Ancient coexistence of norepinephrine, tyramine, and octopamine … · presence of tyramine signaling in S. kowalewskii is also supported by the phyletic distribution of tyrosine

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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.


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EC50 (M)/IC50 (M) Tyramine Octopami

ne Norepinephrine

Epinephrine

Dopamine

Clonidine Yohimbine

Mianserin

P. dumerilii α1-adrenergic KX372342

inactive inactive 2.12E-07 3.79E-07 1.25E-04 inactive 4.41E-06 3.79E-06

P. dumerilii α2-adrenergic KX372343

8.36E-05 2.67E-06 5.25E-09 1.06E-08 1.63E-06 2.57E-06 5.71E-06 2.53E-05

S. kowalevskii α1-adrenergic ALR88680

inactive n/a 1.67E-08 1.94E-08 3.79E-06 inactive 1.32E-05 4.46E-06

S. kovalewskii α2-adrenergic XP_002734932

3.75E-06 1.94E-06 1.16E-13 2.31E-09 5.60E-09 3.61E-08 7.90E-05 n/a

P. caudatus α1-adrenergic XP_014662992

inactive inactive 7.49E-09 inactive inactive inactive n/a n/a

P. caudatus α2-adrenergic XP_014681069

inactive inactive 2.20E-06 4.52E-07 inactive 1.02E-07 n/a 9.85E-07

P. dumerilii Tyramine-1 KP293998

1.12E-08 2.70E-06 n/a n/a 7.79E-06 n/a 2.06E-06 1.05E-05

P. dumerilii Tyramine-2 KU715093

7.02E-09 7.82E-07 n/a n/a 3.89E-06 n/a 5.38E-05 6.36E-06

S. kovalewskii Tyramine-1 XP_002742354

8.40E-05 n/a inactive inactive n/a 2.86E-04 1.68E-06 1.70E-05

S. kovalewskii Tyramine-2A XP_002734062

1.22E-09 6.24E-08 inactive inactive 7.18E-08 1.39E-06 n/a 1.61E-04

S. kovalewskii Tyramine-2B XP_006812999

3.71E-09 1.57E-06 1.15E-04 2.85E-05 1.44E-06 1.60E-05 2.06E-05 1.88E-05

P. dumerilii Octopamine α KU530199

1.34E-05 2.56E-07 n/a n/a inactive n/a 9.02E-09 1.62E-06

S. kowalevskii Octopamine α XP_006823182

1.72E-05 6.89E-07 5.30E-05 1.85E-05 2.65E-04 1.64E-07 7.78E-06 2.16E-05

S. kowalevskii Octopamine β XP_002733926

inactive 6.37E-08 3.49E-06 inactive inactive inactive 1.57E-04 6.45E-06

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.


https://doi.org/10.1101/063743

13

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.

0.5

EFX64650.1_Daphnia_pulex

XP_010205582.1_Colius_striatus

XP_009050255.1_Lottia_gigantea

XP_014662992.1_Priapulus_caudatus

XP_008279073.1_Stegastes_partitus

CDQ72256.1_Oncorhynchus_mykiss

XP_005285240.1_Chrysemys_picta_bellii

XP_003970475.1_Takifugu_rubripes

XP_014743771.1_Sturnus_vulgaris

XP_008334310.1_Cynoglossus_semilaevis

XP_014822169.1_Calidris_pugnax

XP_014051077.1_Salmo_salar


KTG32062.1_Cyprinus_carpio

XP_010788374.1_Notothenia_coriiceps

XP_004460877.1_Dasypus_novemcinctus

XP_013133031.1_Oreochromis_niloticus

NP_001007359.1_Danio_rerio

XP_012687224_Clupea_harengus

XP_003227101.1_Anolis_carolinensis

XP_013867515.1_Austrofundulus_limnaeus

XP_003782039.1_Otolemur_garnettii


KTF87842.1_Cyprinus_carpio


XP_007430469.1_Python_bivittatus

XP_007247270.1_Astyanax_mexicanus

XP_010154880.1_Eurypyga_helias

XP_009993014.1_Chaetura_pelagica

XP_011355023.1_Pteropus_vampyrus

XP_008630655.1_Corvus_brachyrhynchos

NP_001118147.1_Oncorhynchus_mykiss

XP_013091628_Biomphalaria_glabrata

XP_006138206_Pelodiscus_sinensis

XP_008489604.1_Calypte_anna

XP_007896221.1_Callorhinchus_milii

XP_015268171.1_Gekko_japonicus

XP_002935531.2_Xenopus_tropicalis

XP_697043.2_Danio_rerio

XP_007548430.1_Poecilia_formosa

XP_003726237.1_Strongylocentrotus_purpuratus


XP_006211986.1_Vicugna_pacos

XP_007477871.1_Monodelphis_domestica

XP_005142671.1_Melopsittacus_undulatus

XP_005719861.1_Pundamilia_nyererei

XP_012959596.1_Anas_platyrhynchos

XP_006625863.1_Lepisosteus_oculatus

XP_013787844.1_Limulus_polyphemus

XP_004665892.1_Jaculus_jaculus

AAA35496.1_Homo_sapiens_AA1A


XP_012697555.1_Clupea_harengus

XP_003748403.1_Metaseiulus_occidentalis

XP_009566450.1_Cuculus_canorus

ALR88680.1_Saccoglossus_kowalevskii_AA1


XP_015827734.1_Nothobranchius_furzeri

XP_008059269.1_Tarsius_syrichta

XP_010886834.1_Esox_lucius

XP_006276442.2_Alligator_mississippiensis


KX372342_Platynereis_dumerilii_AA1

KFM77668.1_Stegodyphus_mimosarum

ETE57756.1_Ophiophagus_hannah


XP_002197113.1_Taeniopygia_guttata


XP_015598849.1_Cephus_cinctus


XP_015233524.1_Cyprinodon_variegatus

ADK98420.1_Meriones_unguiculatus


XP_010143615.1_Buceros_rhinoceros_silvestris


XP_012709980.1_Fundulus_heteroclitus




KPP75495.1_Scleropages_formosus

XP_004065916.1_Oryzias_latipes

XP_008921792.1_Manacus_vitellinus


XP_009899896.1_Picoides_pubescens



XP_006805960.1_Neolamprologus_brichardi

XP_015152983.1_Gallus_gallus


XP_009951638.1_Leptosomus_discolor


XP_011422115.1_Crassostrea_gigasELU14162.1_Capitella_teleta

XP_010177730.1_Mesitornis_unicolor

XP_006114239.1_Pelodiscus_sinensis


XP_013917984.1_Thamnophis_sirtalis


XP_013419311.1_Lingula_anatina



XP_006252170.1_Rattus_norvegicus


XP_008417330.1_Poecilia_reticulata


XP_012936806.1_Aplysia_californica

XP_014777674.1_Octopus_bimaculoides

XP_006010360.1_Latimeria_chalumnae

XP_010853649.1_Bison_bison_bison

1

100

100

85

83

71

99

80

93

96

95

63

α1-adrenergic receptors


https://doi.org/10.1101/063743

14

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.

0.5


CDJ80122.1_Haemonchus_contortus




Q90WY4.1_Danio_rerio



KTF91499.1_Cyprinus_carpio

KX372343_Platynereis_dumerilii_AA2



XP_010968209.1_Camelus_bactrianus


XP_014938420.1_Acinonyx_jubatus

CAD11981.1_Rhogeessa_tumida

EDL01717.1_Mus_musculus

XP_006778809.1_Myotis_davidii


XP_011573649.1_Aquila_chrysaetos_canadensis



XP_014333406.1_Bos_mutus

XP_010732944.1_Larimichthys_crocea

CAG05052.1_Tetraodon_nigroviridis


KQK80321.1_Amazona_aestiva

XP_010981560.1_Camelus_dromedarius

XP_015350355.1_Marmota_marmota_marmota



XP_014274544.1_Halyomorpha_halys

Q8JG70.1_Danio_rerio

XP_014390455.1_Myotis_brandtii


XP_002734932.1_Saccoglossus_kowalevskii_AA2



XP_012873058.1_Dipodomys_ordii

XP_014242340.1_Cimex_lectularius

XP_009911888.1_Haliaeetus_albicilla


XP_005813740.2_Xiphophorus_maculatus


XP_002644716.1_Caenorhabditis_briggsae

XP_007173405.1_Balaenoptera_acutorostrata_scammoni


XP_011433775.1_Crassostrea_gigas

CAL36767.1_Branchiostoma_floridae

XP_002595000.1_Branchiostoma_floridae

XP_015718435.1_Coturnix_japonica

XP_005308213.1_Chrysemys_picta_bellii

XP_009806348.1_Gavia_stellata

EHH14168.1_Macaca_mulatta


CEF71092.1_Strongyloides_ratti

XP_010733243.1_Larimichthys_crocea







XP_004539207_Maylandia_zebra


XP_012953671.1_Anas_platyrhynchos



XP_013156970.1_Falco_peregrinus


P32251.1_Carassius_auratus




XP_006135062.1_Pelodiscus_sinensis


XP_005446496.1_Falco_cherrug

CAD20302.1_Tachyoryctes_sp.



XP_008538974.1_Equus_przewalskii



XP_015673604.1_Protobothrops_mucrosquamatus



EDL94448.1_Rattus_norvegicus

XP_003755431.1_Sarcophilus_harrisii


XP_014958903.1_Ovis_aries






XP_012579513.1_Condylura_cristata

XP_008198464.2_Tribolium_castaneum

XP_013030003.1_Anser_cygnoides_domesticus


XP_006022896.1_Alligator_sinensis



XP_006750950.1_Leptonychotes_weddellii

XP_012626815.1_Microcebus_murinus

XP_005874627.1_Myotis_brandtii

ERE78076.1_Cricetulus_griseus

XP_008142634.1_Eptesicus_fuscus

XP_005485737.1_Zonotrichia_albicollis


XP_006880000.1_Elephantulus_edwardii

XP_004265893.1_Orcinus_orca


XP_006609424.1_Apis_dorsata


CAC87884.1_Takifugu_rubripes

XP_015390193.1_Panthera_tigris_altaica

KKF18259.1_Larimichthys_crocea


XP_013802685.1_Apteryx_australis_mantelli

XP_010350316.1_Saimiri_boliviensis_boliviensis

NP_001072843.1_Xenopus_tropicalis


XP_010587294.1_Loxodonta_africana




XP_005416433.1_Geospiza_fortis

CAI70426.1_Cricetomys_gambianus

XP_005983264.1_Pantholops_hodgsonii

XP_007120031.1_Physeter_catodon


Q91081.1_Labrus_ossifagus


XP_004942333.2_Gallus_gallus

EPB80586_Ancylostoma_ceylanicum

ELT99717.1_Capitella_teleta

XP_009881752.1_Charadrius_vociferus

XP_013820290.1_Capra_hircus

NP_000672.3_Homo_sapiens_AA2A




AEP17845.1_Anomalurus_beecrofti



KF460458.1_Chilo_suppressalis_Oa






XP_005235799.1_Falco_peregrinus



AAN78417.1_Tupaia_belangeri






XP_008626884.1_Corvus_brachyrhynchos



XP_013096124.1_Biomphalaria_glabrata


XP_004631588.1_Octodon_degus


96

77

95

99

96

100

α2-adrenergic receptors


https://doi.org/10.1101/063743

15

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.

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.

XP_004587615.1_Ochotona_princeps

CAA06540.1_Petromyzon_marinus

XP_005324245.1_Ictidomys_tridecemlineatus

XP_002122923.1_Ciona_intestinalis





XP_006994457.1_Peromyscus_maniculatus_bairdii

XP_004696769.1_Echinops_telfairi


NP_001093397.1_Sus_scrofa


AAQ91625.1_Branchiostoma_floridae




XP_008417789.1_Poecilia_reticulata



XP_014746696.1_Sturnus_vulgaris

AGU01500.1_Sus_scrofa


XP_014708070.1_Equus_asinus


XP_006766281.1_Myotis_davidii

AAA89068.1_Rattus_norvegicus




XP_004620654.1_Sorex_araneus


XP_015678290.1_Protobothrops_mucrosquamatus

XP_002710343.1_Oryctolagus_cuniculus

XP_006784977.1_Neolamprologus_brichardi


CBN81445.1_Dicentrarchus_labrax


XP_008586147.1_Galeopterus_variegatus


XP_009284014.1_Aptenodytes_forsteri

XP_014803719.1_Calidris_pugnax

XP_004384975.1_Trichechus_manatus_latirostris





XP_009474370.1_Nipponia_nippon

XP_015306903.1_Macaca_fascicularis



XP_519708.2_Pan_troglodytes

NP_001117912.1_Oncorhynchus_mykiss


XP_008930097.1_Manacus_vitellinus


XP_008690966.1_Ursus_maritimus



AAF20199.1_Homo_sapiens

XP_008150402.1_Eptesicus_fuscus




ACX46713.1_Pimephales_promelas


XP_009470511.1_Nipponia_nippon

XP_006277924.1_Alligator_mississippiensis

NP_001083418.1_Xenopus_laevis


XP_005719651.1_Pundamilia_nyererei


AAG31164.1_Ovis_aries





XP_008836112.1_Nannospalax_galili

NP_001116897.2_Xenopus_tropicalis


CAA06541.1_Petromyzon_marinus


CAA06539.1_Myxine_glutinosa

XP_008977573.1_Callithrix_jacchus

XP_009069256.1_Acanthisitta_chloris

Q28927.2_sheep

XP_005468045.1_Oreochromis_niloticus

NP_031446.2_Mus_musculus

XP_005614864.1_Equus_caballus


AAA35550.1_Homo_sapiens

XP_007447551.1_Lipotes_vexillifer

XP_015232667.1_Cyprinodon_variegatusXP_014901064.1_Poecilia_latipinna


XP_004664808.1_Jaculus_jaculus

ELW64306.1_Tupaia_chinensis


XP_007516440.1_Erinaceus_europaeus

XP_007188760.1_Balaenoptera_acutorostrata_scammoni

NP_001085791.1_Xenopus_laevis

XP_006052758.1_Bubalus_bubalis



XP_008062588.1_Tarsius_syrichta





ABG56138.1_Bos_taurus




XP_010383600.1_Rhinopithecus_roxellana

XP_009645348.1_Egretta_garzetta


NP_000675.1_Homo_sapiens

ERE85201.1_Cricetulus_griseus


XP_015818613.1_Nothobranchius_furzeri


XP_012597080.1_Microcebus_murinus


XP_004428726.1_Ceratotherium_simum_simum


XP_003477383.1_Cavia_porcellus

AAI60515.1_Xenopus_tropicalis

XP_006863914.1_Chrysochloris_asiatica



XP_010625409.1_Fukomys_damarensis

72

100

30

100

90

0.5

β-adrenergic receptors

0.5

XP_312420.3_Anopheles_gambiae_str._PEST

XP_011339818.1_Cerapachys_biroi

XP_002426131.1_Pediculus_humanus_corporis

XP_014359277.1_Papilio_machaon

KRT86051.1_Oryctes_borbonicus

KOB69231.1_Operophtera_brumata

KFB35059.1_Anopheles_sinensisKFB51134.1_Anopheles_sinensis

XP_013197949.1_Amyelois_transitellaAFG26689.1_Chilo_suppressalis

NP_001164311.1_Tribolium_castaneum

ACJ06651.1_Spodoptera_littoralis

XP_003392960.1_Bombus_terrestris

XP_002742354.2_Saccoglossus_kowalevskii_T1



XP_001863342.1_Culex_quinquefasciatus

XP_012227598.1_Linepithema_humile

XP_013190483.1_Amyelois_transitella

XP_008472990.1_Diaphorina_citri

KPJ14377.1_Papilio_machaon

XP_002415939.1_Ixodes_scapularis


XP_014481134.1_Dinoponera_quadriceps

Q25321.1_Locusta_migratoria_T1

XP_012259361.1_Athalia_rosae

BAB71788.1_Drosophila_melanogaster_T1


XP_014099568.1_Bactrocera_oleae

AKQ63052.1_Platynereis_dumerilii_T1

XP_011501106.1_Ceratosolen_solmsi_marchali

KPI95840.1_Papilio_xuthus


XP_004526236.1_Ceratitis_capitata

XP_001652255.2_Aedes_aegypti

XP_011156273.1_Solenopsis_invicta

EF490687.1_Rhipicephalus_microplus_T1

XP_001958478.1_Drosophila_ananassae

XP_002008573.2_Drosophila_mojavensis

EHJ68870.1_Danaus_plexippus


BAL72847.1_Phormia_regina

NP_001024335.1_Caenorhabitis_elegans_T1

ENN74962.1_Dendroctonus_ponderosae


CAQ48240.1_Periplaneta_americana_T1

ABY71758.1_Macrobrachium_rosenbergii

XP_001352884.2_Drosophila_pseudoobscura_pseudoobscura

KMQ93013.1_Lasius_niger

XP_008206976.1_Nasonia_vitripennis

AHN85845.1_Nicrophorus_vespilloides

XP_014614240.1_Polistes_canadensis

AFX62896.1_Pieris_rapae

ALM55746.1_Sitophilus_oryzae

CAB76374.1_Apis_mellifera_T1

XP_011562493.1_Plutella_xylostella

EGT40715_Caenorhabditis_brenneri

XP_011202418.1_Bactrocera_dorsalis

XP_013116014.1_Stomoxys_calcitrans

XP_011194697.1_Bactrocera_cucurbitae

XP_001985558.1_Drosophila_grimshawi


XP_002068046.2_Drosophila_willistoni

XP_011312300.1_Fopius_arisanus


CAA64865.1_Bombyx_mori_T1


ALC43118.1_Drosophila_busckii

KDR16545.1_Zootermopsis_nevadensis

ETN58983.1_Anopheles_darlingi

XP_006822832.1_Saccoglossus_kowalevskii

XP_014218105.1_Copidosoma_floridanum

XP_005185605.2_Musca_domesticaKNC24708.1_Lucilia_cuprina


XP_014235818.1_Trichogramma_pretiosum


81

73

98

74

95

Tyramine-1 receptors


https://doi.org/10.1101/063743

16

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.

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.

0.5

DAA64505.1_Tribolium_castaneum

XP_002056769.2_Drosophila_virilis

XP_013142077.1_Papilio_polytes


KPJ03809.1_Papilio_xuthus

KU715093_Platynereis_dumerilii_T2


XP_011154023.1_Harpegnathos_saltator


XP_015174118.1_Polistes_dominula

XP_011069002.1_Acromyrmex_echinatior


XP_012281361.1_Orussus_abietinus






XP_011875691.1_Vollenhovia_emeryi

KNC22325.1_Lucilia_cuprina

XP_001954230.1_Drosophila_ananassaeNP_650652.1_Drosophila_melanogaster_T2


XP_008475339.1_Diaphorina_citri

XP_005185145.1_Musca_domestica














XP_003702699.1_Megachile_rotundata

ERL83362.1_Dendroctonus_ponderosae




XP_008188216.1_Acyrthosiphon_pisum


XP_013165484.1_Papilio_xuthus




KOF79848.1_Octopus_bimaculoides

XP_006812999.1_Saccoglossus_kowalevskii_T2B

KFB41294.1_Anopheles_sinensis


XP_002734062.1_Saccoglossus_kowalevskii_T2A

BAI52937.1_Bombyx_mori_T2

XP_011633420.1_Pogonomyrmex_barbatus


ADK91078.1_Chilo_suppressalis_T2



KOC70478.1_Habropoda_laboriosa



XP_011184895.1_Bactrocera_cucurbitaeXP_013112132.1_Stomoxys_calcitrans




ELU14919.1_Capitella_teleta

XP_002096110.1_Drosophila_yakuba


KOX76145.1_Melipona_quadrifasciata

KPJ21156.1_Papilio_machaon



XP_015368899.1_Diuraphis_noxia





XP_012257334.1_Athalia_rosae98

77

92

100

100

98

Tyramine-2 receptors

0.5




AFG22597.1_Mythimna_separata



XP_002408812.1_Ixodes_scapularis


AAP93817.1_Periplaneta_americana_Oa

XP_011264281.1_Camponotus_floridanus

XP_015116423.1_Diachasma_alloeum




XP_008558339.1_Microplitis_demolitor












GU074418.1_Balanus_improvisus_Oa


KOC61009.1_Habropoda_laboriosa

EGK96547.1_Anopheles_gambiae_Oa







JN641302.1_Chilo_suppressalis_Oa




U62771.1_Lymnaea_stagnalis_Oa









XP_011702891.1_Wasmannia_auropunctata

ERL88449.1_Dendroctonus_ponderosae


KU530199_Platynereis_dumerilii_Oa

CAD67999.1_Apis_mellifera_Oa

ABI33825.1_Manduca_sexta

AAC17442.1_Drosophila_melanogaster_Oa


XM_013530280.1_Lingula_anatina

XP_006823182.1_Saccoglossus_kowalevskii_OA1

BAF33393.1_Bombyx_mori_OaXP_013183250.1_Amyelois_transitella

84

9883

77

Octopamine−α receptors


https://doi.org/10.1101/063743

17

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.

0.5

FC563777.1_Lottia_gigantea


AF117654.1_Aplysia_kurodai_Ob

HF548211.1_Apis_mellifera_Ob

XP_396348.4_Apis_mellifera


XP_011702778.1_Wasmannia_auropunctata






XP_002733926.1_Saccoglossus_kowalevskii_Ob




JN620367.1_Chilo_suppressalis_Ob




HF548212.1_Apis_mellifera_Ob


GU074422.1_Balanus_improvisus_Ob

AEO17899.1_Drosophila_melanogaster


CCO13922.1_Apis_mellifera_Ob

XP_012281474.1_Orussus_abietinusAB470228.1_Bombyx_mori_Ob






XP_011166275.1_Solenopsis_invicta




XP_011630965.1_Pogonomyrmex_barbatus

XP_013165427.1_Papilio_xuthus




XP_015180910.1_Polistes_dominula





AY055377.1_Spisula_solidissima_Ob

AGV79326.1_Chilo_suppressalis


XP_011263631.1_Camponotus_floridanus











XP_011053483.1_Acromyrmex_echinatiorXP_011870420.1_Vollenhovia_emeryi




XP_004922133.2_Bombyx_mori

XP_012343074.1_Apis_florea


EFX87996.1_Daphnia_pulexXP_014671143.1_Priapulus_caudatus




KNC25006.1_Lucilia_cuprinaXP_014254146.1_Cimex_lectularius

XP_001955623.2_Drosophila_ananassae


Q4LBB9.2_Drosophila_melanogaster_Ob

AF222978.1_Aplysia_californica_Ob



KU886229_Platynereis_dumerilii


87

80

86

71

99

73

74100

Octopamine−β receptors


https://doi.org/10.1101/063743

18

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.

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]P. dumerilii α1-adrenergic

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

S. kowalevskii α1-adrenergic

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

P. caudatus α1-adrenergic

log conc (log[M])

activ

atio

n [%

]

Norepinephrine

Dopamine

Epinephrine

Yohimbine

Mianserin

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]Yohimbine

Mianserin

Yohimbine

Mianserin

Octopamine

Norepinephrine

Dopamine

Epinephrine

Norepinephrine

A

B

C


https://doi.org/10.1101/063743

19

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.

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-20 -15 -10 -5 00

50

100

150

log conc (log[M])

activ

atio

n [%

]

-15 -10 -5 00

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

Tyramine

Octopamine

Norepinephrine

Epinephrine

Yohimbine

Mianserin

Yohimbine

Mianserin

Yohimbine

Mianserin

Octopamine

Norepinephrine

Epinephrine

Tyramine

Clonidine

Dopamine

Clonidine

Norepinephrine

Epinephrine

Clonidine

Dopamine

P. dumerilii α2-adrenergic

S. kowalevskii α2-adrenergic

P. caudatus α2-adrenergic

A

B

C


https://doi.org/10.1101/063743

20

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.

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -2 00

50

100

150

log conc (log[M])

activ

atio

n [%

]

-14 -12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-14 -12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

Tyramine

Octopamine

Dopamine

Yohimbine

Mianserin

Yohimbine

Mianserin

Tyramine

Octopamine

Dopamine

Tyramine

Octopamine

Clonidine

Dopamine

Tyramine

Octopamine

Tyramine

Octopamine

Norepinephrine

Epinephrine

Clonidine

Dopamine

Clonidine

Dopamine

Yohimbine

Mianserin

Yohimbine

Mianserin

Yohimbine

Mianserin

P. dumerilii Tyramine-1

P. dumerilii Tyramine-2

S. kowalevskii Tyramine-1

A

B

C

S. kowalevskii Tyramine-2AD

S. kowalevskii Tyramine-2BE


https://doi.org/10.1101/063743

21

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.

-12 -10 -8 -6 -4 -2 00

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

-12 -10 -8 -6 -4 -20

50

100

150

log conc (log[M])

activ

atio

n [%

]

Tyramine

Octopamine

Yohimbine

Mianserin

Yohimbine

Mianserin

Yohimbine

Mianserin

Tyramine

Octopamine

Norepinephrine

Dopamine

Clonidine

Epinephrine

Octopamine

Norepinephrine

P. dumerilii Octopamine α

S. kowalevskii Octopamine α

S. kowalevskii Octopamine β

A

B

C


https://doi.org/10.1101/063743

22

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).

0.2

XP_010219192.1_Tinamus_guttatus

XP_001899866.1_Brugia_malayi







AAI30528.1_Homo_sapiens

lcl|Contig8616_Platynereis_dumerilii

XP_004739444.1_Mustela_putorius_furo


XP_006875343.1_Chrysochloris_asiatica

XP_012534953.1_Monomorium_pharaonis




XP_012506091.1_Propithecus_coquereli


XP_003896028.1_Papio_anubisELV13924.1_Tupaia_chinensis

XP_010006646.1_Chaetura_pelagica





CEF60746.1_Strongyloides_ratti

NP_001161568.1_Saccoglossus_kowalevskii

BAO52000.1_Gryllus_bimaculatus


XP_004630583.1_Octodon_degus

NP_001191536.1_Aplysia_californica

NP_495744.1_Caenorhabditis_elegans

XP_009028665.1_Helobdella_robusta






BAP16218.1_Gallus_gallus



XP_014649145.1_Ceratotherium_simum_simum


NP_058712.2_Rattus_norvegicus

XP_003762585.1_Sarcophilus_harrisii




XP_005396658.1_Chinchilla_lanigera

XP_010138453.1_Buceros_rhinoceros_silvestris

XP_848285.2_Canis_lupus_familiaris

EKC41301.1_Crassostrea_gigas


CAJ38793.1_Platynereis_dumerilii





BAM35936.1_Lymnaea_stagnalis








XP_015508789.1_Parus_major



KRT84890.1_Oryctes_borbonicus





XP_004839911.1_Heterocephalus_glaber









BAQ94593.1_Ambigolimax_valentianus



XP_005434516.1_Falco_cherrug

EFO20782.2_Loa_loa

XP_012025784.1_Ovis_aries_musimon


XP_004010670.1_Ovis_aries





ADP08788.1_Azumapecten_farreri


XP_008847491.1_Nannospalax_galili

XP_010022195.1_Nestor_notabilis


XP_013000851.1_Cavia_porcellus


KHN71251.1_Toxocara_canis


XP_005949669.1_Haplochromis_burtoni










XP_001379620.3_Monodelphis_domestica





XP_004415117.1_Odobenus_rosmarus_divergens


XP_004582273.2_Ochotona_princeps

XP_003744997.1_Metaseiulus_occidentalis



XP_006030466.1_Alligator_sinensis



XP_012055250.1_Atta_cephalotes


lcl|comp422145_c0_seq4_Platynereis_dumerilii

XP_009680213.1_Struthio_camelus_australis




XP_007942142.1_Orycteropus_afer_afer


XP_009023368.1_Helobdella_robusta

ACJ13262.1_Drosophila_melanogaster



XP_011061414.1_Acromyrmex_echinatior

XP_005278593.1_Chrysemys_picta_belliiXP_004708097.1_Echinops_telfairi

XP_002050467.1_Drosophila_virilisXP_001360141.2_Drosophila_pseudoobscura_pseudoobscura






KFB44891.1_Anopheles_sinensis

XP_008768473.1_Rattus_norvegicus


XP_007061422.1_Chelonia_mydas


XP_006570903.1_Apis_mellifera

XP_544676.3_Canis_lupus_familiaris


XP_002630249.1_Caenorhabditis_briggsae


96

100

100

52100

100

95

100

91

arom

atic

am

ino

acid

dec

arbo

xyla

sety

rosi

ne d

ecar

boxy

lase


https://doi.org/10.1101/063743

Documents

Ancient coexistence of norepinephrine, tyramine, and octopamine … · presence of tyramine signaling in S. kowalewskii is also supported by the phyletic distribution of tyrosine