Institute for Molecular Bioscience, University of Queensland 15 6Centre for Advanced Imaging, University of Queensland 16 7Centre for Microscopy and Microanalysis, University of Queensland 17 *Correspondence: [email protected]; 18 Molecular Ecology Page 2 of 50 19 Abstract 20 Members of phylum Cnidaria are an ancient group of venomous animals and rely on a number 21 of specialised tissues to produce toxins in order to fulfil a range of ecological roles including prey 22 capture, defence against predators, digestion, and aggressive encounters. However, limited 23 comprehensive analyses of the evolution and expression of toxin genes currently exists for cnidarian 24 species. In this study, we use genomic and transcriptomic sequencing data to examine gene copy 25 number variation and selective pressure on toxin gene families in phylum Cnidaria. Additionally, we 26 use quantitative RNA-seq and mass spectrometry imaging to understand expression patterns and 27 tissue localisation of toxin productionFor Review in sea anemones. UsingOnly genomic data, we demonstrate that the 28 first large scale expansion and diversification of known toxin genes occurs in phylum Cnidaria, a 29 process we also observe in other venomous lineages, which we refer to as convergent amplification. 30 Our analyses of selective pressure on sea anemone toxin gene families reveal that purifying selection 31 is the dominant mode of evolution for these genes and that phylogenetic inertia is an important 32 determinant of toxin gene complement in this group. The gene expression and tissue localisation data 33 revealed that specific genes and proteins from toxin gene families show strong patterns of tissue and 34 developmental-phase specificity in sea anemones. Overall, convergent amplification and phylogenetic 35 inertia has strongly influenced the distribution and evolution of the toxin complement observed in sea 36 anemones, while the production of venoms with different compositions across tissues is related to 37 the functional and ecological roles undertaken by each tissue type. 38 Keywords 39 Venom, Cnidaria, RNA-seq, phylogenetics, mass spectrometry imaging, selective pressure 40 1 Page 3 of 50 Molecular Ecology 41 1. Introduction 42 Venomous animals rely on their toxins for a range of ecological processes, including prey 43 capture, defence against predators, and intra and interspecific aggression (Casewell, Wüster, Vonk, 44 Harrison, & Fry, 2013; Fry et al., 2009). Toxins are primarily gene-encoded peptides and proteins that 45 evolved from ancestral “house-keeping” molecules that perform functions unrelated to venom 46 production in the body (Casewell et al., 2013). Venomous taxa have evolved multiple times during 47 metazoan evolution, and the genes that encode peptide and protein toxins are often considered to 48 evolve rapidly under positiveFor Darwinian Review selection, enhanced Only by a genetic redundancy generated 49 through gene duplication events (Casewell et al., 2013; Fry et al., 2009; Sunagar & Moran, 2015). 50 New evidence suggests that the evolution of toxin and toxin-like (TTL) genes is dominated by 51 purifying selection in ancient venomous lineages such as cnidarians, coleoids, and arthropods (Jouiaei 52 et al., 2015; Pineda et al., 2014; Ruder et al., 2013; Sunagar & Moran, 2015; Sunagar et al., 2013; 53 Undheim et al., 2014a, 2014b). This observation, however, does not account for gene age within these 54 taxa. Consequently, this calls for a comprehensive analysis of selective pressures on widespread gene 55 families (i.e., those shared in venomous lineages across a broad taxonomic distribution) versus those 56 that are lineage-specific (i.e., gene families restricted to particular phylum or order) to better 57 understand venom evolution in ancient lineages. Importantly, a lack of positive selection on TTL genes 58 indicates other evolutionary processes may play a key role in venom evolution in ancient taxa. 59 Cnidarians are the oldest venomous metazoan lineage (Erwin et al., 2011; Menon, McIlroy, & 60 Brasier, 2013; Park et al., 2012) and they are defined by their envenomation system, which consists of 61 specialised cells called cnidocytes (Fautin, 2009; Fautin & Mariscal, 1991; Kass-Simon & Scappaticci, 62 2002). Cnidocytes are distributed throughout the body, but they vary in density and morphology 63 across tissues (Beckmann & Özbek, 2012; David et al., 2008; Fautin, 2009; Fautin & Mariscal, 1991; 64 Özbek, 2010). This envenomation system is unique, and it allows cnidarians to produce toxins across 65 multiple tissues (Macrander, Broe, & Daly, 2016), whereas most venomous lineages show restricted 2 Molecular Ecology Page 4 of 50 66 expression of toxin genes within one or more isolated gland (Dutertre et al., 2014; Fingerhut et al., 67 2018; Gao et al., 2018; Modica, Lombardo, Franchini, & Oliverio, 2015; Undheim et al., 2015; Walker 68 et al., 2018). In sea anemone species, three tissue types have become highly specialised for different 69 ecological roles associated with venom delivery: acrorhagi are inflatable aggressive organs used in 70 intraspecific aggressive encounters, tentacles are used in prey capture and defence, and mesenteric 71 filaments are multifunctional morphological structures used principally in digestion and killing of prey 72 (Fautin & Mariscal, 1991; Kass-Simon & Scappaticci, 2002; Macrander, Brugler, & Daly, 2015; Prentis, 73 Pavasovic, & Norton, 2018). While venoms in sea anemones are by far the most well studied among 74 cnidarians, toxin gene expressionFor and Review protein localisation Only patterns across these functionally distinct 75 tissues remains largely unexplored. Significantly, as evidence supports that changes in TTL gene 76 expression may generate different venom profiles (Amazonas et al., 2018), we hypothesise that in sea 77 anemones, TTL gene expression varies among tissue types and correlates with their distinct ecological 78 functions. 79 In this paper, we comprehensively surveyed the TTL gene complement in both venomous and 80 non-venomous taxa using comparative genomics, which revealed that the total TTL gene repertoire 81 has expanded in the majority of known venomous lineages investigated. We refer to this process as 82 convergent amplification and it is the result of convergent recruitment (Fry et al., 2009) followed by 83 an increase in copy number of toxin-encoding genes. Our results indicate that the first evidence of 84 convergent amplification is observed in phylum Cnidaria, the oldest extant venomous lineage. As with 85 all cnidarians, sea anemones (actiniarians) share a common venomous ancestor, and their TTL genes 86 are the best studied among all cnidarian groups. Consequently, we performed a fine scale comparative 87 analysis on actiniarian transcriptomes, to systematically investigate the toxin gene complement and 88 selective forces acting on widespread and lineage-specific TTL gene families in this group. Finally, 89 functional genomic analyses were performed on a candidate sea anemone, Actinia tenebrosa, using 90 quantitative RNA-seq and mass spectrometry imaging (MSI), to investigate whether functionally 3 Page 5 of 50 Molecular Ecology 91 distinct tissue types generate different venom profiles consistent with their ecological functions. 92 93 2. Materials and methods 94 2.1 Identification of TTL genes 95 The first aim of this study was to comprehensively investigate the distribution, copy number 96 and evolution of TTL genes and gene