bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Title: Phylogenetic inference identifies two eumetazoan TRPM clades and an 8th family of TRP channel, TRP soromelastatin (TRPS)

Authors and Affiliations: Nathaniel J. Himmel1,*, Thomas R. Gray1, and Daniel N. Cox1,*

1 – Neuroscience Institute, Georgia State University, Atlanta, GA, 30303 * - Corresponding authors

Authors for Correspondence:

Nathaniel J. Himmel Daniel N. Cox Neuroscience Institute Neuroscience Institute Georgia State University Georgia State University P.O. Box 5030 P.O. Box 5030 Atlanta, GA 30302-5030 Atlanta, GA 30302-5030 [email protected] [email protected] bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Abstract: 2 TRP melastatins (TRPMs) are most well-known as cold and menthol sensors, but are in fact 3 broadly critical for life, from ion homeostasis to reproduction. Yet the evolutionary relationship 4 between TRPM channels remains largely unresolved, particularly with respect to the placement 5 of several highly divergent members. To characterize the evolution of TRPM and like channels, 6 we performed a large-scale phylogenetic analysis of >1,300 TRPM-like sequences from 14 phyla 7 (Annelida, Arthropoda, Brachiopoda, Chordata, Cnidaria, Echinodermata, Hemichordata, 8 Mollusca, Nematoda, Nemertea, Phoronida, Priapulida, Tardigrada, and Xenacoelomorpha), 9 including sequences from a variety of recently sequenced genomes that fill what would 10 otherwise be substantial taxonomic gaps. These findings suggest: (1) The previously recognized 11 TRPM family is in fact two distinct families, including canonical TRPM channels, and an 8th 12 major, previously undescribed family of animal TRP channel, TRP soromelastatin (TRPS); (2) 13 two TRPM clades predate the last bilaterian-cnidarian ancestor; and (3) the vertebrate-centric 14 trend of categorizing TRPM channels as 1-8 is inappropriate for most phyla, including other 15 chordates. 16 17 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Main Text: 2 Introduction 3 Transient receptor potential (TRP) channels are a superfamily of ion channel commonly 4 characterized by their 6 transmembrane segments and broad sensory capacity. Among animals, 5 TRP channels have been canonically divided into 7 families (Venkatachalam and Montell 2007; 6 Peng, et al. 2015): TRPA (ankyrin), TRPC (canonical), TRPM (melastatin), TRPML 7 (mucolipin), TRPN (no mechanoreceptor potential C), TRPP (polycystin, or polycystic kidney 8 disease), and TRPV (vanilloid; and a proposed sister family, TRPVL). These TRP channels vary 9 substantially, but TRPM has arguably diversified the most with respect to function, participating 10 in at least cardiac activity (Yue, et al. 2015), magnesium homeostasis (Schlingmann, et al. 2007; 11 Hofmann, et al. 2010), egg activation (Carlson 2019), sperm thermotaxis (De Blas, et al. 2009), 12 cell adhesion (Su, et al. 2006), apoptosis (Driscoll, et al. 2017), inflammation (Ramachandran, et 13 al. 2013), and most famously, cold (Bautista, et al. 2007; Turner, et al. 2016) and menthol 14 (McKemy, et al. 2002; Peier, et al. 2002; Himmel, et al. 2019) sensing. 15 TRPM channels are also thought to be incredibly ancient, predating the emergence of metazoans 16 (>1000Ma) (Peng, et al. 2015; Himmel, et al. 2019). In some species, single moonlighting 17 proteins carry out several functions (e.g. Drosophila melanogaster Trpm), while in others, 18 functions are compartmentalized in a set of diverse paralogues (e.g. human TRPM1-8). 19 However, little is known about the evolutionary history of TRPMs, or to what degree channels 20 are related across taxa. Our understanding of TRPM evolution is additionally clouded by the 21 existence of several highly divergent putative TRPM channels with uncertain origins (Teramoto, 22 et al. 2005; Peng, et al. 2015; Kozma, et al. 2018; Himmel, et al. 2019). Here, we made use of 23 the rapidly growing body of genomic data in order to better characterize the evolution of the 24 TRPM family. 25 Via a stringent screening process, we assembled a database of >1,300 predicted TRPM-like 26 sequences from 14 diverse eumetazoan phyla (Fig. 1). In this database we gave particular 27 attention to underrepresented taxa, as well as included TRP genes identified in a number of 28 recently sequenced genomes (Table S1; including, but not limited to, acoel flatworm, moon 29 jelly, and great white shark). Herein, we elucidate the evolutionary history and familial 30 organization of both TRP melastatin, and a previously unrecognized sister family that predates 31 the Cnidaria-Bilateria split, TRP soromelastatin. 32 33 Materials & Methods 34 Data Collection & Curation 35 Starting with previously characterized TRPM sequences from human (NCBI CCDS), mouse 36 (NCBI CCDS), Drosophila melanogaster (FlyBase), and Caenorhabditis elegans (WormBase), a 37 TRPM-like protein sequence database was assembled by performing BLASTp against NCBI 38 collections of non-redundant protein sequences, with D. melanogaster Trpm (isoform RE, 39 FlyBase ID: FBtr0339077) serving as the bait sequence. In order to maximize useful 40 phylogenetic information, only BLAST hits >300 amino acids in length with an E-value less than 41 1E-30 were retained. As we were interested in the origins of TRPM channels, and in less-studied 42 taxa, only three tetrapod sequence-sets were included, from human, mouse, and chicken. bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 In order to expand the taxa sampled, tBLASTn and BLASTp were used to search publically 2 available, genomically-informed gene models for 11 cnidarians, 2 xenacoelomorphs, 1 3 hemichordate, 1 nemertean, 1 phoronid, 2 agnathans, and 4 chondrichthyes (Table S1). 4 We used several methods in order to validate and improve the quality of the initial database. 5 First, CD-HIT (threshold 90% similarity) was used to identify and remove duplicate sequences 6 and predicted isoforms, retaining the longest isoform (Li and Godzik 2006; Huang, et al. 2010; 7 Fu, et al. 2012). Phobius was then used to predict transmembrane topology (Käll, et al. 2004, 8 2007); sequences which did not have at least 6 predicted transmembrane (TM) segments, which 9 is typical of TRP channels, were removed. Sequences with more than the 6 predicted TM 10 segments were analyzed via InterProScan (Mitchell, et al. 2019), and those with more than 1 ion- 11 transport domain were removed. More than 90% of the remaining sequences contained a highly 12 conserved glycine residue in the predicted TM domain (corresponding to D. melanogaster G- 13 1049); the vast majority of those missing this residue had large gaps in an initial alignment and 14 were subsequently removed. 15 Searches for TRPS (ced-11-like), TRPN, and TRPC sequences followed the same protocol. For 16 TRPS, sequences from Caenorhabditis elegans, Strigamia maritima, and Octopus vulgaris were 17 used as bait. For TRPN and TRPC datasets, Drosophila melanogaster nompC (isoform PA, 18 FlyBase ID: FBpp0084879) and Trp (isoform PA, Flybase ID: FBpp0084879) served as bait 19 sequences, respectively. 20 Principal Component Analysis 21 Principal component analysis (PCA) was used to help resolve protein families. TRPC, TRPN, 22 and TRPM/TRPS database sequences were aligned by MAFFT. A pairwise sequence identity 23 matrix was then computed, and PCA performed against it in Jalview (Waterhouse, et al. 2009). 24 Data were exported from Jalview and visualized and edited in GraphPad Prism and Adobe 25 Illustrator CS6. 26 Phylogenetic Tree Estimation 27 For the maximum likelihood approach, sequences were first aligned using MAFFT with default 28 settings (Rozewicki, et al. 2019). Gap rich sites and poorly-aligned sequences were trimmed 29 with TrimAl (Capella-Gutiérrez, et al. 2009). IQ-Tree (Nguyen, et al. 2014) was then used to 30 generate trees by the maximum likelihood approach, using the best models automatically 31 selected by ModelFinder (Kalyaanamoorthy, et al. 2017). Branch support was calculated by 32 ultrafast bootstrapping (UFBoot, 2000 bootstraps) (Hoang, et al. 2017). 33 In order to test the alternative hypothesis that some trees formed due to long-branch attraction, 34 gs2 was used to generate trees by the Graph Splitting method (Matsui and Iwasaki 2019). 35 Branch support values were computed by the packaged edge perturbation method (EP, 2000 36 iterations). All trees were visualized and edited in iTOL and Adobe Illustrator CS6. 37 Homologue Prediction via Tree Reconciliation 38 In order to identify duplication events, TRPS and TRPM phylograms were reconciled using 39 NOTUNG 2.9.1 (Durand, et al. 2006; Vernot, et al. 2008; Stolzer, et al. 2012). Edge weight 40 threshold was set to 1.0, and the costs of duplications and losses were set to 1.5 and 1.0, 41 respectively. In order to formulate the most parsimonious interpretation of the resulting trees, 42 weak branches were rearranged (UFboot 95 cutoff) against a cladogram based in an NCBI 43 taxonomic tree, wherein we placed Xenacoelomorpha (represented by acoel flatworms) as the bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 sister group to all other bilaterians (Cannon, et al. 2016), and Priapulida as an outgroup to all 2 other ecdysozoans (Yamasaki, et al. 2015). All other polytomies were randomly resolved using 3 the ape package in R (Paradis and Schliep 2018). 4 5 6 Results 7 An ancient, unrecognized sister family to TRPM – TRP soromelastatin (TRPS) 8 Proteins within the same family typically have a high degree of sequence similarity, yet highly 9 divergent TRPM-like proteins have been catalogued, a notable example being Caenorhabditis 10 elegans cell death abnormal 11 (ced-11). The canonical C. elegans TRPMs gtl-1, gtl-2, and gon- 11 2 share roughly 40% sequence identity with each other. However, ced-11—often considered a 12 fourth C. elegans TRPM—shares approximately 18% sequence identity with the 3 canonical 13 paralogues. Given this substantial difference, it seemed plausible that ced-11, and like proteins, 14 had been errantly included in the TRPM family. 15 The TRPC and TRPN families are typically thought to be most closely related to TRPM (Peng, 16 et al. 2015), and therefore constituted hypothetical homes for ced-11. ced-11, however, shares 17 only 15% sequence identity with known C. elegans TRPC paralogues (trp-1 and trp-2), and 14% 18 sequence identity with C. elegans TRPN (trp-4). Yet sequence identity between trp-4 and its 19 TRPC counterparts is approximately 20%. In other words, ced-11 is less similar to TRPMs than 20 TRPNs and TRPCs are to each other. 21 In order to clarify the relationship of ced-11-like proteins to canonical TRPM channels, we 22 collected those sequences most similar to it from our initial TRPM-like sequence database, and 23 phylogenetically characterized them. BLASTing our database with ced-11-like sequences 24 recovered a number of sequences restricted to several protostome taxa and lancelets 25 (Cephalochordata). 26 For any species with a ced-11-like protein, we assembled a database of putative TRPC and 27 TRPN channel sequences. These sequences were then phylogenetically characterized alongside 28 cnidarian, xenacoelomorph, insect (D. melanogaster), and human sequences. In the resulting 29 tree, ced-11-like proteins formed a sister clade to the more traditional TRPM clade, the latter 30 including cnidarian TRPM-like channels (Fig. 2 and Fig. S1). 31 Two competing hypotheses could explain these findings: (1) ced-11-like proteins constitute a 32 distinct family of TRP channel which predates the cnidarian-bilaterian split, or (2) a variety of 33 TRPM channels emerged independently in various taxa and diversified extremely rapidly, 34 resulting in a clade which formed as a result of long-branch attraction, an artifact of many 35 phylogenetic analyses (Bergsten 2005). 36 Hypothesis 2 appears highly unlikely. Most importantly, while C. elegans ced-11 itself has a 37 relatively long branch, when qualitatively compared to other clades, the branches within the ced- 38 11-like clade were not unusually long (Fig. 2 and Fig. S1). Additionally, principal component 39 analysis of a pairwise sequence identity matrix revealed that ced-11-like sequences cluster 40 together independent of TRPM-like sequences, suggesting they cluster in the phylogram due to 41 sequence similarity (Fig. 3A). We tested the long-branch hypothesis by estimating trees which 42 excluded Cnidaria and Xenacoelomorpha, which had particularly long branches and could serve bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 to exacerbate long-branch attraction, were it present. The resultant phylogram still evidenced the 2 split between ced-11-like and TRPM-like channels, with high branch confidence (Fig. S2). 3 Moreover, we generated a phylogram by the Graph Splitting method, which is reported to be 4 extremely robust when faced with the possibility of long-branch attraction in superfamily-level 5 datasets (Matsui and Iwasaki 2019). This method likewise reproduced the ced-11-like-TRPM 6 split with high edge perturbation branch support (Fig. S3). 7 These results strongly indicate that these two lineages diverged in or prior to the last cnidarian- 8 bilaterian ancestor, and that ced-11-like proteins constitute an 8th family of metazoan TRP 9 channel. We have thus named the ced-11-like family of TRP channels TRP soromelastatin 10 (soro-, sister), or TRPS (Fig. 2 and Fig. 3). 11 The structure of TRPS channels suggests a SLOG- and Nudix-linked ancestor 12 While the function of TRPS channels remains unknown, domain prediction reveals that both 13 TRPM and TRPS channels share an N-terminal SMF/DprA-LOG (SLOG) domain (which is 14 hypothesized to function in ligand sensing) and a C-terminal ADP-ribose phosphohydrolase 15 (Nudix) domain (Fig. 3B, top). These results indicate that the ancestral TRPM-TRPS channel 16 was likely both SLOG- and Nudix-linked, and that the Ankyrin repeats typical of TRPC and 17 TRPNs were lost prior to the TRPM-TRPS split. The TRPM alpha kinase domain (typical of 18 human TRPM6 and TRPM7), however, appears to have arisen specifically in the TRPM lineage. 19 These findings are consistent with previous findings suggesting that the TRPM ancestor was 20 Nudix-linked (Schnitzler, et al. 2008), but pushes the origins of this domain further back in 21 evolutionary history. 22 A notable difference between TRPM and TRPS channels lies in the TRP domain, a highly 23 conserved, hydrophobic region located C-terminally to the transmembrane domain of TRPC, 24 TRPN, and TRPM channels (Venkatachalam and Montell 2007). Consensus sequences for 25 TRPM and TRPS (Fig. 2), while identical in TRP box 1, are divergent in TRP box 2 and the 26 intermediate TRP segment (Fig. 3B, bottom). The functional consequences of these changes, if 27 any, are unknown. 28 The TRPS family is largely restricted to protostomes 29 Having established that these TRPS sequences constitute a distinct set of channels, we assembled 30 a more complete TRPS sequence database and phylogenetically characterized the channel 31 family. These data suggest that, among Eumetazoa, TRPS genes are only present in some 32 protostomes and lancelets (Fig. 4 and Fig. S4). The lack of widespread conservation among 33 deuterostomes (most notably vertebrates) and insects likely explains why the family had gone 34 unnoticed until now. 35 TRPS was likely lost early in deuterostome evolution – among the ambulacrarians (echinoderms 36 and hemichordates), and in early Olfactores (tunicates and vertebrates) following the olfactore- 37 lancelet split (Fig. S5, left). A recent study evidences that Ambulacraria and Xenacoelomorpha 38 might form sister clades (Philippe, et al. 2019) – if this is the case, it may be more likely that 39 TRPS was lost early in so-called “xenambulacrarian” evolution (Fig. S5, right). 40 TRPS duplication appears to have been limited during early animal evolution. While the number 41 of TRPS paralogues varies by species (Fig. S4), duplication events occurred only after major 42 taxa emerged, independently in molluscs, nematodes, tardigrades, and chelicerates (including 43 arachnids and horseshoe crabs). Molluscs have two TRPS paralogues, but present lack of bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 evidence for lophotrochozoan TRPS outside of molluscs makes it difficult to predict at what 2 point in spiralian evolution the duplication event occurred. The simplest explanation is that it 3 occurred specifically in molluscs, and that a single TRPS copy was lost among other 4 lophotrochozoan taxa. 5 Among Euarthropoda, TRPS appears in chelicerates and myriapods, but there is no evidence for 6 TRPS in crustaceans, springtails, or insects, suggesting that the single arthropod TRPS was lost 7 in Pancrustacea, conserved in Myriapoda, and expanded independently in Chelicerata (Fig. S6). 8 Two TRPM clades predate the Cnidaria-Bilateria split 9 We next phylogenetically characterized and reconciled TRPM sequences among major taxa. 10 Each set of sequences was initially assessed alongside cnidarian, xenacoelomorph, Drosophila, 11 and human sequences, and rooted with TRPS sequences. 12 The general consensus of these analyses indicates that the TRPM family is made up of two 13 distinct clades, here and previously deemed αTRPM and βTRPM (Himmel, et al. 2019), which 14 emerged prior to the Cnidaria-Bilateria split (Fig. 5 and Figs. S7-S16). What might have 15 constituted a previously described basal clade can be almost wholly explained by the discovery 16 of TRPS (Peng, et al. 2015; Himmel, et al. 2019). In some of our initial phylograms, a basal or 17 separate clade did appear, yet it always included Xenacoelomorpha and was inconsistent in its 18 topology across analyses (Figs. S7-S11), suggesting that Xenacoelomorpha acted as a 19 phylogenetically unstable rogue taxon (Thomson and Shaffer 2010). In order to assess this 20 possibility, we performed a second set of analyses which excluded Xenacoelomorpha. This 21 resulted in trees with largely consistent topology despite differing taxon sampling, indicating that 22 xenacoelomorph sequences are in fact problematic (Figs. S12-S16). 23 Due to the overwhelming consistency of trees with different taxon sampling, and the 24 inconsistency seen in trees including Xenacoelomorpha, xenacoelomorph TRPM sequences were 25 treated as rogue taxa. In addition, an extremely small subset of arthropod TRPMs (12 sequences 26 restricted to chelicerates and crustaceans; Fig. S11 and Fig. S16) may be part of a previously 27 described Crustacea-specific TRPM sub-family (Kozma, et al. 2018). These trees suggest that 28 these sequences are βTRPM-like and related to a subset of cnidarian sequences, yet this 29 Cnidaria-inclusive clade is not strongly evidenced in phylograms with different taxon sampling. 30 Like Xenacoelomorph sequences, the evolutionary histories of these sequences are left incertae 31 sedis. 32 In summary, these results strongly support two duplication events predating the Cnidaria- 33 Bilateria split: the TRPS-TRPM split and the α-β TRPM split. 34 TRPM1-8 expansion occurred early in vertebrate evolution, and constitutes a poor 35 standard for TRPM family organization 36 The vertebrate TRPM1-8 expansion has been the focus of the majority of TRPM literature, and 37 has been the principal basis for characterizing TRPM channels (Samanta, et al. 2018; Zhang, et 38 al. 2018; Chen, et al. 2019). However, these trees evidence that the TRPM1-8 expansion 39 occurred after the vertebrate-tunicate split, and before agnathans (jawless fish; lampreys and 40 hagfish) split from the ancestor of all other vertebrates (Fig. 5, Fig. 6, and Fig. S13). Although 41 immunohistochemical evidence has previously suggested that TRPM8 is present in teleost fish 42 (Majhi, et al. 2015), we found no evidence of it in available sequences for ray-finned fish, 43 cartilaginous fish, or agnathans (Fig. 6 and Fig. S17). While the simplest naive hypothesis bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 would be that TRPM8 did not emerge until lobe-finned fish emerged, these phylogenetic 2 analyses indicate that TRPM8 was independently lost in the indicated taxa, and conserved in the 3 lobe-finned vertebrate lineage (including tetrapods). 4 Moreover, the 1-8 nomenclature may under-describe TRPMs among one of the most abundant 5 vertebrate clades – the teleost fish. While basal ray-finned fish (e.g., Erpetoichthys calabaricus, 6 the freshwater snakefish, or reedfish) have a TRPM topology that closely matches other 7 vertebrates, the emergence of teleosts came with TRPM expansion. For example, there are as 8 many as three teleost TRPM4 paralogues (Fig. S17). 9 10 11 Discussion 12 The evolutionary history of TRPM channels has been clouded by divergent sequences, making it 13 uncertain if an ancestral clade of TRPMs had survived in species like C. elegans, or if these 14 species had independently evolved rapidly changing TRPM paralogues (Teramoto, et al. 2005; 15 Peng, et al. 2015; Kozma, et al. 2018; Himmel, et al. 2019). By taking advantage of the 16 abundance of publicly available genomic data, we have demonstrated that the difficulty in 17 phylogenetically characterizing TRPM channels is the result of an ancient, hidden family of 18 channels that appeared before the Cnidaria-Bilateria split – the TRPS. By recognizing and 19 characterizing this family, we now better understand not only the evolution and diversification of 20 TRPM, but also the evolution of the broader TRP superfamily. 21 While some have been careful in describing TRP channels in taxon-specific ways (Saito and 22 Shingai 2006; Hofmann, et al. 2010; Peng, et al. 2015), these findings are the strongest challenge 23 to the pervasive, vertebrate-centric dogma that the TRPM family is constituted by 8 distinct 24 paralogues organized into four subfamilies (Samanta, et al. 2018; Zhang, et al. 2018; Chen, et al. 25 2019). These results instead support that the eumetazoan TRPM family consists of two distinct 26 radiations (αTRPM and βTRPM) which themselves predate the Cnidaria-Bilateria split. 27 Importantly, these findings support that TRPM diversification occurred independently among 28 cnidarians, ambulacrarians, lophotrochozoans, and other taxa, and that the TRPM1-8 expansion 29 is specific to vertebrates. Based on these findings, we conclude that the TRPM1-8 nomenclature 30 is at best evolutionarily uninformative (e.g. insect channels being simply TRPM1- or 3-like), and 31 at worst grossly inaccurate (e.g. cnidarian TRPMs belonging to the TRPM2/8 subfamily) for 32 describing members of this diverse family of critically important ion channels. 33 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Acknowledgments: We thank Dr. Charles Derby and Mihika Kozma for critically assessing the 2 original manuscript, and the PhyloPic repository, the source for many of the animal silhouettes 3 used throughout (distributed in public domain). We also thank all the investigators who made 4 sequence information public, which made this work possible. 5 Funding: This work is supported by the National Institute of Neurological Disorders and Stroke 6 at the National Institutes of Health (R01NS115209 to DNC); the National Institute of General 7 Medical Sciences at the National Institutes of Health (R25GM109442-01A1); a GSU Brains & 8 Behavior Fellowship (to NJH); a GSU Brains & Behavior Seed Grant (to DNC); and a Kenneth 9 W. and Georgeanne F. Honeycutt Fellowship (to NJH). 10 Author contributions: Conceptualization and methodology, NJH; sequence collection, NJH; 11 database curation, NJH and TRG; domain homology analysis, NJH and TRG; phylogenetic and 12 other formal analyses, NJH; prepared the original draft, NJH; reviewed and edited the final draft, 13 NJH, TRG, and DNC; visualization, NJH; supervision, DNC; funding acquisition, DNC. 14 Data and materials availability: The TRPN, TRPC, TRPM, and TRPS sequence databases 15 have been deposited on Dryad in the FASTA format (doi:10.5061/dryad.kwh70rz03). 16 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Fig. 1. Major, widely-recognized taxa included in these phylogenetic analyses, and relationships 2 assumed throughout. TRPM and TRPM-like sequences were collected for 318 species, from 14 3 diverse eumetazoan phyla (Annelida, Arthropoda, Brachiopoda, Chordata, Cnidaria, 4 Echinodermata, Hemichordata, Mollusca, Nematoda, Nemertea, Phoronida, Priapulida, 5 Tardigrada, and Xenacoelomorpha).

6 Fig. 2. Caenorhabditis elegans ced-11, and ced-11-like sequences, belong to a previously 7 unrecognized family of TRP channels, the TRP soromelastatins (TRPS). TRPM-like and ced- 8 11-like sequences form two distinct clades, with the topology suggesting divergence prior to the 9 Cnidaria-Bilateria split. Left, maximum likelihood tree showing the relationship between 10 traditional TRPM and TRPS/ced-11-like sequences among those species that have TRPS/ced-11- 11 like species. Right, summary and branch support (UFboot) for indicated clades.

12 Fig. 3. The predicted structures of putative TRPS channels suggest a SLOG- and Nudix-linked, 13 Ankyrin-free TRPS-TRPM ancestor. (A) Principal component analyses of pairwise sequence 14 identity for alignment of TRPC, TRPN, TRPM, and ced-11-like (TRPS) sequences show 4 15 distinct clusters. 3-dimensional PCA plot extracted from Jalview and plotted in two transformed 16 dimensions. (B) Like TRPM channels, TRPS channels have both SLOG and Nudix domains, but 17 lack the kinase domain associated with some TRPM channels. Moreover, these TRPS channels 18 have a divergent consensus sequence in the highly conserved TRP domain.

19 Fig. 4. The evolution of TRPS channels. The TRPS family is largely restricted to protostome 20 lineages. Figure derived from reconciled maximum likelihood tree of TRPS sequences (Fig. S3). 21 Red dots and colored branches indicate phylum-specific duplication events. Grey and dashed 22 branches indicate that no TRPS sequences were found for the indicated phylum, and were 23 inferred to be loss events.

24 Fig. 5. Summary of the evolution of TRPM channels. The TRPM family is widely conserved, 25 and is present in all phyla surveyed except Tardigrada. Figure derived from consensus 26 topologies in trees generated against TRPM database sequences, with branch support for 27 duplication branches extracted from phylograms without Xenacoelomorpha (Fig. S12-S16). 28 Asterisk (*) indicates that a duplication branch most frequently formed due to rearrangement 29 (initial UFboot branch support <95). Here, phyla were expanded/collapsed to more easily show 30 TRPM diversification: Chordata was expanded into Cephalochordata (lancelets), Tunicata, and 31 Vertebrata; Echinodermata and Hemichordata were collapsed into Ambulacraria; and Annelida, 32 Nemertea, Brachiopoda, Phoronida, and Mollusca were collapsed into Lophotrochozoa. Red 33 dots indicate duplication events. Dashed lines in color indicate sequences considered incertae 34 sedis. Dashed lines in grey indicate that no sequences were found for the indicated taxon, and 35 were inferred to be loss events.

36 Fig. 6. TRPM1-8 diversified soon after the vertebrate-tunicate split, and there is no evidence for 37 TRPM8 in jawless, cartilaginous, or ray-finned fish. Figure derived from phylogram of chordate 38 TRPM sequences (Fig. S17). An “X” indicates inferred gene loss, or lack of evidence of that 39 gene in the indicated taxon. 40 41 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1 explore the adaptability and resilience of coral holobionts to environmental change. Frontiers in 2 Marine Science 2. 3 4 Voolstra CR, Li Y, Liew YJ, Baumgarten S, Zoccola D, Flot J-F, Tambutté S, Allemand D, 5 Aranda M. 2017. Comparative analysis of the genomes of Stylophora pistillata and Acropora 6 digitifera provides evidence for extensive differences between species of corals. Scientific 7 Reports 7:17583. 8 9 Wang X, Liew YJ, Li Y, Zoccola D, Tambutte S, Aranda M. 2017. Draft genomes of the 10 corallimorpharians Amplexidiscus fenestrafer and Discosoma sp. Molecular Ecology Resources 11 17:e187-e195. 12 13 Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. 2009. Jalview Version 2--a 14 multiple sequence alignment editor and analysis workbench. Bioinformatics (Oxford, England) 15 25:1189-1191. 16 17 Yamasaki H, Fujimoto S, Miyazaki K. 2015. Phylogenetic position of Loricifera inferred from 18 nearly complete 18S and 28S rRNA gene sequences. Zoological letters 1:18-18. 19 20 Yue Z, Xie J, Yu AS, Stock J, Du J, Yue L. 2015. Role of TRP channels in the cardiovascular 21 system. American journal of physiology -- Heart and circulatory physiology 308:H157-H182. 22 23 Zhang Z, Tóth B, Szollosi A, Chen J, Csanády L. 2018. Structure of a TRPM2 channel in 24 complex with Ca2+ explains unique gating regulation. eLife 7:e36409. 25 26 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Echinodermata

(echinoderms)

(acoel flatworns) (anemones and corals)

Acoela Anthozoa

Hemichordata (acorn worms)

Tunicata (sea squirts)

Hydrozoa Cephalochordata (lancelets)

Ambulacraria

(hydra) Xenacoelomorpha Agnatha (jawless fish) Scyphozoa (true jellyfish) Chondrichthyes (cartilaginous fish)

Cnidaria Insecta Actinopterygii (insects) Chordata (ray-finned fish) Bilateria Coelacanthiformes Collembola (coelacanths) (springtails) Eumetazoa Nephrozoa Tetrapoda (tetrapods) Crustacea (crustaceans) Annelida Ecdysozoa (segmented worms) Lophrotrochozoa Myriapoda (myriapods) Cephalopoda (cephalopod molluscs)

Gastropoda Arachnida (gastropod molluscs) (arachnids)

Nemertea (ribbon worms)

Xiphosura Brachiopoda (brachiopods)

Phoronida (horseshoe worms)

(horseshoe crabs) Tardigrada

(tardigrades)

Nematoda

Priapulida (roundworms)

(penis worms)

Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available Phylogeny of TRPN, TRPC,under TRPM, aCC-BY-NC-ND and 4.0 International TRPS license channels.

C. elegans gtl-1, gtl-2, and gon-2 C. elegans ced-11 100 TRPS ced-11-like nephrozoan 100

99 TRPM TRPM-like eumetazoan 100 TRPN eumetazoan

100 TRPC eumetazoan

Tree scale: 1 Tree scale: 0.5

Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available A Sequence Identity under aCC-BY-NC-ND 4.0 InternationalB Domain license. Homology

10 TRPC A A A A TRANSMEMBRANE TRP TRPS ced-11-like TRPN ▼ A A A A A A A A TRANSMEMBRANE TRP

TRPM SLOG TRANSMEMBRANE TRP NUDIX KINASE

PC2 TRPS SLOG TRANSMEMBRANE TRP NUDIX PC1 0 PC3

TRP Box 1 TRP Box 2 A.U. TRPC WKF A R T KLWM S YF DEGST LPPPF N TRPM TRPC TRPN WKF G R A KL - IR N M N K TSST P S PL N

TRPM WKF Q RY Q LIM E Y HS R - PVLPPPFI

▼ ▼ TRPS ▼WKF ▼ ▼Q ▼RY Q ▼LVV D F EN R - LRLPPPL N -10

TRPN Small nonpolar Negatively charged

Hydrophobic Positively charged ▼ -20 Polar Sequence identity -15-10 -5 0510 A.U.

Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The evolution of TRP soromelastatinunder aCC-BY-NC-ND (TRPS) 4.0 International license. Cnidaria Xenacoelomorpha Chordata Echinodermata Hemichordata Annelida Nemertea Brachiopoda Phoronida Mollusca Priapulida Nematoda Tardigrada Arthropoda

100 100 100

Gene loss or no evidence Gene duplication

Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The evolution of TRP melastatinunder aCC-BY-NC-ND (TRPM) 4.0 International license. Ambulacraria Vertebrata Vertebrata Lophotrochozoa Ambulacraria Arthropoda Arthropoda Cnidaria Xenacoelomorpha Cephalochordata Tunicata Lophotrochozoa Priapulida Nematoda Tardigrada Cnidaria Xenacoelomorpha Cephalochordata Tunicata Priapulida Nematoda Tardigrada

100 100 100 100 100 100

100 100 100 * * * *

Uncertain 100 α β Gene loss or no evidence Gene duplication

Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/860445; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 Internationalα licenseTRPM. βTRPM

Cephalochordata Chordata (lancelets)

Tunicata Olfactores (sea squirts)

Agnatha 13 6 7 28 4 5 Vertebrata (jawless fish) XXXX X X Chondrichthyes (cartilaginous fish) X Coelacanthiformes (Latimera chalumnae)

Tetrapoda

Actinopterygii (ray-finned fish) X

Figure 6