bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Ancient coexistence of norepinephrine, tyramine, and octopamine signaling in bilaterians

Philipp Bauknecht1 and Gáspár Jékely1*

1Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany *Correspondence to: [email protected]

Abstract Norepinephrine/noradrenaline is a neurotransmitter implicated in arousal and other aspects of vertebrate behavior and physiology. In invertebrates, adrenergic signaling is considered absent and analogous functions are attributed to the biogenic amine octopamine. Here we describe the coexistence of signaling by norepinephrine, octopamine, and its precursor tyramine in representatives of the two major clades of Bilateria, the protostomes and the deuterostomes. Using phylogenetic analysis and receptor pharmacology we show that six receptors coexisted in the protostome-deuterostome last common ancestor, two each for the three ligands. All receptors were retained in the genomes of the deuterostome Saccoglossus kowalewskii (a hemichordate) and the protostomes Platynereis dumerilii (an annelid) and Priapulus caudatus (a priapulid). Adrenergic receptors were lost from most insects and nematodes and tyramine and octopamine receptors were lost from most deuterostomes. These results clarify the history of monoamine signaling in animals and highlight the importance of studying slowly evolving marine taxa.

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Introduction

Norepinephrine is a major neurotransmitter in vertebrates with a variety of functions including roles in promoting wakefulness and arousal (Singh et al., 2015), regulating aggression (Marino et al., 2005), and autonomic functions such a heart beat (Kim et al., 2002). Signaling by the monoamine octopamine in protostome invertebrates is often considered equivalent to vertebrate adrenergic signaling (Roeder, 2005) with analogous roles in promoting aggression and wakefulness in flies (Crocker and Sehgal, 2008; Zhou et al., 2008), or the regulation of heart rate in annelids and arthropods (Crisp et al., 2010; Florey and Rathmayer, 1978). Octopamine is synthesized from tyramine (Figure 1A) which itself also acts as a neurotransmitter or neuromodulator in arthropods and nematodes (Jin et al., 2016; Kutsukake et al., 2000; Nagaya et al., 2002; Rex and Komuniecki, 2002; Roeder, 2005; Saudou et al., 1990; Selcho et al., 2012). Octopamine and norepinephrine are chemically similar, are synthesized by homologous enzymes (Monastirioti et al., 1996; Wallace, 1976), and signal by similar G-protein coupled receptors (GPCRs) (Evans and Maqueira, 2005; Roeder, 2005), further arguing for their equivalence. However, the precise evolutionary relationship of these transmitter systems is unclear. The evolution of neurotransmitter systems has been analyzed by studying the distribution of monoamines or biosynthetic enzymes in different organisms (Gallo et al., 2016). This approach has limitations, however, because some of the biosynthetic enzymes are not specific to one substrate (Wallace, 1976) and because trace amounts of several monoamines are found across many organisms, even if specific receptors are often absent. For example, even if invertebrates can synthesize trace amounts of norepinephrine and vertebrates can synthesize tyramine and octopamine, these are not considered to be active neuronal signaling molecules, since the respective receptors are lacking. Consequently, the presence of specific neuronally expressed monoamine receptors is the best indicator that a particular monoamine is used in neuronal signaling (Arakawa et al., 1990; Saudou et al., 1990). We thus decided to analyze the evolution of adrenergic, octopamine, and tyramine receptors in bilaterians.

Results

Using database searches, sequence-similarity-based clustering, and phylogenetic analysis, we identified a few invertebrate GPCR sequences that were similar to vertebrate adrenergic α1 and α2 receptors (Figure 1B). An adrenergic α1 receptor ortholog is present in the sea urchin Strongylocentrotus purpuratus. Adrenergic α1 and α2 receptors were both present in Saccoglossus kowalewskii, a deuterostome hemichordate (Figure 1B and Supplementary figures 1-2), as previously reported (Krishnan et al., 2013). We also identified adrenergic α1 and α2 receptor orthologs in annelids and mollusks (members of the Lophotrochozoa), including Aplysia californica, and in the priapulid worm Priapulus caudatus (member of the Ecdysozoa)(Figure 1B). Adrenergic α receptors are also present in a few arthropods, including the crustacean Daphnia pulex and the moth Chilo suppressalis (the Chilo receptor was first described as an octopamine receptor (Wu et al., 2012)), but are absent from most other insects (Supplementary figures 1-2). We also identified α2 receptors in some nematodes. The identification of adrenergic α1 and α2 receptor orthologs in ambulacrarians, lophotrochozoans, and ecdysozoans indicates that both families were present in the protostome-deuterostome last common ancestor. We could not identify adrenergic β receptors outside chordates (Supplementary figure 3).

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Figure 1. Biosynthesis of monoamines and cluster map of GPCR sequences. (A) Biosynthesis of tyramine, octopamine, norepinephrine and epinephrine from tyrosine. The enzymes catalyzing the reaction steps are indicated. (B) Sequence-similarity-based cluster map of bilaterian octopamine, tyramine, and adrenergic GPCRs. Nodes correspond to individual GPCRs and are colored based on taxonomy. Edges correspond to BLAST connections of P value >1e-20.

To characterize the ligand specificities of these putative invertebrate adrenergic receptors we cloned them from S. kowalewskii, P. caudatus, and the marine annelid Platynereis dumerilii. We performed in vitro GPCR activation experiments using a Ca2+-mobilization assay (Bauknecht and Jékely, 2015; Tunaru et al., 2005). We found that norepinephrine activated both the adrenergic α1 and α2 receptors from all three species with EC50 values in the nanomolar range. In contrast, tyramine, octopamine, and dopamine were either inactive or only activated the receptors at higher concentrations (Figure 2, Table 1, and Supplementary figures 4-5). These phylogenetic and pharmacological results collectively establish these invertebrate receptors as bona fide adrenergic α receptors. To investigate if adrenergic signaling coexists with octopamine and tyramine signaling in protostomes we searched for octopamine and tyramine receptors in P. dumerilii and P. caudatus. In phylogenetic and clustering analyses, we identified orthologs for tyramine type 1 and type 2 and octopamine α and β receptors in both species (Figure 1B and Supplementary figures 6-9). We performed activation assays with the P. dumerilii receptors. The tyramine type 1 and type 2 receptors orthologs were preferentially activated by tyramine with EC50 values in the nanomolar range (Figure 2, Table 1, and Supplementary figures 10-11). The P. dumerilii octopamine α receptor was activated by octopamine at a lower concentration than by tyramine and dopamine. The P. dumerilii octopamine β receptor was not active in our assay. These results show that specific receptor systems for norepinephrine, octopamine, and tyramine coexist in P. dumerilii and very likely also P. caudatus.

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Figure 2. Dose-Response curves of selected monoamine GPCRs from P. dumerilii, P. caudatus, and S. kowalevskii treated with varying concentrations of ligand. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3) for selected monoamine GPCRs. All dose-response curves with diverse agonists and antagonists are shown in the Supplementary material. EC50 values are listed in Table 1.

When did tyramine and octopamine signaling originate? To answer this, we surveyed available genome sequences for tyramine and octopamine receptors. As expected, we identified several receptors across the protostomes, including ecdysozoans and lophotrochozoans (Supplementary figures 6-9). However, chordate genomes lacked clear orthologs of these receptors. Strikingly, we identified tyramine type 1 and 2 and octopamine α and β receptor orthologs in the genome of the hemichordate S. kowalewskii (Figure 1B). In phylogenetic analyses, we recovered at least one S. kowalewskii sequence in each of the four receptor clades (one octopamine α, one octopamine β, two tyramine type 1, and two tyramine type 2 receptors), establishing these sequences as deuterostome orthologs of these predominantly protostome GPCR families (Supplementary figures 6-9). We cloned the candidate S. kowalewskii tyramine and octopamine receptors and performed ligand activation experiments. The S. kowalewskii type 2 receptors were preferentially activated by tyramine in the nanomolar range. The type 1 receptor was only activated at very high ligand concentrations. The octopamine α and β receptors were preferentially activated by octopamine in the nanomolar, and low micromolar range, respectively (Figure 2, table 1, and Supplementary figures 10-11). These data show that octopamine and tyramine signaling also

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coexists with adrenergic signaling in this deuterostome, as in P. dumerilii and P. caudatus. The presence of tyramine signaling in S. kowalewskii is also supported by the phyletic distribution of tyrosine decarboxylase, a specific enzyme for tyramine synthesis (Alkema et al., 2005). Tyrosine decarboxylase is present in protostomes and S. kowalewskii but is absent from other deuterostomes (Supplementary figure 12). We also tested the α adrenergic agonist clonidine and the GPCR antagonists mianserin and yohimbine on several receptors from all three species. These chemicals did not show specificity for any of the receptor types, suggesting these chemicals may not be useful for studying invertebrate neurotransmission (Table 1, and Supplementary figures 4-5, Supplementary figures 9-10).

Discussion

Our results established the coexistence of adrenergic, octopaminergic, and tyraminergic signaling in the deuterostome S. kowalewskii and the protostomes P. dumerilii and P. caudatus. The discovery of adrenergic signaling in some protostomes and octopamine and tyramine signaling in a deuterostome changes our view on the evolution of monoamine signaling in bilaterians (Figure 3). It is clear from the phyletic distribution of orthologous receptor systems that two families of receptors per each ligand were present in the protostome-deuterostome last common ancestor. These include the adrenergic α1 and α2 receptors, the tyramine type 1 and type 2 receptors, and the octopamine α and β receptors. From the six ancestral families, the octopamine and tyramine receptors were lost from most deuterostomes, and the adrenergic receptors were lost from most ecdysozoans.

Figure 3. Evolution of adrenergic, octopamine, and tyramine signaling in bilaterians. (A) Phylogenetic tree of major clades of bilaterian animals with the loss of specific GPCR families indicated. (B) Phyletic distribution of adrenergic, octopamine, and tyramine GPCR families across major bilaterian clades.

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We consider the measured in vitro ligand preferences for these receptors as indicative of their physiological ligands. In general, there is a two orders of magnitude difference in the EC50 values between the best ligand and other related ligands, indicating the high specificity of the receptors. The preferred ligand of all six orthologous receptor families is the same across protostomes and deuterostomes, indicating the evolutionary stability of ligand-receptor pairs, similar to the long-term stability of neuropeptide GPCR ligand-receptor pairs (Jékely, 2013; Mirabeau and Joly, 2013). Understanding the ancestral role of these signaling systems and why they may have been lost differentially in different animal groups will require functional studies in organisms where all three neurotransmitter systems coexist. Signaling by norepinephrine in vertebrates has often been considered as equivalent to signaling by octopamine in invertebrates. Our results change this view and show that these signaling systems coexisted ancestrally and still coexist in some bilaterians, and thus cannot be equivalent. This has important implications for our interpretation of comparative studies of neural circuits and nervous system evolution.

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Acknowledgments

We thank John Gerhart for Saccoglossus DNA and the image of Saccoglossus. We thank Mattias Hogvall for Priapulus DNA and the image of Priapulus. We also thank Elizabeth Williams for comments on the manuscript. The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ European Research Council Grant Agreement 260821. P.B. is supported by the International Max Planck Research School (IMPRS) ‘‘From Molecules to Organisms.’’

Materials and Methods

Gene identification and receptor cloning Platynereis protein sequences were collected from a Platynereis mixed stages transcriptome assembly (Conzelmann et al., 2013). The accession numbers of all newly identified GPCRs tested here are GenBank: KX372342, KX372343, KP293998, KU715093, KU530199, KU886229). GPCR sequences from other species were downloaded from NCBI. GPCRs were cloned into pcDNA3.1(+) (Thermo Fisher Scientific, Waltham, USA) as described before (Bauknecht and Jékely, 2015). Forward primers consisted of a spacer (ACAATA) followed by a

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BamHI or EcoRI restriction site, the Kozak consensus sequence (CGCCACC), the start codon (ATG) and a sequence corresponding to the target sequence. Reverse primers consisted of a spacer (ACAATA), a NotI restriction site, a STOP codon, and reverse complementary sequence to the target sequence. Primers were designed to end with a C or G with 72°C melting temperature. PCR was performed using Phusion polymerase (New England Biolabs GmbH, Frankfurt, Germany). The sequences of all Platynereis GPCRs tested here were deposited in GenBank (accession numbers: α1-adrenergic receptor, KX372342; α2-adrenergic receptor, KX372343 Tyramine-1 receptor, KP293998; Tyramine-2 receptor, KU715093; Octopamine α receptor, KU530199; Octopamine β receptor, KU886229). Tyramine receptor 1 has been previously published (Bauknecht and Jékely, 2015) as Pdu orphan GPCR 48.

Cell culture and receptor deorphanization Cell culture assays were done as described before (Bauknecht and Jékely, 2015). Briefly, CHO- K1 cells were kept in Ham’s F12 Nut Mix medium (Thermo Fisher Scientific, Waltham, USA) with 10 % fetal bovine serum and penicillin-streptomycin (PenStrep, Invitrogen). Cells were seeded in 96-well plates (Thermo Fisher Scientific, Waltham, USA) at approximately 10,000 cells/well. After 1 day, cells were transfected with plasmids encoding a GPCR, the promiscuous Gα-16 protein (Offermanns and Simon, 1995), and a reporter construct GFP-apoaequorin (Baubet et al., 2000) (60 ng each) using 0.375 µl of the transfection reagent TurboFect (Thermo Fisher Scientific, Waltham, USA). After two days of expression, the medium was removed and replaced with Hank’s Balanced Salt Solution (HBSS) supplemented with 1.8 mM Ca2+, 10 mM glucose and 1 mM coelenterazine h (Promega, Madison, USA). After incubation at 37°C for 2 hours, cells were tested by adding synthetic monoamines (Sigma, St. Louis, USA) in HBSS supplemented with 1.8 mM Ca2+ and 10 mM glucose. Solutions containing norepinephrine, epinephrine or dopamine were supplemented with 100 µM ascorbic acid to prevent oxidation. Luminescence was recorded for 45 seconds in a plate reader (BioTek Synergy Mx or Synergy H4, BioTek, Winooski, USA). For inhibitor testing, the cells were incubated with yohimbine or mianserin (Sigma, St. Louis, USA) for 1 hour. Then, synthetic monoamines were added to yield in each case the smallest final concentration expected to elicit the maximal response in the absence of inhibitor and luminescence was recorded for 45 seconds. Data were integrated over the 45-second measurement period. Data for dose-response curves were recorded in triplicate for each concentration. Dose-response curves were fitted with a four-parameter curve using Prism 6 (GraphPad, La Jolla, USA). The curves were normalized to the calculated upper plateau values (100% activation).

Bioinformatics Protein sequences were downloaded from the NCBI. Redundant sequences were removed from the collection using CD-HIT (Li and Godzik, 2006) with an identity cutoff of 95%. Sequence cluster maps were created with CLANS2 (Frickey and Lupas, 2004) using the BLOSUM62 matrix and a P-value cutoff of 1E-70. For phylogenetic trees, protein sequences were aligned with MUSCLE (Edgar, 2004). Alignments were trimmed with TrimAI (Capella-Gutiérrez et al., 2009) in “Automated 1” mode. The best amino acid substitution model was selected using ProtTest 3 (Darriba et al., 2011). Maximum likelihood trees were calculated with RAxML (Stamatakis, 2014) using the CIPRES Science Gateway (Miller et al., 2010). Bootstrap analysis was done and automatically stopped (Pattengale et al., 2010) when the Majority Rule Criterion (autoMRE) was met. The resulting trees were visualized with FigTree

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(http://tree.bio.ed.ac.uk/software/figtree/). The identifiers of deorphanized octopamine and tyramine receptors (Balfanz et al., 2014; Blais et al., 2010; Chang et al., 2000; Chen et al., 2010; Gerhardt et al., 1997; Gross et al., 2015; Huang et al., 2012; Jezzini et al., 2014; Kastner et al., 2014; Lind et al., 2010; Rex and Komuniecki, 2002; Verlinden et al., 2010; Wu et al., 2012; Wu et al., 2014; Wu et al., 2015) were tagged with _Oa, _Ob, _T1, or _T2.

11 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

EC50 (M)/IC50 (M) Tyramine Octopami Norepine Epinephri Dopamin Clonidine Yohimbin Mianserin ne phrine ne e e P. dumerilii α1- inactive inactive 2.12E-07 3.79E-07 1.25E-04 inactive 4.41E-06 3.79E-06 adrenergic KX372342 P. dumerilii α2- 8.36E-05 2.67E-06 5.25E-09 1.06E-08 1.63E-06 2.57E-06 5.71E-06 2.53E-05 adrenergic KX372343 S. kowalevskii α1- inactive n/a 1.67E-08 1.94E-08 3.79E-06 inactive 1.32E-05 4.46E-06 adrenergic ALR88680 S. kovalewskii α2- 3.75E-06 1.94E-06 1.16E-13 2.31E-09 5.60E-09 3.61E-08 7.90E-05 n/a adrenergic XP_002734932 P. caudatus α1- inactive inactive 7.49E-09 inactive inactive inactive n/a n/a adrenergic XP_014662992 P. caudatus α2- inactive inactive 2.20E-06 4.52E-07 inactive 1.02E-07 n/a 9.85E-07 adrenergic XP_014681069 P. dumerilii 1.12E-08 2.70E-06 n/a n/a 7.79E-06 n/a 2.06E-06 1.05E-05 Tyramine-1 KP293998 P. dumerilii 7.02E-09 7.82E-07 n/a n/a 3.89E-06 n/a 5.38E-05 6.36E-06 Tyramine-2 KU715093 S. kovalewskii 8.40E-05 n/a inactive inactive n/a 2.86E-04 1.68E-06 1.70E-05 Tyramine-1 XP_002742354 S. kovalewskii 1.22E-09 6.24E-08 inactive inactive 7.18E-08 1.39E-06 n/a 1.61E-04 Tyramine-2A XP_002734062 S. kovalewskii 3.71E-09 1.57E-06 1.15E-04 2.85E-05 1.44E-06 1.60E-05 2.06E-05 1.88E-05 Tyramine-2B XP_006812999 P. dumerilii 1.34E-05 2.56E-07 n/a n/a inactive n/a 9.02E-09 1.62E-06 Octopamine α KU530199 S. kowalevskii 1.72E-05 6.89E-07 5.30E-05 1.85E-05 2.65E-04 1.64E-07 7.78E-06 2.16E-05 Octopamine α XP_006823182 S. kowalevskii inactive 6.37E-08 3.49E-06 inactive inactive inactive 1.57E-04 6.45E-06 Octopamine β XP_002733926 Table 1. EC50 (M)/IC50 (M) values of all tested GPCRs with the indicated ligands or inhibitors. The most effective ligand for each receptor is shown in bold.

12 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

α1-adrenergic receptors

XP_014662992.1_Priapulus_caudatus XP_014663408.1_Priapulus_caudatus EFX64650.1_Daphnia_pulex XP_015598849.1_Cephus_cinctus 71 XP_013787844.1_Limulus_polyphemus XP_003748403.1_Metaseiulus_occidentalis KFM77668.1_Stegodyphus_mimosarum XP_012936806.1_Aplysia_californica 80 XP_013091628_Biomphalaria_glabrata XP_009050255.1_Lottia_gigantea KX372342_Platynereis_dumerilii_AA1 XP_014777674.1_Octopus_bimaculoides XP_011422115.1_Crassostrea_gigas ELU14162.1_Capitella_teleta XP_013419311.1_Lingula_anatina ALR88680.1_Saccoglossus_kowalevskii_AA1 XP_003726237.1_Strongylocentrotus_purpuratus XP_006010360.1_Latimeria_chalumnae XP_006625863.1_Lepisosteus_oculatus XP_007247270.1_Astyanax_mexicanus XP_008417330.1_Poecilia_reticulata XP_008334310.1_Cynoglossus_semilaevis XP_006805960.1_Neolamprologus_brichardi 85 XP_013867515.1_Austrofundulus_limnaeus XP_015827734.1_Nothobranchius_furzeri XP_015233524.1_Cyprinodon_variegatus XP_012736728.1_Fundulus_heteroclitus XP_010788374.1_Notothenia_coriiceps 93 XP_008279073.1_Stegastes_partitus 100 CDQ56739.1_Oncorhynchus_mykiss CDQ56384.1_Oncorhynchus_mykiss XP_002933012.2_Xenopus_tropicalis XP_010177730.1_Mesitornis_unicolor XP_015152983.1_Gallus_gallus XP_010143615.1_Buceros_rhinoceros_silvestris XP_008489604.1_Calypte_anna 83 1 XP_008921792.1_Manacus_vitellinus XP_009899896.1_Picoides_pubescens XP_012959596.1_Anas_platyrhynchos XP_009566450.1_Cuculus_canorus XP_010205582.1_Colius_striatus 96 XP_002197113.1_Taeniopygia_guttata XP_008630655.1_Corvus_brachyrhynchos XP_005142671.1_Melopsittacus_undulatus XP_006114239.1_Pelodiscus_sinensis XP_005285240.1_Chrysemys_picta_bellii XP_006276442.2_Alligator_mississippiensis XP_007424603.1_Python_bivittatus XP_003227101.1_Anolis_carolinensis XP_015268171.1_Gekko_japonicus XP_004460877.1_Dasypus_novemcinctus 99 XP_004460873.1_Dasypus_novemcinctus XP_008059269.1_Tarsius_syrichta XP_003793998.1_Otolemur_garnettii XP_004665892.1_Jaculus_jaculus ADK98420.1_Meriones_unguiculatus XP_006252170.1_Rattus_norvegicus XP_007477871.1_Monodelphis_domestica AAA35496.1_Homo_sapiens_AA1A XP_007896221.1_Callorhinchus_milii XP_002935531.2_Xenopus_tropicalis XP_006629253.1_Lepisosteus_oculatus XP_004065916.1_Oryzias_latipes CDQ72256.1_Oncorhynchus_mykiss 95 XP_014051077.1_Salmo_salar 100 XP_007235606.1_Astyanax_mexicanus KTG32062.1_Cyprinus_carpio XP_010795024.1_Notothenia_coriiceps XP_697043.2_Danio_rerio XP_012687224_Clupea_harengus XP_010886834.1_Esox_lucius XP_009993014.1_Chaetura_pelagica XP_008499885.1_Calypte_anna XP_010154880.1_Eurypyga_helias XP_009951638.1_Leptosomus_discolor XP_014822169.1_Calidris_pugnax XP_007430469.1_Python_bivittatus XP_013917984.1_Thamnophis_sirtalis ETE57756.1_Ophiophagus_hannah XP_001922013.3_Danio_rerio 63 KTF87842.1_Cyprinus_carpio XP_013864145.1_Austrofundulus_limnaeus XP_012709980.1_Fundulus_heteroclitus XP_007548430.1_Poecilia_formosa XP_015255918.1_Cyprinodon_variegatus XP_004073535.1_Oryzias_latipes XP_011478573.1_Oryzias_latipes NP_001007359.1_Danio_rerio KTG31243.1_Cyprinus_carpio KTG02316.1_Cyprinus_carpio XP_012697555.1_Clupea_harengus XP_008325595.1_Cynoglossus_semilaevis XP_010795800.1_Notothenia_coriiceps XP_008282667.1_Stegastes_partitus XP_003970475.1_Takifugu_rubripes XP_005719861.1_Pundamilia_nyererei KPP75495.1_Scleropages_formosus XP_013133031.1_Oreochromis_niloticus NP_001118147.1_Oncorhynchus_mykiss CDQ95487.1_Oncorhynchus_mykiss CDQ73039.1_Oncorhynchus_mykiss XP_014068076.1_Salmo_salar XP_010900547.1_Esox_lucius XP_006138206_Pelodiscus_sinensis XP_006211986.1_Vicugna_pacos XP_011355023.1_Pteropus_vampyrus XP_010853649.1_Bison_bison_bison XP_003782039.1_Otolemur_garnettii XP_014743771.1_Sturnus_vulgaris

0.5

Supplementary figure 1. Maximum likelihood tree of α1-adrenergic receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs.

13 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

α2-adrenergic receptors

CEF71092.1_Strongyloides_ratti CDJ80122.1_Haemonchus_contortus XP_002644716.1_Caenorhabditis_briggsae EPB80586_Ancylostoma_ceylanicum XP_002595000.1_Branchiostoma_floridae XP_014767930.1_Octopus_bimaculoides XP_009067029.1_Lottia_gigantea XP_012943687.1_Aplysia_californica XP_013096124.1_Biomphalaria_glabrata XP_011433775.1_Crassostrea_gigas XP_013383118.1_Lingula_anatina KX372343_Platynereis_dumerilii_AA2 ELT99717.1_Capitella_teleta XP_014681069.1_Priapulus_caudatus KPP71495.1_Scleropages_formosus XP_015718435.1_Coturnix_japonica XP_013156970.1_Falco_peregrinus XP_005446496.1_Falco_cherrug XP_005485737.1_Zonotrichia_albicollis XP_008626884.1_Corvus_brachyrhynchos 96 AAN78417.1_Tupaia_belangeri XP_006750950.1_Leptonychotes_weddellii XP_012579513.1_Condylura_cristata EHH14168.1_Macaca_mulatta XP_015390193.1_Panthera_tigris_altaica XP_013820290.1_Capra_hircus XP_012879340.1_Dipodomys_ordii XP_007903477.1_Callorhinchus_milii XP_008325312.1_Cynoglossus_semilaevis P32251.1_Carassius_auratus XP_012675626.1_Clupea_harengus Q8JG70.1_Danio_rerio XP_010895107.1_Esox_lucius XP_014055357.1_Salmo_salar CDQ75057.1_Oncorhynchus_mykiss 96 CDQ85392.1_Oncorhynchus_mykiss XP_004073361.1_Oryzias_latipes XP_004912925.1_Xenopus_tropicalis XP_015267985.1_Gekko_japonicus XP_007420380.1_Python_bivittatus XP_003217630.1_Anolis_carolinensis XP_005998869.1_Latimeria_chalumnae CAG05052.1_Tetraodon_nigroviridis XP_013857577.1_Austrofundulus_limnaeus XP_008280550.1_Stegastes_partitus XP_014010145.1_Salmo_salar XP_014059545.1_Salmo_salar XP_013986063.1_Salmo_salar CDQ94371.1_Oncorhynchus_mykiss KTG45995.1_Cyprinus_carpio Q90WY4.1_Danio_rerio KTF91499.1_Cyprinus_carpio XP_015463451.1_Astyanax_mexicanus XP_013868957.1_Austrofundulus_limnaeus XP_012722422.1_Fundulus_heteroclitus XP_015228090.1_Cyprinodon_variegatus XP_008296024.1_Stegastes_partitus XP_008315656.1_Cynoglossus_semilaevis XP_010784224.1_Notothenia_coriiceps XP_010733243.1_Larimichthys_crocea CAC87884.1_Takifugu_rubripes XP_004539207_Maylandia_zebra XP_004066404.1_Oryzias_latipes CAG01293.1_Tetraodon_nigroviridis XP_015202895.1_Lepisosteus_oculatus KQK80321.1_Amazona_aestiva XP_005308213.1_Chrysemys_picta_bellii XP_006022896.1_Alligator_sinensis XP_003755431.1_Sarcophilus_harrisii XP_006880000.1_Elephantulus_edwardii XP_010968209.1_Camelus_bactrianus XP_015350355.1_Marmota_marmota_marmota XP_006778809.1_Myotis_davidii XP_005983264.1_ EDL01717.1_Mus_musculus Pantholops_hodgsonii EDL94448.1_Rattus_norvegicus XP_010981560.1_Camelus_dromedarius XP_005874627.1_Myotis_brandtii 95 XP_008538974.1_Equus_przewalskii XP_008142634.1_Eptesicus_fuscus NP_000672.3_Homo_sapiens_AA2A XP_012626815.1_Microcebus_murinus XP_003787216.1_Otolemur_garnettii XP_014938420.1_Acinonyx_jubatus XP_012873058.1_Dipodomys_ordii XP_004265893.1_Orcinus_orca XP_007120031.1_Physeter_catodon XP_007173405.1_Balaenoptera_acutorostrata_scammoni XP_010587294.1_Loxodonta_africana XP_004631588.1_Octodon_degus XP_014333406.1_Bos_mutus XP_014958903.1_Ovis_aries ERE78076.1_Cricetulus_griseus XP_006135062.1_Pelodiscus_sinensis XP_013802685.1_Apteryx_australis_mantelli XP_005416433.1_Geospiza_fortis XP_009806348.1_Gavia_stellata XP_003218553.1_Anolis_carolinensis XP_015262234.1_Gekko_japonicus XP_007437437.1_Python_bivittatus XP_015673604.1_Protobothrops_mucrosquamatus ETE69512.1_Ophiophagus_hannah XP_013920993.1_Thamnophis_sirtalis XP_013030003.1_Anser_cygnoides_domesticus XP_004942333.2_Gallus_gallus XP_009911888.1_Haliaeetus_albicilla NP_001072843.1_Xenopus_tropicalis XP_012953671.1_Anas_platyrhynchos XP_009881752.1_Charadrius_vociferus XP_005235799.1_Falco_peregrinus 100 XP_011573649.1_Aquila_chrysaetos_canadensis XP_015262427.1_Gekko_japonicus XP_014390455.1_Myotis_brandtii CAD11981.1_Rhogeessa_tumida CAI70426.1_Cricetomys_gambianus XP_010350316.1_Saimiri_boliviensis_boliviensis AEP17845.1_Anomalurus_beecrofti CAD20302.1_Tachyoryctes_sp. XP_015738423.1_Coturnix_japonica XP_008334393.1_Cynoglossus_semilaevis KPP60422.1_Scleropages_formosus XP_012685854.1_Clupea_harengus CDQ73934.1_Oncorhynchus_mykiss XP_014026328.1_Salmo_salar XP_002663705.3_Danio_rerio CDQ90138.1_Oncorhynchus_mykiss XP_007234270.1_Astyanax_mexicanus XP_007905605.1_Callorhinchus_milii XP_006011954.1_Latimeria_chalumnae XP_015200296.1_Lepisosteus_oculatus KPP70140.1_Scleropages_formosus XP_014011189.1_Salmo_salar XP_014062277.1_Salmo_salar XP_010886159.1_Esox_lucius XP_012730184.1_Fundulus_heteroclitus 77 XP_013875010.1_Austrofundulus_limnaeus XP_015252539.1_Cyprinodon_variegatus XP_010777801.1_Notothenia_coriiceps XP_008296631.1_Stegastes_partitus KKF18259.1_Larimichthys_crocea XP_003966929.1_Takifugu_rubripes CAG00720.1_Tetraodon_nigroviridis XP_008320162.1_Cynoglossus_semilaevis Q91081.1_Labrus_ossifagus XP_012697346.1_Clupea_harengus XP_007236033.1_Astyanax_mexicanus KTG06372.1_Cyprinus_carpio XP_012692326.1_Clupea_harengus NP_997522.1_Danio_rerio XP_015244492.1_Cyprinodon_variegatus XP_005813740.2_Xiphophorus_maculatus XP_008315824.1_Cynoglossus_semilaevis XP_003972567.1_Takifugu_rubripes XP_010732944.1_Larimichthys_crocea XP_010778794.1_Notothenia_coriiceps XP_008276148.1_Stegastes_partitus XP_010886433.1_Esox_lucius XP_014046094.1_Salmo_salar XP_002734932.1_Saccoglossus_kowalevskii_AA2 CAL36767.1_Branchiostoma_floridae XP_002605761.1_Branchiostoma_floridae EFX76926.1_Daphnia_pulex XP_014242340.1_Cimex_lectularius 99 XP_008198464.2_Tribolium_castaneum KF460458.1_Chilo_suppressalis_Oa XP_006609424.1_Apis_dorsata XP_014274544.1_Halyomorpha_halys

0.5 Supplementary figure 2. Maximum likelihood tree of α2-adrenergic receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs.

14 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

β-adrenergic receptors

AAQ91625.1_Branchiostoma_floridae CAA06539.1_Myxine_glutinosa NP_001085791.1_Xenopus_laevis NP_001083418.1_Xenopus_laevis AAI60515.1_Xenopus_tropicalis NP_001116897.2_Xenopus_tropicalis XP_015262106.1_Gekko_japonicus XP_003217407.2_Anolis_carolinensis XP_015678290.1_Protobothrops_mucrosquamatus XP_013912817.1_Thamnophis_sirtalis ETE71498.1_Ophiophagus_hannah XP_007421173.1_Python_bivittatus XP_006277924.1_Alligator_mississippiensis XP_008930097.1_Manacus_vitellinus XP_014803719.1_Calidris_pugnax XP_009645348.1_Egretta_garzetta XP_006052758.1_Bubalus_bubalis XP_004686925.1_Condylura_cristata 72 XP_007188760.1_Balaenoptera_acutorostrata_scammoni XP_004620654.1_Sorex_araneus XP_007447551.1_Lipotes_vexillifer XP_004384975.1_Trichechus_manatus_latirostris XP_006863914.1_Chrysochloris_asiatica XP_004696769.1_Echinops_telfairi XP_006893779.1_Elephantulus_edwardii XP_003404888.1_Loxodonta_africana XP_008062588.1_Tarsius_syrichta XP_008690966.1_Ursus_maritimus XP_003782105.1_Otolemur_garnettii XP_012597080.1_Microcebus_murinus XP_003934046.2_Saimiri_boliviensis_boliviensis XP_015306903.1_Macaca_fascicularis AAF20199.1_Homo_sapiens XP_004460730.1_Dasypus_novemcinctus XP_006766281.1_Myotis_davidii XP_008150402.1_Eptesicus_fuscus XP_012880977.1_Dipodomys_ordii 90 XP_014708070.1_Equus_asinus XP_002710343.1_Oryctolagus_cuniculus XP_003477383.1_Cavia_porcellus AGU01500.1_Sus_scrofa XP_010625409.1_Fukomys_damarensis ERE85201.1_Cricetulus_griseus AAA89068.1_Rattus_norvegicus NP_031446.2_Mus_musculus XP_006985221.1_Peromyscus_maniculatus_bairdii XP_008586147.1_Galeopterus_variegatus XP_005324245.1_Ictidomys_tridecemlineatus XP_008836112.1_Nannospalax_galili XP_004587615.1_Ochotona_princeps XP_004664808.1_Jaculus_jaculus XP_004428726.1_Ceratotherium_simum_simum ELW64306.1_Tupaia_chinensis XP_007516440.1_Erinaceus_europaeus 30 XP_014746696.1_Sturnus_vulgaris XP_009069256.1_Acanthisitta_chloris XP_015731815.1_Coturnix_japonica XP_009284014.1_Aptenodytes_forsteri XP_009470511.1_Nipponia_nippon KPP58190.1_Scleropages_formosus XP_007232946.1_Astyanax_mexicanus NP_001096122.1_Danio_rerio KTG34400.1_Cyprinus_carpio KTG06855.1_Cyprinus_carpio XP_014054344.1_Salmo_salar XP_010895941.1_Esox_lucius XP_008277801.1_Stegastes_partitus XP_008325872.1_Cynoglossus_semilaevis XP_011609112.1_Takifugu_rubripes XP_005795585.2_Xiphophorus_maculatus XP_008417789.1_Poecilia_reticulata XP_014901064.1_Poecilia_latipinna XP_015232667.1_Cyprinodon_variegatus XP_012707242.1_Fundulus_heteroclitus XP_004073575.1_Oryzias_latipes XP_013870150.1_Austrofundulus_limnaeus XP_005719651.1_Pundamilia_nyererei XP_005468045.1_Oreochromis_niloticus KKF21487.1_Larimichthys_crocea KPP77020.1_Scleropages_formosus ACX46713.1_Pimephales_promelas KTG42897.1_Cyprinus_carpio NP_001082940.1_Danio_rerio XP_007231763.1_Astyanax_mexicanus NP_001117912.1_Oncorhynchus_mykiss XP_010869486.2_Esox_lucius KKF11668.1_Larimichthys_crocea XP_015229372.1_Cyprinodon_variegatus XP_014326866.1_Xiphophorus_maculatus XP_007553424.1_Poecilia_formosa XP_013873112.1_Austrofundulus_limnaeus XP_006784977.1_Neolamprologus_brichardi XP_012691165.1_Clupea_harengus XP_015204860.1_Lepisosteus_oculatus XP_007906212.1_Callorhinchus_milii XP_007260201.1_Astyanax_mexicanus XP_006000436.1_Latimeria_chalumnae XP_008977573.1_Callithrix_jacchus XP_003796342.1_Otolemur_garnettii XP_010383600.1_Rhinopithecus_roxellana AAA35550.1_Homo_sapiens XP_519708.2_Pan_troglodytes 100 NP_001093397.1_Sus_scrofa AAG31164.1_Ovis_aries XP_006994457.1_Peromyscus_maniculatus_bairdii XP_015356717.1_Marmota_marmota_marmota XP_005614864.1_Equus_caballus 100 XP_015202883.1_Lepisosteus_oculatus XP_012669957.1_Clupea_harengus NP_001122161.1_Danio_rerio XP_004080293.1_Oryzias_latipes XP_013863035.1_Austrofundulus_limnaeus XP_015818613.1_Nothobranchius_furzeri XP_015245990.1_Cyprinodon_variegatus XP_007574343.1_Poecilia_formosa CAG12607.1_Tetraodon_nigroviridis CBN81445.1_Dicentrarchus_labrax XP_008275795.1_Stegastes_partitus NP_000675.1_Homo_sapiens XP_006879982.1_Elephantulus_edwardii ABG56138.1_Bos_taurus Q28927.2_sheep XP_008488388.1_Calypte_anna XP_009474370.1_Nipponia_nippon XP_007435758.1_Python_bivittatus XP_013918020.1_Thamnophis_sirtalis XP_007903898.1_Callorhinchus_milii XP_006002873.1_Latimeria_chalumnae CAA06541.1_Petromyzon_marinus CAA06540.1_Petromyzon_marinus XP_002122923.1_Ciona_intestinalis

0.5

Supplementary figure 3. Maximum likelihood tree of β-adrenergic receptors. Bootstrap support values are shown for some nodes of interest. This tree is part of a larger tree containing all investigated GPCRs.

Tyramine-1 receptors

XP_002742354.2_Saccoglossus_kowalevskii_T1 XP_006822832.1_Saccoglossus_kowalevskii EF490687.1_Rhipicephalus_microplus_T1 XP_013791379.1_Limulus_polyphemus XP_013784920.1_Limulus_polyphemus XP_002415939.1_Ixodes_scapularis ABY71758.1_Macrobrachium_rosenbergii XP_014278335.1_Halyomorpha_halys KOB67833.1_Operophtera_brumata KOB69231.1_Operophtera_brumata 98 AFG26689.1_Chilo_suppressalis XP_013197949.1_Amyelois_transitella KPI95840.1_Papilio_xuthus KPJ14377.1_Papilio_machaon XP_014359277.1_Papilio_machaon CAA64865.1_Bombyx_mori_T1 EHJ68870.1_Danaus_plexippus AFX62896.1_Pieris_rapae ACJ06651.1_Spodoptera_littoralis KOB71731.1_Operophtera_brumata XP_011562493.1_Plutella_xylostella XP_013190483.1_Amyelois_transitella XP_002426131.1_Pediculus_humanus_corporis XP_008472990.1_Diaphorina_citri Q25321.1_Locusta_migratoria_T1 XP_003392960.1_Bombus_terrestris XP_011312300.1_Fopius_arisanus XP_014235818.1_Trichogramma_pretiosum XP_014218105.1_Copidosoma_floridanum XP_008206976.1_Nasonia_vitripennis XP_011501106.1_Ceratosolen_solmsi_marchali XP_012227598.1_Linepithema_humile KMQ93013.1_Lasius_niger XP_011339818.1_Cerapachys_biroi 95 XP_014481134.1_Dinoponera_quadriceps XP_011156273.1_Solenopsis_invicta XP_012259361.1_Athalia_rosae XP_014614240.1_Polistes_canadensis CAB76374.1_Apis_mellifera_T1 NP_001164311.1_Tribolium_castaneum AHN85845.1_Nicrophorus_vespilloides ENN74962.1_Dendroctonus_ponderosae ENN72328.1_Dendroctonus_ponderosae ALM55746.1_Sitophilus_oryzae KRT86051.1_Oryctes_borbonicus CAQ48240.1_Periplaneta_americana_T1 KDR16544.1_Zootermopsis_nevadensis KDR16545.1_Zootermopsis_nevadensis XP_005185605.2_Musca_domestica KNC24708.1_Lucilia_cuprina XP_013116014.1_Stomoxys_calcitrans BAL72847.1_Phormia_regina XP_001352884.2_Drosophila_pseudoobscura_pseudoobscura XP_002008573.2_Drosophila_mojavensis XP_001985558.1_Drosophila_grimshawi XP_002068046.2_Drosophila_willistoni ALC43118.1_Drosophila_busckii BAB71788.1_Drosophila_melanogaster_T1 74XP_001958478.1_Drosophila_ananassae 73 XP_011194697.1_Bactrocera_cucurbitae XP_014099568.1_Bactrocera_oleae XP_004526236.1_Ceratitis_capitata XP_011202418.1_Bactrocera_dorsalis XP_001863342.1_Culex_quinquefasciatus XP_001652255.2_Aedes_aegypti ETN58983.1_Anopheles_darlingi XP_312420.3_Anopheles_gambiae_str._PEST 81 KFB35059.1_Anopheles_sinensis KFB51134.1_Anopheles_sinensis XP_014240675.1_Cimex_lectularius NP_001024335.1_Caenorhabitis_elegans_T1 EGT40715_Caenorhabditis_brenneri ELT94407.1_Capitella_teleta AKQ63052.1_Platynereis_dumerilii_T1 XP_014672793.1_Priapulus_caudatus XP_011451309.1_Crassostrea_gigas XP_014776496.1_Octopus_bimaculoides

0.5 Supplementary figure 4. Maximum likelihood tree of Tyramine-1 receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized tyramine receptors were tagged with _T1.

15 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Tyramine-2 receptors

XP_013786964.1_Limulus_polyphemus XP_013785118.1_Limulus_polyphemus XP_008475339.1_Diaphorina_citri XP_015368899.1_Diuraphis_noxia XP_008188216.1_Acyrthosiphon_pisum KFB41294.1_Anopheles_sinensis ETN64509.1_Anopheles_darlingi XP_001649033.2_Aedes_aegypti XP_001998428.2_Drosophila_mojavensis 98 XP_001994504.1_Drosophila_grimshawi XP_015026222.1_Drosophila_virilis ALC46687.1_Drosophila_busckii XP_005185145.1_Musca_domestica XP_013112132.1_Stomoxys_calcitrans XP_011184895.1_Bactrocera_cucurbitae XP_004529355.2_Ceratitis_capitata KNC22325.1_Lucilia_cuprina 100 XP_002072092.2_Drosophila_willistoni 77 XP_001359694.4_Drosophila_pseudoobscura_pseudoobscura NP_650652.1_Drosophila_melanogaster_T2 XP_001954230.1_Drosophila_ananassae XP_011184892.1_Bactrocera_cucurbitae XP_004529353.2_Ceratitis_capitata XP_011203887.1_Bactrocera_dorsalis XP_014097299.1_Bactrocera_oleae KDR18992.1_Zootermopsis_nevadensis XP_002429476.1_Pediculus_humanus_corporis AHN85846.1_Nicrophorus_vespilloides XP_008197781.1_Tribolium_castaneum DAA64505.1_Tribolium_castaneum ERL83362.1_Dendroctonus_ponderosae EHJ64085.1_Danaus_plexippus XP_011555966.1_Plutella_xylostella ADK91078.1_Chilo_suppressalis_T2 XP_013188063.1_Amyelois_transitella BAI52937.1_Bombyx_mori_T2 XP_013165484.1_Papilio_xuthus XP_013142077.1_Papilio_polytes KPJ03809.1_Papilio_xuthus KPJ21156.1_Papilio_machaon XP_014356598.1_Papilio_machaon XP_014207104.1_Copidosoma_floridanum XP_012257334.1_Athalia_rosae 98 XP_012281361.1_Orussus_abietinus XP_011154023.1_Harpegnathos_saltator XP_006608318.1_Apis_dorsata 100 XP_003702699.1_Megachile_rotundata KOX76145.1_Melipona_quadrifasciata KOC70478.1_Habropoda_laboriosa XP_003393336.1_Bombus_terrestris XP_011633420.1_Pogonomyrmex_barbatus XP_011875691.1_Vollenhovia_emeryi XP_011069002.1_Acromyrmex_echinatior XP_012224475.1_Linepithema_humile XP_014488846.1_Dinoponera_quadriceps XP_015174118.1_Polistes_dominula XP_014235895.1_Trichogramma_pretiosum XP_003426713.1_Nasonia_vitripennis XP_011500363.1_Ceratosolen_solmsi_marchali XP_001994503.1_Drosophila_grimshawi XP_002056769.2_Drosophila_virilis XP_001998427.2_Drosophila_mojavensis ALC46686.1_Drosophila_busckii XP_011293189.1_Musca_domestica KNC22330.1_Lucilia_cuprina XP_013112135.1_Stomoxys_calcitrans XP_002096110.1_Drosophila_yakuba 92 XP_011184896.1_Bactrocera_cucurbitae XP_004529356.1_Ceratitis_capitata XP_011203890.1_Bactrocera_dorsalis XP_014097290.1_Bactrocera_oleae XP_001649034.2_Aedes_aegypti ETN64745.1_Anopheles_darlingi XP_309588.5_Anopheles_gambiae_str._PEST XP_014678717.1_Priapulus_caudatus KOF79848.1_Octopus_bimaculoides XP_014778476.1_Octopus_bimaculoides ELU14919.1_Capitella_teleta KU715093_Platynereis_dumerilii_T2 XP_006812999.1_Saccoglossus_kowalevskii_T2B XP_002734062.1_Saccoglossus_kowalevskii_T2A

0.5 Supplementary figure 5. Maximum likelihood tree of Tyramine-2 receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized tyramine receptors were tagged with _T2.

Octopamine−α receptors

XP_014675454.1_Priapulus_caudatus XM_013530280.1_Lingula_anatina XP_014772693.1_Octopus_bimaculoides XP_009058244.1_Lottia_gigantea XP_012937854.1_Aplysia_californica U62771.1_Lymnaea_stagnalis_Oa XP_013079946.1_Biomphalaria_glabrata KU530199_Platynereis_dumerilii_Oa GU074418.1_Balanus_improvisus_Oa AAP93817.1_Periplaneta_americana_Oa XP_002429940.1_Pediculus_humanus_corporis XP_014291339.1_Halyomorpha_halys XP_014253839.1_Cimex_lectularius NP_001280520.1_Tribolium_castaneum AHN85844.1_Nicrophorus_vespilloides ERL88449.1_Dendroctonus_ponderosae XP_013110682.1_Stomoxys_calcitrans XP_003736341.2_Drosophila_pseudoobscura_pseudoobscura XP_002096452.2_Drosophila_yakuba XP_002072172.2_Drosophila_willistoni XP_011291813.1_Musca_domestica AAC17442.1_Drosophila_melanogaster_Oa 77 XP_001994470.1_Drosophila_grimshawi XP_011185894.1_Bactrocera_cucurbitae XP_012156222.1_Ceratitis_capitata XP_004523572.1_Ceratitis_capitata XP_014091610.1_Bactrocera_oleae XP_011185898.1_Bactrocera_cucurbitae XP_311113.4_Anopheles_gambiae_str._PEST XP_001869401.1_Culex_quinquefasciatus XP_001648244.2_Aedes_aegypti EGK96547.1_Anopheles_gambiae_Oa EHJ74631.1_Danaus_plexippus XP_011555307.1_Plutella_xylostella ABI33825.1_Manduca_sexta JN641302.1_Chilo_suppressalis_Oa 98 BAF33393.1_Bombyx_mori_Oa XP_013183250.1_Amyelois_transitella 83 AFG22597.1_Mythimna_separata XP_014362386.1_Papilio_machaon XP_011505609.1_Ceratosolen_solmsi_marchali XP_014225936.1_Trichogramma_pretiosum XP_008558339.1_Microplitis_demolitor 84 XP_015116423.1_Diachasma_alloeum XP_011303410.1_Fopius_arisanus XP_014486016.1_Dinoponera_quadriceps XP_011147506.1_Harpegnathos_saltator XP_012261366.1_Athalia_rosae XP_011264281.1_Camponotus_floridanus XP_012223081.1_Linepithema_humile XP_011872000.1_Vollenhovia_emeryi XP_011702891.1_Wasmannia_auropunctata XP_012136296.1_Megachile_rotundata KOX67468.1_Melipona_quadrifasciata CAD67999.1_Apis_mellifera_Oa KOC61009.1_Habropoda_laboriosa XP_014613083.1_Polistes_canadensis XP_002408812.1_Ixodes_scapularis XP_013790352.1_Limulus_polyphemus XP_013772246.1_Limulus_polyphemus XP_006823182.1_Saccoglossus_kowalevskii_OA1

0.5 Supplementary figure 6. Maximum likelihood tree of Octopamine-α receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized octopamine receptors were tagged with _Oa.

16 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Octopamine−β receptors

XP_002733926.1_Saccoglossus_kowalevskii_Ob Q4LBB9.2_Drosophila_melanogaster_Ob XP_011294329.1_Musca_domestica KNC25006.1_Lucilia_cuprina XP_014254146.1_Cimex_lectularius JN620367.1_Chilo_suppressalis_Ob AB470228.1_Bombyx_mori_Ob XP_012281474.1_Orussus_abietinus 71 XP_014207204.1_Copidosoma_floridanum XP_011504152.1_Ceratosolen_solmsi_marchali XP_014221613.1_Trichogramma_pretiosum XP_008207151.1_Nasonia_vitripennis XP_011140150.1_Harpegnathos_saltator XP_011166275.1_Solenopsis_invicta XP_011876313.1_Vollenhovia_emeryi XP_011344277.1_Cerapachys_biroi XP_012261826.1_Athalia_rosae XP_015180910.1_Polistes_dominula XP_396348.4_Apis_mellifera XP_003402376.1_Bombus_terrestris XP_003701983.1_Megachile_rotundata XP_012343074.1_Apis_florea AHN85842.1_Nicrophorus_vespilloides GU074419.1_Balanus_improvisus_Ob GU074420.1_Balanus_improvisus_Ob GU074421.1_Balanus_improvisus_Ob XP_001948521.2_Acyrthosiphon_pisum NP_001280505.1_Tribolium_castaneum HF548211.1_Apis_mellifera_Ob HF548212.1_Apis_mellifera_Ob XP_012269188.1_Athalia_rosae XP_008548361.1_Microplitis_demolitor XP_011312392.1_Fopius_arisanus 100 74 XP_015118835.1_Diachasma_alloeum XP_012281328.1_Orussus_abietinus XP_014467276.1_Dinoponera_quadriceps XP_011263631.1_Camponotus_floridanus XP_011702778.1_Wasmannia_auropunctata XP_011053483.1_Acromyrmex_echinatior XP_011870420.1_Vollenhovia_emeryi XP_011630965.1_Pogonomyrmex_barbatus XP_012215458.1_Linepithema_humile 87 XP_011349656.1_Cerapachys_biroi XP_014611330.1_Polistes_canadensis XP_003706543.1_Megachile_rotundata CCO13922.1_Apis_mellifera_Ob XP_012166217.1_Bombus_terrestris XP_001947781.1_Acyrthosiphon_pisum XP_013165427.1_Papilio_xuthus XP_004922133.2_Bombyx_mori XP_011557485.1_Plutella_xylostella EHJ66744.1_Danaus_plexippus 80 73 AGV79326.1_Chilo_suppressalis XP_013185050.1_Amyelois_transitella AHN85841.1_Nicrophorus_vespilloides NP_001280514.1_Tribolium_castaneum KDR19125.1_Zootermopsis_nevadensis XP_014241423.1_Cimex_lectularius XP_002422997.1_Pediculus_humanus_corporis XP_014088159.1_Bactrocera_oleae XP_004523493.1_Ceratitis_capitata XP_013099911.1_Stomoxys_calcitrans 86 XP_001357862.3_Drosophila_pseudoobscura_pseudoobscura XP_001955623.2_Drosophila_ananassae AEO17899.1_Drosophila_melanogaster XP_002074057.1_Drosophila_willistoni KNC25005.1_Lucilia_cuprina XP_002054844.1_Drosophila_virilis XP_002000086.1_Drosophila_mojavensis XP_001994735.1_Drosophila_grimshawi GU074422.1_Balanus_improvisus_Ob EFX87996.1_Daphnia_pulex XP_014671143.1_Priapulus_caudatus ELT86969.1_Capitella_teleta KU886229_Platynereis_dumerilii XP_014771877.1_Octopus_bimaculoides AY055377.1_Spisula_solidissima_Ob 99 XP_013096128.1_Biomphalaria_glabrata AF117654.1_Aplysia_kurodai_Ob AF222978.1_Aplysia_californica_Ob XP_009052442.1_Lottia_gigantea FC563777.1_Lottia_gigantea XP_011448928.1_Crassostrea_gigas XP_013389322.1_Lingula_anatina

0.5 Supplementary figure 7. Maximum likelihood tree of Octopamine-β receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized octopamine receptors were tagged with _Ob.

17 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A P. dumerilii α1-adrenergic

Norepinephrine 150 Yohimbine 150 Epinephrine Mianserin ] Dopamine ] % % [ [ 100 100 ion ion t t va va i i t 50 t 50 ac ac

0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M])

B S. k owalevskii α1-adrenergic Octopamine Epinephrine 150 Yohimbine 150 150 Norepinephrine Mianserin Dopamine ] ] ] % % % [ [ [ 100 100 100 ion ion ion t t t va va va i i i t t 50 50 t 50 ac ac ac

0 0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M]) log conc (log[M])

C P. caudatus α1-adrenergic

150 150 Norepinephrine Yohimbine Mianserin ] ] % % [ 100 [ 100 ion ion t t va va i i t 50 t 50 ac ac

0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M]) Supplementary figure 8. Dose-response curves of α1-adrenergic receptors from P. dumerilii, P. caudatus, and S. kowalevskii treated with varying concentrations of ligand or inhibitor. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 and IC50 values are listed in Table 1.

18 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A P. dumerilii α2-adrenergic

Tyramine Dopamine 150 Yohimbine 150 Octopamine Clonidine Mianserin 150 ] ] Norepinephrine ] % % % [ [ Epinephrine [ 100 100 ion ion ion 100 t t t va va va i i i t t t 50 50 ac ac ac 50

0 0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M]) log conc (log[M])

B S. kowalevskii α2-adrenergic

150 Octopamine 150 Tyramine 150 Yohimbine Norepinephrine Dopamine Mianserin Epinephrine Clonidine ] ] ] % % % [ [ [ 100 100 100 ion ion ion t t t va va va i i i t t t 50 50 50 ac ac ac

0 0 0 -20 -15 -10 -5 0 -15 -10 -5 0 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M]) log conc (log[M])

C P. caudatus α2-adrenergic

Norepinephrine Yohimbine 150 150 Epinephrine Mianserin Clonidine ] ] % % [ 100 [ 100 ion ion t t va va i i t 50 t 50 ac ac

0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M]) Supplementary figure 9. Dose-response curves of α2-adrenergic receptors from P. dumerilii, P. caudatus, and S. kowalevskii treated with varying concentrations of ligand or inhibitor. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 and IC50 values are listed in Table 1.

19 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A P. dumerilii Tyramine-1

150 150 Tyramine Yohimbine Octopamine Mianserin ] ] Dopamine % % [ [ 100 100 ion ion t t va va i i t t 50 50 ac ac

0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M])

B P. dumerilii Tyramine-2

150 150 Tyramine Yohimbine Octopamine Mianserin ] ] Dopamine % % [ [ 100 100 ion ion t t va va i i t t 50 50 ac ac

0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M])

C S. kowalevskii Tyramine-1

150 150 Tyramine Yohimbine Octopamine Mianserin ] Dopamine ] % % [ 100 [ 100 Clonidine ion ion t t va va i i t 50 t 50 ac ac

0 0 -12 -10 -8 -6 -4 -2 0 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M])

D S. kowalevskii Tyramine-2A

150 150 150 Tyramine Dopamine Yohimbine Octopamine Clonidine Mianserin ] ] ] % % % [ [ 100 100 [ 100 ion ion ion t t t va va va i i i t t 50 50 t 50 ac ac ac

0 0 0 -14 -12 -10 -8 -6 -4 -2 -14 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M]) log conc (log[M])

E S. kowalevskii Tyramine-2B

150 150 Tyramine 150 Dopamine Yohimbine Octopamine Clonidine Mianserin ] ] Norepinephrine ] % % % [ [ [ 100 100 Epinephrine 100 ion ion ion t t t va va va i i i t t 50 t 50 50 ac ac ac

0 0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M]) log conc (log[M])

Supplementary figure 10. Dose-response curves of tyramine receptors from P. dumerilii and S. kowalevskii treated with varying concentrations of ligand or inhibitor. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 and IC50 values are listed in Table 1.

20 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A P. dumerilii Octopamine α

150 150 Tyramine Yohimbine Octopamine Mianserin ] ] % %

[ 100 [ 100 ion ion t t va va i i t 50 t 50 ac ac

0 0 -12 -10 -8 -6 -4 -2 0 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M])

B S. kowalevskii Octopamine α

150 150 150 Tyramine Epinephrine Yohimbine Octopamine Clonidine Mianserin ] ] ] % Norepinephrine % % [ [ [ 100 100 100 Dopamine ion ion ion t t t va va va i i i t 50 t 50 t 50 ac ac ac

0 0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M]) log conc (log[M])

C S. kowalevskii Octopamine β

150 Octopamine Yohimbine Norepinephrine 150 Mianserin ] ] % %

[ 100 [ 100 ion ion t t va va i i t 50 t 50 ac ac

0 0 -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 -2 log conc (log[M]) log conc (log[M])

Supplementary figure 11. Dose-response curves of octopamine receptors from P. dumerilii and S. kowalevskii treated with varying concentrations of ligand or inhibitor. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 and IC50 values are listed in Table 1.

21 bioRxiv preprint doi: https://doi.org/10.1101/063743; this version posted July 13, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

XP_014769443.1_Octopus_bimaculoides XP_011441559.1_Crassostrea_gigas EKC37654.1_Crassostrea_gigas NP_001191536.1_Aplysia_californica XP_012940696.1_Aplysia_californica XP_013070812.1_Biomphalaria_glabrata BAQ94593.1_Ambigolimax_valentianus XP_013401543.1_Lingula_anatina XP_014666248.1_Priapulus_caudatus XP_013781988.1_Limulus_polyphemus XP_014245782.1_Cimex_lectularius XP_008202389.1_Nasonia_vitripennis XP_012274121.1_Orussus_abietinus XP_006570903.1_Apis_mellifera XP_006621116.1_Apis_dorsata KFB44891.1_Anopheles_sinensis XP_011290217.1_Musca_domestica ACJ13262.1_Drosophila_melanogaster XP_002731843.1_Saccoglossus_kowalevskii lcl|comp422145_c0_seq4_Platynereis_dumerilii ELU11164.1_Capitella_teleta XP_009023368.1_Helobdella_robusta 91 XP_014774406.1_Octopus_bimaculoides XP_009048193.1_Lottia_gigantea XP_005107794.1_Aplysia_californica XP_011449820.1_Crassostrea_gigas ADP08788.1_Azumapecten_farreri 100 XP_013404119.1_Lingula_anatina EFX81779.1_Daphnia_pulex AHN85839.1_Nicrophorus_vespilloides XP_308519.3_Anopheles_gambiae_str._PEST XP_001656857.1_Aedes_aegypti XP_001870804.1_Culex_quinquefasciatus XP_002050467.1_Drosophila_virilis XP_001360141.2_Drosophila_pseudoobscura_pseudoobscura XP_015054436.1_Drosophila_yakuba XP_002074575.1_Drosophila_willistoni XP_004526583.1_Ceratitis_capitata 100 XP_011196622.1_Bactrocera_cucurbitae KNC26593.1_Lucilia_cuprina XP_005180269.1_Musca_domestica XP_002426536.1_Pediculus_humanus_corporis XP_008198031.1_Tribolium_castaneum ENN79134.1_Dendroctonus_ponderosae XP_014241491.1_Cimex_lectularius XP_014291344.1_Halyomorpha_halys XP_014291341.1_Halyomorpha_halys BAO52000.1_Gryllus_bimaculatus XP_012265736.1_Athalia_rosae XP_003699867.1_Megachile_rotundata XP_012534953.1_Monomorium_pharaonis XP_011061414.1_Acromyrmex_echinatior XP_015122001.1_Diachasma_alloeum XP_011297268.1_Fopius_arisanus 96 XP_008548921.1_Microplitis_demolitor XP_972728.2_Tribolium_castaneum KRT84890.1_Oryctes_borbonicus 100 KDR12158.1_Zootermopsis_nevadensis XP_014227242.1_Trichogramma_pretiosum XP_012265735.1_Athalia_rosae XP_008548824.1_Microplitis_demolitor XP_011297267.1_Fopius_arisanus XP_014489121.1_Dinoponera_quadriceps

XP_012055250.1_Atta_cephalotes tyrosine decarboxylase XP_012272741.1_Orussus_abietinus XP_014605048.1_Polistes_canadensis XP_012147325.1_Megachile_rotundata KOX68436.1_Melipona_quadrifasciata XP_013789180.1_Limulus_polyphemus XP_003744997.1_Metaseiulus_occidentalis XP_002630249.1_Caenorhabditis_briggsae NP_495744.1_Caenorhabditis_elegans XP_001899866.1_Brugia_malayi EFO20782.2_Loa_loa KHN71251.1_Toxocara_canis CEF60746.1_Strongyloides_ratti XP_014671545.1_Priapulus_caudatus XP_005108337.1_Aplysia_californica XP_011443238.1_Crassostrea_gigas EKC25403.1_Crassostrea_gigas XP_002735383.1_Saccoglossus_kowalevskii XP_007895676.1_Callorhinchus_milii XP_006635648.1_Lepisosteus_oculatus XP_012680382.1_Clupea_harengus XP_010895280.1_Esox_lucius XP_005949669.1_Haplochromis_burtoni XP_015245016.1_Cyprinodon_variegatus 95 XP_003966032.1_Takifugu_rubripes KTG31909.1_Cyprinus_carpio 100 XP_010138453.1_Buceros_rhinoceros_silvestris XP_005434516.1_Falco_cherrug XP_009680213.1_Struthio_camelus_australis 52XP_010219192.1_Tinamus_guttatus XP_010022195.1_Nestor_notabilis XP_010006646.1_Chaetura_pelagica XP_008489540.1_Calypte_anna XP_015508789.1_Parus_major XP_007061422.1_Chelonia_mydas XP_005278593.1_Chrysemys_picta_bellii XP_003407186.1_Loxodonta_africana XP_004708097.1_Echinops_telfairi XP_006875343.1_Chrysochloris_asiatica XP_007942142.1_Orycteropus_afer_afer XP_004739444.1_Mustela_putorius_furo XP_004415117.1_Odobenus_rosmarus_divergens XP_848285.2_Canis_lupus_familiaris XP_014649145.1_Ceratotherium_simum_simum XP_012025784.1_Ovis_aries_musimon XP_004694560.1_Condylura_cristata XP_010339695.1_Saimiri_boliviensis_boliviensis XP_003896028.1_Papio_anubis ELV13924.1_Tupaia_chinensis XP_004582273.2_Ochotona_princeps XP_012879949.1_Dipodomys_ordii XP_008847491.1_Nannospalax_galili XP_008768473.1_Rattus_norvegicus XP_006973100.1_Peromyscus_maniculatus_bairdii XP_005396658.1_Chinchilla_lanigera XP_013000851.1_Cavia_porcellus XP_004630583.1_Octodon_degus XP_004839911.1_Heterocephalus_glaber XP_015345826.1_Marmota_marmota_marmota XP_003797782.1_Otolemur_garnettii XP_012506091.1_Propithecus_coquereli XP_003762585.1_Sarcophilus_harrisii XP_001379620.3_Monodelphis_domestica XP_006000049.1_Latimeria_chalumnae XP_014667849.1_Priapulus_caudatus XP_012942331.1_Aplysia_californica XP_012942336.1_Aplysia_californica XP_013088909.1_Biomphalaria_glabrata BAM35936.1_Lymnaea_stagnalis EKC41301.1_Crassostrea_gigas XP_014775466.1_Octopus_bimaculoides XP_009050823.1_Lottia_gigantea XP_013772406.1_Limulus_polyphemus XP_012226027.1_Linepithema_humile XP_002002172.1_Drosophila_mojavensis XP_001950555.2_Acyrthosiphon_pisum

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0.2 Supplementary figure 12. Maximum likelihood tree of tyrosine decarboxylase and aromatic amino acid decarboxylase enzymes. Bootstrap support values are shown for selected nodes. P. dumerilii, P. caudatus, and S. kowalevskii sequences are highlighted in color. The Caenorhabditis elegans tyrosine decarboxylase was experimentally shown to be required for tyramine biosynthesis (Alkema et al., 2005).

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