Nicotine Stimulates Peristalsis in N. Vectensis: From Behavior to Nicotinic Receptor Genes

Nicotinic acetylcholine receptors (nAChRs) play a pivotal role in the nervous system, and there is great interest in understanding their evolution. Cnidarians, an ancestral group of metazoans, display an early nervous system utilizing complex neural architecture that provides insight into the early evolution and function of nAChRs. Using the anthozoan Nematostella vectensis, we found that the rate of peristalsis, a radial constriction of the body column, is modulated by nicotinic agonists. Nicotine caused a robust, dose dependent increase in the rate of peristalsis. The peristalsis rate also increased in response to ACh in the presence of edrophonium, an acetylcholine esterase inhibitor. Thus, N. vectensis possesses functional nAChRs. We developed a phylogeny of nAChRs from 15 cnidarian and 16 bilaterian species. Our results indicate that the large number of cnidarian and bilaterian nAChR gene copies is convergent, resulting from gene expansions after the two lineages diverged. These independent expansions led to an average of 31 and 34 paralogous nAChR gene copies in sampled bilaterians and cnidarians, respectively. The N. vectensis proteome contains 49 paralogous nAChR copies. The independent expansions indicate that nAChRs receptors in cnidarians may have distinct pharmacological and biochemical characteristics in comparison to their bilaterian counterparts. to explore both experimental and phylogenomic evidence for the functionality of nAChR receptors. We provide clear evidence N. vectensis has a robust physiologic response to nicotine and present a detailed phylogeny of the nAChR in cnidarians and bilaterians. Although a number of different behaviors have been identied in N. vectensis including feeding, swimming, sudden contraction 20 and peristalsis 21 , only tentacle contraction has been shown to be under neuronal control 7 . Here we demonstrate that peristalsis, a periodic radial constriction of the body column progressing as a wave that begins approximately half-way down the body column and continues to the foot or physa, can be modulated by nicotinic cholinergic agonists. Specically, we show that the rate of peristalsis is dramatically increased in a dose-dependent way by nicotine. Our results suggest that peristaltic waves are coupled to nicotinic cholinergic receptors in N. vectensis and that this behavior can be used to better characterize the function of these receptors. In addition, we examine the phylogenetic relationship of cnidarian nAChRs in comparison to other eumetazoan receptors, infer dramatic parallel evolution in gene copy number between the cnidarians For three concentrations of nicotine, 93 µM (63 animals), 185 µM (169 animals) and 370 µM (55 animals) and the corresponding controls, the mean frequency of waves increased by factors of 1.8, 2.4 and 4.3, respectively. Linear regression of the waves vs. nicotine concentration data yielded a slope of 0.015 ± 0.002 waves/ µM (95% condence limits) indicating that the dose-dependence is highly signicant (Supplementary Figure S2).


Introduction
The phylum Cnidaria is made up of a diverse group of organisms including jelly sh, corals, hydroids and sea anemones. A recent phylogenetic analysis of all metazoan phyla places Cnidaria as sister to Bilateria 1 . An important feature in these organisms is the presence of a well-de ned nervous system: nerve nets in cnidarians and a central nervous system in bilaterians. On a molecular level, both groups possess voltage-gated and ligand-gated ion channels which suggests similar neuronal signaling processes 2 . Thus, cnidarians have the potential to provide insight into the evolution of the nervous system.
Elucidation of which neurochemical signals cnidarians utilize has been hampered by the paucity of clear physiologic and behavioral responses to these molecules. Species for which meaningful behavioral assays exist include luminescence in Renilla kollikeri 3,4 , photic behavior in Tripedalia cystophora 5 and tentacle contraction in Hydra vulgaris 6 and Nematostella vectensis 7 . A challenge in studying cnidarians has been the lack of representative species with fully sequenced genomes. Until 2019, only the genomes of N. vectensis 8 , Acropora digitifera 9 , H. vulgaris 10 , and Exaiptasia diaphana 11 , were available.
The sea anemone N. vectensis has great potential to unlock some of the secrets of cnidarian neurotransmission. This anthozoan cnidarian has emerged as an important model organism 12 . In addition to having a fully sequenced genome 8 , it is easy to cultivate and propagate. Furthermore, it has been shown to possess the complete repertoire of nervous system proteins associated with synaptic transmission in vertebrates 13 . The N. vectensis genome contains genes that encode receptors and enzymes related to classic neurotransmitters such as acetylcholine, glutamate, GABA, and ATP; proteins associated with biogenic amines and other non-peptidergic transmitters (such as DOPA and adrenaline); as well as sequences that code for proteins associated with neuropeptides (such as RFamide) 13 . Here, we focus on the nicotinic acetylcholine receptor (nAChR) ion channel protein.
nAChRs are members of the pentameric ligand-gated ion channel superfamily. These proteins possess an extracellular stretch of 13 amino acids anked by a cysteine at each end (cys-loop), an extracellular ligand binding site, four transmembrane helices (TM1-TM4), and exhibit a pentameric quaternary structure with a central pore through which ions can pass 14 . Receptors are composed of ve subunits, which may be encoded by one or multiple paralogous gene copies. Nicotinic receptors, along with 5-HT3 and Zinc-activated channels form cationic pores, while GABA A and glycine receptors form anionic channels. In the case of the nAChR, the endogenous ligand is acetylcholine, but nicotine (from which the receptor gets its name) is also a potent ligand. In vertebrates, nAChRs participate in various functions including fast synaptic transmission at the neuromuscular junction and synapses in the central nervous system, cognitive and pathophysiological effects of nicotine addiction, learning and memory, and the immune system (reviewed in 15 .
nAChRs have been studied in other cnidarians where behavioral or physiologic assays exist. Cholinergic agonists induce muscle contractions and bioelectric activity in H. vulgaris 16 and the anemone Bunodosoma caissarum 17 . Biochemical studies have provided evidence that acetylcholinesterases are present in the anenome Actinia equina 18 . Recent evidence shows that both ACh and nicotine induce tentacle contractions in N. vectensis and that this effect can be blocked by nicotinic antagonists such as mecamylamine 7 .
The presence of genes that code for cholinergic system proteins in N. vectensis was rst detailed by 13 . He described 20 paralogous cholinergic genes, including 12 nAChR genes, 3 choline acetyltransferase genes, and 5 acetylcholinesterase genes.
Further molecular evidence for cholinergic function was provided by a study demonstrating that nAChRs are expressed during N. vectensis development 19 . This work presented evidence that these receptors can be found in the ectoderm and are enriched in the apical organ in planulae. A recent extension of this work showed that N. vectensis possesses 26 nAChR gene paralogs and that the transcriptional products of these genes are expressed during development in neurons, muscles, and gonads 7 .
The goal of the work presented here was to explore both experimental and phylogenomic evidence for the functionality of nAChR receptors. We provide clear evidence N. vectensis has a robust physiologic response to nicotine and present a detailed phylogeny of the nAChR in cnidarians and bilaterians. Although a number of different behaviors have been identi ed in N. vectensis including feeding, swimming, sudden contraction 20 and peristalsis 21 , only tentacle contraction has been shown to be under neuronal control 7 . Here we demonstrate that peristalsis, a periodic radial constriction of the body column progressing as a wave that begins approximately half-way down the body column and continues to the foot or physa, can be modulated by nicotinic cholinergic agonists. Speci cally, we show that the rate of peristalsis is dramatically increased in a dose-dependent way by nicotine. Our results suggest that peristaltic waves are coupled to nicotinic cholinergic receptors in N. vectensis and that this behavior can be used to better characterize the function of these receptors. In addition, we examine the phylogenetic relationship of cnidarian nAChRs in comparison to other eumetazoan receptors, infer dramatic parallel evolution in gene copy number between the cnidarians and eumetazoans, and evaluate evidence for the closest related bilaterian receptors.

Animal Care
Cultures of N. vectensis were maintained in 12 ppt arti cial sea water (ASW) at 18℃ under arti cial light (12 h light:12 h dark).
They were fed three times per week with freshly hatched Artemia salina nauplii and were cleaned after each feeding 20,22 . Sexual reproduction was induced by the method of 23 . Fertilized eggs were removed from adult cultures and placed in separate 39mm culture dishes in 12 ppt ASW. Eggs were incubated at 18℃ for 6-8 d until the 4 tentacle juvenile polyps appeared. Polyps 8-21 d old were used in experiments on peristalsis. Developing polyps were not fed until day 21 post fertilization.

Behavioral Experiments
Polyps were placed in 6 well culture plates 24 h before experiments. Approximately 20-25 polyps were added to 8 ml of 12 ppt ASW in each well. Animals were carefully transferred using a 12 cm glass pipette so that 8-12 animals were visible in the eld of view on a Zeiss Stemi Stereo Microscope. Experiments were initiated by recording a 5 min video under control conditions. Next, the appropriate dose of chemical treatment was added to the well and a 5 min equilibration period was allowed. Finally, a second 5 min video was recorded with the animals still in their original relative positions. This before and after treatment design made it possible for each animal to act as its own control. The sequence of treatment conditions was varied randomly. In some experiments a second chemical treatment was added to the well. In these cases, a 5 min equilibration period preceded a 5 min video of the stimulated polyps.
Videos were recorded using an Excelis digital camera attached to the Zeiss microscope. Videos were converted to MP4 format using HandBrake for analysis. Control and treatment videos were opened side by side using two windows on the computer monitor to ensure the identity of control and stimulated animals. In order to count the number of waves, videos were watched at 10X speed. Waves that began before the 5 min recording session were counted but waves that did not complete before the end of the session were not. Animals were excluded from analysis if their position changed so that they were not in the eld of view during both their control and treatment states. Each animal was used for only one experiment and only two wave frequency values are obtained from each animal, one for control and one for treatment. The speed of peristaltic waves in a small set of animals was determined by measuring the locations of the animal's mouth and the constricted region at 0.8 s intervals using the video analysis program Tracker v4.96 (http://physlets.org/tracker/, a project of Open Source Physics http://www.compadre.org/OSP/). A plot of the distance between the mouth and constriction vs time was t by linear regression. The slope of the regression line provided the wave speed.

Statistical Analysis
Histograms of the number of peristaltic waves during a 5 min interval do not exhibit a normal distribution. Because each animal served as its own control, we used the nonparametric, Wilcoxon signed rank test, which is used for paired samples to test for differences in a repeated measures design. In cases where we compared waves under three conditions, the Friedman test (the nonparametric equivalent of ANOVA) was employed and was followed by Dunn's Multiple Comparison Test. Differences were considered signi cant when p < 0.05. Prism v6 (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical tests and Igor Pro v8.04 (Wavemetrics, Inc., Oswego, OR, USA) was used for graphing of behavioral data. Violin plots were constructed as boxes showing the 25, 50 and 75 percentiles, the feathers showing the full range of the data points and the solid shapes showing a smoothed representation of the distribution.

Gene Family Identi cation, Alignment, and Phylogeny
We created a seed alignment for studying two cationic pentameric ligand gated channels by selecting 14 well-characterized human nAChR proteins (each encoded by a distinct gene) and two Drosophila melanogaster nAChR proteins, as well as 3 human 5HT3 receptor proteins (Supplementary Table S1). We aligned these using t_coffee v12 24 in accurate mode, and then trimmed the terminal ends to retain conserved regions, including loop A, the cys-loop, loop B, loop C, and transmembrane domains 1-3 (but excluding the poorly conserved transmembrane domain 4). We built a hidden Markov model (HMM) using this seed alignment using hmmbuild in hmmer v3.1b2 25 .
For a query database, we examined 15 cnidarian predicted proteomes, in addition to selecting representatives from available xenocoelemorph, protostome, echinoderm, and hemichordate proteomes (31 proteomes; Supplementary Table S2). When available, we used proteomes with only a single isoform per gene, as curated by uniprot 26 . For all Genbank proteomes, we used SeqKit 0.12.1 27 to retain the longest isoform per gene. We only included proteomes with at least 80% 28 completeness according to the eukaryota_odb9 reference set in BUSCO v3.0.2 28 . For reference, we constructed a species tree based on relationships reported in the literature [29][30][31][32] , although some of these relationships, such as the position of Xenacoelomorpha, are unsettled.
We queried these proteomes using hmmsearch from hmmer and kept only the top scoring domain if there were multiple hits. We only retained proteins with an independent e-value less than 1x10 − 50 , and where the full protein length is at least 400 amino acids.
We also removed any protein that did not have a cysteine at both the beginning and end of the cys-loop (positions 119 and 133 in our HMM) according to the hmmer alignment. We aligned proteins that passed these lters with muscle 33 , and then used the seq_reformat function in t_coffee to remove any sites with greater than 50% gaps.
We used IQ-TREE v.2.0-rc1 34 to produce a phylogeny based on this alignment using the WAG substitution matrix 35 and FreeRate model of substitution heterogeneity 36 with 10 categories. To avoid local minima, we chose the optimal tree from six independent runs, including four optimized for short input sequences with a smaller than default perturbation strength (0.2) and larger number (500) of stop iterations. Node support was estimated using the ultrafast bootstrap 37 and SH-aLRT test 38 and 10,000 replicates.
To assess alternative topologies, we swapped fully-resolved branches on the optimal maximum likelihood tree in TreeGraph 39 and performed AU 40 and ELW 41 tests with 10,000 replicates in IQ-TREE. We identi ed α-like proteins by the presence of doublecysteine at positions 182-183 of the HMM based alignment. We reconstructed the ML ancestral states of α or non-α status using the fastAnc function in phytools 42 . The species and gene trees were visualized with ggtree 43 .

Results And Discussion
Contractions in 12ppt ASW. N. vectensis polyps occasionally undergo a slow peristaltic contraction that starts about halfway down the body column and ends at the foot or physa ( Fig. 1a; Supplementary Video 1). The waves travel at 9.7 ± 2.0 µm/s (6 animals, 17 measurements) (Fig. 1b). In no case did we observe a second wave begin in an animal before the rst wave had ended. Occasionally, we observed a wave traveling in the reverse direction (physa to mid-column).
Polyps underwent an average of 2.5 peristaltic waves per 5 min interval (pooled measurements from 808 animals that were subsequently used in experiments with different treatments). However, the number of waves varied from zero to nine with a median value of 2 and was not normally distributed (Fig. 2a). We tted the distribution to the sum of two weighted Poisson distributions. The means and 95% con dence limits of the t were 0.47 ± 0.11 waves per 5 min (0.41 ± 0.05 weight) and 3.94 ± 0.34 waves per 5 min. This suggests that N. vectensis polyps normally have two activity levels: low and high, with slightly more than half of the polyps being in the high activity group.
Wave frequency is increased by nicotine. The addition of nicotine ( nal concentration 185µM) to the solution bathing the polyps dramatically increased the number of waves to an average of 6.0 waves per 5 min (median of 6 waves per 5 min, 187 animals, Supplementary Video 2). We tted the distribution as the sum of two Poisson distributions but the tted weight of one component was zero; the remaining Poisson component had a mean of 6.5 ± 0.83 waves per 5 min (Fig. 2b). Thus, all of the polyps contracted at an activity level higher than that of the high activity in the control. The speed of the contractions in the presence of 185 µM nicotine was twice as fast as those under control conditions ( Fig. 2c; 19.3 ± 3.7 µm/s, 5 animals, 21 measurements, p < 0.001, ttest, the same set of animals as were used to obtain the control speed, Supplementary Figure S1).
The excitation induced when nicotine is added to the well is not due to the animals being disturbed by the addition of uid. Figure 3 shows the results for a set of 56 animals recorded under control conditions and then again ve minutes after the addition of 0.10 ml of 12 ppt ASW. There was no signi cant change in the distribution of waves. These same animals responded to the subsequent addition of nicotine to the well ( nal concentration 370 µM).
The stimulating effect of nicotine was dose dependent (Fig. 4). For three concentrations of nicotine, 93 µM (63 animals), 185 µM (169 animals) and 370 µM (55 animals) and the corresponding controls, the mean frequency of waves increased by factors of 1.8, 2.4 and 4.3, respectively. Linear regression of the waves vs. nicotine concentration data yielded a slope of 0.015 ± 0.002 waves/ µM (95% con dence limits) indicating that the dose-dependence is highly signi cant (Supplementary Figure S2).
Contractions in the presence of acetylcholine (ACh). Surprisingly, ACh did not stimulate peristalsis. At concentrations ranging from 10 µM to 5 mM, ACh appeared to inhibit peristalsis, but the results were inconsistent. In the example shown in Fig. 5a, 500 µM ACh had no effect on the frequency of waves. One possible explanation for the lack of a stimulatory effect of ACh is that acetylcholinesterases in N. vectensis may rapidly hydrolyze exogenously applied ACh. We tested this idea by rst exposing polyps to the acetylcholinesterase inhibitor, edrophonium and then added ACh. 500 µM edrophonium increased the frequency of waves from 1.50 per 5 min to 3.32 per 5 min (Fig. 5b). Subsequent addition of 500 µM ACh increased the frequency of waves even further to 5.21 per 5 min. This suggests that inhibition of acetylcholinesterase by 500 µM edrophonium increases the effectiveness of both endogenous and exogenously applied ACh.
N. vectensiscontains a large number of nAChR paralogs. We hypothesized that our behavioral observation of dramatic, dosedependent peristalsis in response to nicotine is due to the presence of nAChRs. To test this hypothesis, we rst identi ed all potential nAChR paralogs in the N. vectensis genome. To determine the extent that the details of this type of neurochemical signaling may be homologous or convergent with bilaterian systems, we determined the phylogenetic relationship of the N. vectensis nAChRs to other characterized nAChR genes. According to our conservative estimate, the N. vectensis proteome contains 49 nAChR copies (Supplementary Table S3), the most of any cnidarian sampled to date. All of these encoded proteins have the characteristic structural features found in mammalian nAChRs (Supplementary Figure S3) and many of the amino acid residues within the agonist/antagonist binding loops are identical to those found in human nAChR proteins (Supplementary Table  S4). These paralogous proteins are encoded on 27 different scaffolds. Queries into previous expression sets indicate that all 49 genes examined are expressed with varying temporal dynamics and magnitude 44-46 . The 15 species of cnidarians we analyzed average 34 nAChR paralogs, ranging from 13 in H. vulgaris to 46 in Pocillopora damicornis (Supplementary Table S2). The sampled bilaterian proteomes contain, on average, 31 nAChR paralogs (including Zinc/5HT3 receptors), ranging from 7 in Ciona intestinalis (ascidian) to 68 in Mizuhopecten yessoensis (scallop). Humans have 16 nAChR paralogs. We veri ed that the number of predicted nAChR genes in a genome is not strongly predicted by the completeness of the genome annotation (Supplementary Figure S4). In total, our nal protein alignment includes 502 cnidarian genes and 498 bilaterian genes and contains 435 positions (Supplementary Table S5).
A single lineage of nAChR genes underwent a dramatic expansion in Cnidaria. Our phylogeny is rooted with a zinc/5HT3 outgroup, the paralogous sister group to all other cationic subunits ("Gene Tree" in Fig. 6; Supplementary Figure S5). In this tree, the relationships among the bilaterian nAChRs generally agrees with typical relationships described in recent literature (e.g. 47 ). Within this phylogeny, we observe the 502 cnidarian gene copies occur in two distinct clades. The rst cnidarian clade contains genes present in a single copy in just ve of the anthozoans (Fungia spp., Stylophora pistillata, Pocillopora damicornis, Acropora tenuis, and Porites asteroides). These ve genes ("Cnidaria-2" in Fig. 6) are part of the divergent group (Branch E) related to Drosophila β3 and found in xenoceolemorphs, protostomes, echinoderms, and hemichordates, but not in chordates.
We tested our con dence in the placement of the Cnidaria-group as sister (closest relatives) to the B1 + α7 group using alternative topology tests (Supplementary Table S6), and were able to reject alternative placement (c-ELW and p-AU < 0.01 for both; joining along branches B or C in Fig. 6). All other sister relationships to major groups we tested cannot be rejected by either test (p > 0.05). We cannot reject the Cnidaria-group as sister to the α9/10 group (branch D; found in protostomes, echinoderms, hemichordates, and chordates), the Dβ3/divergent group 48 (branch E; found in protostomes, echinoderms, hemichordates, and some cnidarians; see below), B1 + α7 + α9/10 (branch F), or B1 + α7 + α9/10 + Dβ3 (branch G).
It appears that the 497 gene copies in the Cnidaria-group all expanded from a single common ancestral gene in ancient cnidarians. Gene duplications led to increases in nAChR copy number especially in anthozoans, including the Hexacorallia, and Scleractinia cndiarians. Intriguingly, this expansion in cnidarians tentatively appears to be the result of an expansion of the same stem lineage that also exhibited a large expansion in bilaterian lineages (B1-group; although we cannot reject some alternatives). This brings about several important questions. First, why was this speci c lineage subject to dramatic expansion, independently but in parallel (convergently), in bilaterians and cnidarians? Second, in how many ways did the nAChR systems in bilaterians and cnidarians evolve in parallel? Here, we have shown that they evolved in parallel in terms of expansion of the gene family copy number, suggesting the convergent evolution of a complex nAChR system. However, do the same types of subunits exist? Are they assembled into homopentamers and heteropentamers of similar types to those of bilaterians? Are they equivalently tissue speci c? And do the serve the same range of functions, or do they have new and marvelous functions to discover? Answers to these questions will require further phylogenetic and functional characterization.
The observation that various types of complexity has arisen in animals multiple times, and is not speci c to bilaterians, is now becoming well-appreciated 49 . Indeed, the independent expansion of nAChR gene family copy number in bilaterians and cnidarians that we observe here is consistent with a similar inference for the entire ligand-gated, cys-loop receptor superfamily (which also includes GABA A and Glycine anionic receptors; not considered here) in a study using two cnidarian genomes 50 . That study also inferred a similar pattern for glutamate-gated channels and voltage gated potassium channels, also suggesting repeated and independent occurrences of the expansion of nervous system complexity.
To consider the question of how early nAChRs evolved, we also searched in proteomes from earlier diverging metazoan lineages. We found no evidence of nAChRs in the poriferans Xestospongia testudinaria, Tethya wilhelma, Stylissa carteri, Ephydatia muelleri, Stylissa carteri, and Amphimedon queenslandica, or in the ctenophores Mnemiopsis leidyi and Pleurobrachia bachei (not shown). Accumulating phylogenomic evidence suggests that Bilateria and Cnidaria form a monophyletic group that does not include the Porifera and Ctenophora (e.g. 51-53 ; although other work places Porifera as sister to cnidarians, as in 54 . The presence of nAChRs in Bilateria and Cnidaria supports their sister relationship and monophyly, with respect to poriferans and ctenophores; this distribution is the same as for the entire cys-loop receptor superfamily as well as acid sensing channel 50 , and has also been observed for other types as genes, such as for Hox/ParaHox, S50 and K50 PRD and HNF class homeodomains 55 . Repeated evolution of non-αsubunits. Of the 49 nAChR genes in N. vectensis, 44 encode α-like subunits (90%), as de ned by the presence of a double-cysteine in loop C (Supplementary Figure S3). The ve genes encoding non-α subunits come from four clades that each evolved independently into the non-α state (Fig. 6, Supplementary Figure S5). Among all cnidarian nAChR genes, 83% (417/502) are α-like. In contrast, for bilaterians, only 57% are α-like (256/453). In both cases, the non-α state has arisen independently numerous times from the ancestral, α-like nAChRs ancestor. Like Dβ3, the Cnidaria-2 group is non-α.
There are no muscarinic cholinergic receptors inN. vectensis. To rule out the possibility that the behavioral, dose dependent peristalsis we observed is due to the presence of muscarinic cholinergic receptors (mAChRs), we searched for the closest related genes to mAChRs within N. vectensis and determined their phylogenetic placement among rhodopsin-like G-protein-coupled receptors. Previous work has shown that anthozoans like N. vectensis do not possess mAChRs (Anctil, 2009; Faltine-Gonzalez and Layden, 2019) whereas hydrozoans such as H. vulgaris do (Collins, 2013.) We therefore conducted a phylogenetic analysis of mAChRs and other related rhodopsin-like G-protein-coupled muscarinic receptors. We identi ed the 10 top matching genes to a mAChR-speci c HMM from each H. vulgaris and N. vectensis (Supplementary Figure S6). We found genes from H. vulgaris are sister to the bilaterian mAChRs. In contrast, the N. vectensis genes are related to octopamine receptors, rather than mAChRs, consistent with a lack of mAChRs. We also compared these cnidarian sequences at the 14 amino acid sites known to be critical to ACh binding (Supplementary Table S7). We found that the H. vulgaris genes have an average of 8 identical amino acids (range 7-14) while N. vectensis genes have an average of only 3 critical amino acids (range 2-5). We therefore infer that the response we see in N. vectensis is mediated by nAChRs, rather than mAChRs.

Summary
We have studied the effect of cholinergic agonists on peristalsis in N. vectensis. In other cnidarians, peristalsis has been shown to play important physiologic roles such as uid circulation 56 , digestion 57 and locomotion 58 . We found that the rate of peristalsis in N. vectensis exhibited a dramatic and dose-dependent increase in response to nicotine. Additionally, in the presence of the acetylcholinesterase inhibitor, edrophonium, acetylcholine increased the rate of peristalsis. Our ndings are largely in agreement with previous work that showed that nicotine and acetylcholine induce tentacle contraction and that nicotine induces radial contractions in N. vectensis polyps 7 . In that work, concentrations of nicotine from 2.5 mM to 10 mM were used. The behavioral method for assessing contractions described here is much more sensitive, showing a clear dose response from 90 µM to 370 µM.
These observations lead us to infer that peristalsis in N. vectensis is mediated by nicotinic cholinergic receptors.
We then focused on the relationship between cnidarian nAChR genes and the well-studied genes expressed by bilaterians. To do this, we identi ed nAChR copies encoded in the genomes of 16 bilaterians and 15 cnidarians. We found that bilaterians possess roughly equivalent numbers of paralogous nAChR gene copies, with N. vectensis encoding the highest number. A detailed gene tree suggests that the major nAChR cnidarian lineage came from a single stem lineage which has since undergone dramatic expansion. All 49 of the N. vectensis nAChR genes fall into this clade, which is sister to one of the stem bilaterian lineages. Independently, the bilaterian nAChR gene family also expanded to a high copy number from this and other stem lineages. Taken together, the work presented here shows that the high copy number of nAChR subunits in both Cnidaria and Bilateria is due to convergent and parallel expansion. In other words, there is no one-to-one relationship between the many subunits in bilaterians and the many subunits in cnidarians, since the cnidarian-group subunits all expanded from a single ancestral cnidarian copy. This convergent expansion is likely to have produced a set of cnidarian proteins that have distinct pharmacological and biochemical properties from those of their bilaterian counterparts.
Declarations Supplementary Table S6. Alternative topology test results, which assess con dence in the phylogenetic placement of the Cnidariagroup Supplementary Table S7. Residues found sites known to be critical to ACh binding in the closest related genes to mAChRs in Hydra vulgaris and Nematostella vectensis.  indicate times when the animal was undergoing a contraction. Linear regression of these sections was used to obtain the slope which is the contraction speed. For animal 1, the contraction speed was 10.2 µm/s; for animal 2 the speeds were 7.9 and 7.7 µm/s. During other parts of the record, the animal was either not contracting, was contracting while bending, or was not orientated horizontally. The distribution is best t by the sum of two weighted Gaussian functions (see text). b. Histogram of the number of waves per 5 min for 187 animals exposed to 185 µM nicotine. c. The distributions from A and B shown as violin plots (see Methods for details).  The frequency of waves increases with increasing concentrations of nicotine. *** indicates difference with Wilcoxon signed rank test p < 0.0001 a. 93 µM nicotine (63 animals, mean frequency = 1.64 waves per 5 min for control, mean frequency = 2.98 waves per 5 min for nicotine). b. 185µM nicotine (169 animals, mean frequency = 2.48 waves per 5 min for control, mean frequency = 6.02 waves per 5 min for nicotine). c. 370µM nicotine (55 animals, mean frequency = 1.53 waves per 5 min for control, mean frequency = 6.62 waves per 5 min for nicotine). the frequency further. (153 animals, mean frequency = 1.50 waves per 5 min for control, 3.32 waves per 5 min for 500 µM edrophonium, 5.21 waves per 5 min for 500 µM edrophonium + 500 µM ACh). Dunn's multiple comparison after a Friedman test shows p<0.001 for control vs. 500µM edrophonium (***), p<0.001 for control vs. 500 µM edrophonium + 500 µM ACh (***), and p<0.001 for 500 µM edrophonium vs. 500 µM edrophonium + 500 µM ACh ( † † †).

Figure 6
Phylogenetic relationships of nAChR genes in select cnidarian and bilaterian genomes shows parallel expansion. Species Tree (top left) illustrates included species with literature relationships (some nodes are unsettled; see Methods). Species names are colored by class, phylum, or superphylum. Number of nAChR gene copies identi ed in each genome are indicated for each tip. The protein-based Gene Tree (right) for these copies includes genes identi ed with a nAChR-speci c hidden Markov model, with relationships calculated via maximum likelihood (see Methods). The tree is rooted with 5HT3/Zinc receptors. Almost all cnidarian receptors (light and dark blue tip labels) are part of a single, large lineage ("Cnidaria-group", indicated by *) that is sister to a large linage of bilaterian receptors (branches B and C). An additional group of cnidarian orthologs ("Cnidaria-2") is found in the Dβ3 group (branch E). Lineages that encode α-like subunits are colored black, while non-α lineages are colored tan. Strong ultra-fast bootstrap (> 95%) and SH-aLRT (>80%) support is indicated by black dots at nodes. The gene tree with labels is provided as Supplementary Figure S5. Con dence in the Placement of the Cnidarian group (bottom left) was tested using ELW and AU topology tests (see Methods). Letters in the summary diagram correspond to branches in the gene tree. In the optimal tree, the Cnidaria-group attaches to branch A; both tests reject attachment of the Cnidaria-group to branches B and C (Supplementary Table   S6). However, attachment to branches D,E,F, or G cannot be rejected from the 0.95 con dence set.

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