Smýkal et al. Complex evolution of insect insulin receptors and two homologous decoy receptors SUPPLEMENTARY MATERIAL ONLINE Sycon raphanus CAC14731 98 Sycon raphanus CAC14730 Porifera 100 Sycon raphanus CAC14729 100 Echinococcus granulosus EUB62503 Schistosoma japonicum ACT20714 100 100 Echinococcus granulosus EUB63052 Platyhelminthes Schistosoma japonicum ACT20715 100 Caenorhabditis elegans AAC47715 54 100 Trichinella spiralis 1EFV59569 Nematoda Trichinella spiralis EFV52508 Biomphalaria glabrata NP_001298211 98 100 Lymnaea stagnalis Q25410.1 100 Pinctada fucata AGA94627.1 Mollusca Crassostrea gigas XP_011456321 Strongylocentrotus purpuratus XP_011668294 Echinodermata Xenopus leavis NP_001081702 89 100 100 Mus musculus NP_034698.2 Homo sapiens AAA59174.1 InR 96 Xenopus leavis BC060457.1 100 100 Mus musculus NP_035962.2 IRR Vertebrata 96 Homo sapiens NP_055030 100 Mus musculus NP_034643.2 97 Homo sapiens AAB22215 IGF1R Xenopus leavis NP_001081734.1 87 Metaseiulus occidentalis XP_003739590 100 Ixodes scapularis XP_002416224.1 Arachnida Ornithodoros rostratus GCJJ01004115.1 Daphnia pulex EFX85280.1 100 Daphnia pulex EFX62637.1 100 Daphnia pulex EFX63421.1 Crustacea Daphnia pulex EFX85381.1 Tetrodontophora bielanensis GAXI01021207 53 100 Anurida maritima GAUE01001371 99 Pogonus chalceus JU418048 Tribolium castaneum EFA02828 100 Apis mellifera XP_394771 Solenopsis invicta JF304723 75 Acyrthosiphon pisum XP_001942660 Nilaparvata lugens AIY24639 97 Homalodisca vitripennis HVIT005312-RA Homalodisca vitripennis HVIT005314-RA Pyrrhocoris apterus KX087105 73 100 Medauroidea extradentata GAWD01045541 Extatosoma tiaratum GAWG01048820 84 Tanzaniophasma sp. GAXB01155547 55 Blattella germanica KN196784_2 100 51 Zootermopsis nevadensis KDR21367 67 69 Leuctra sp. GAUF01012297 100 Extatosoma tiaratum GAWG01064895 92 Medauroidea extradentata GAWD01037141 Tetrix subulata GASQ01015035 55 100 Blattella germanica KN196784_1 Zootermopsis nevadensis KDR21366 100 Eurylophella sp. GAZG01015571 Ephemera danica GAUK01018742 Lepisma saccharina KX087106 100 Pyrrhocoris apterus KX087104 Pyrrhocoris apterus KX087103 Hexapoda Nilaparvata lugens AIY24638 55 Homalodisca vitripennis HVIT002229-RA Ephemera danica GAUK01018485 74 88 Epiophlebia superstes GAVW01013590 Tanzaniophasma sp. GAXB0203043 100 Medauroidea extradentata GAWD01065534 45 Extatosoma tiaratum GAWG01064894 51 Leuctra sp. GAUF01084507 Blattella germanica HG518668 100 Zootermopsis nevadensis KDR13786 60 Tetrix subulata GASQ02008536 Boreus hyemalis GAYK01004971 78 Tribolium castaneum XP_008199415 99 Pogonus chalceus JU427775 100 Nasonia vitripennis XP_008203941 Solenopsis invicta JF304722 100 Apis mellifera NP_001233596 100 Liposcelis bostrychophila GAYV01001422 Pediculus humanus corporis XP_002430961 Plutella xylostella XP_011567916 100 Bombyx mori NP_001037011 83 Danaus plexippus EHJ65074 Musca domestica XP_005178583 72 97 100 Ceratitis capitata XP_004518075 100 Bactrocera dorsalis XP_011210333 Drosophila melanogaster AAF55903 100 Aedes aegypti Q93105 Anopheles sinensis KFB49958 0.5 Acyrthosiphon pisum XP_008185917 Supplementary Figure 1. Phylogenetic tree of insulin receptor protein sequences identified across Animalia, including selected representatives of Insecta. Sycon raphanus InR sequences were set as outgroup. Duplication events in non-insect phyla are marked with arrowheads. The topology and branching supports were inferred using RAxML maximum likelihood algorithm with WAG + Γ model (-ln = 131220.705824), the bootstrap values calculated from 1000 replicates are shown for nodes represented in more than 50% of trees. InR, insulin receptor; IRR, insulin receptor-related receptor; IGF1R, insulin-like growth factor 1 receptor. 1 Smýkal et al. Complex evolution of insect insulin receptors and two homologous decoy receptors SUPPLEMENTARY MATERIAL ONLINE Sarcophaga peregrina GGEP01039589 >96 Musca domestica XP_011291787 >90 Ceratitis capitata XP_004527212 bootstrap >80 Drosophila melanogaster NP_724157 value (%) Clunio marinus CRL06839 Diptera >65 Aedes aegypti FAA01101 >50 Anopheles sinensis KFB49143 Liogma simplicicornis GEMK01029352 0.3 Plutella xylostella XP_011567028 Danaus plexippus EHJ77075 Lepidoptera InR2 InR1 Bombyx mori XP_004925605 Armisén et al. Kremer et al. GASS02028789 DR2 Platycentropus radiatus 2018 2018 Annulipalpia sp. GATX01005845_ Trichoptera Annulipalpia sp. GATX01012065 Boreus hyemalis GAYK01108088 Mecoptera Tribolium castaneum EFA01771 * Tribolium castaneum EFA02828 Dendroctonus ponderosae XP_019755840 Coleoptera Leptinotarsa decemlineata XP_023030119 InR-1 Pogonus chalceus JU418048 Lu & Pietrantonio Chrysopa pallens AVK43098 Neuroptera 2011 Xanthostigma xanthostigma GAUI02048109 Raphidioptera Fopius arisanus XP_011310956 Cephus cinctus XP_024940155 Athalia rosae XP_012259845 Hymenoptera Apis mellifera XP_394771 Solenopsis invicta JF304723 Pyrrhocoris apterus KX087105 (InR2) Jadera haematoloma AVT56265 Oncopeltus fasciatus AVT56270 Podisus maculiventris GFUB01178628 Halyomorpha halys XP_024217440 Lygus hesperus JAG20929 Rhodnius prolixus GECK01019799 Pagasa sp. GCXU01024823 Cimex lectularius XP_014242611 Heteroptera Rhagovelia antilleana INR2 Microvelia longipes INR2 Gerris buenoi INR2 Limnoporus dissortis INR2 Aquarius paludum INR2 Hydrometra cumata INR2 Mesovelia furcata INR2 Hebrus sp. INR2 Nilaparvata lugens AIY24639 Euscelidius variegatus GFTU01002760 Homalodisca vitripennis HVIT005314-RA Diceroprocta seminicta GGPH01050080 Neotibicen dorsatus GCYV01053567 * Clastoptera arizonana GEDC01010167 Mapuchea sp. GCXN01044447_1045662 Homalodisca liturata GECU01010637 Graphocephala atropunctata GEBQ01003303 Campylenchia latipes GCWI01051784 Auchenorrhyncha Euscelidius variegatus GFTU01002463 Graminella nigrifrons GAQX01001711 * Euscelidius variegatus GFTU01007873 Graphocephala atropunctata GEBQ01020846_01005496 * Cuerna arida GECZ01003109 Homalodisca liturata GECU01004804 Homalodisca vitripennis HVIT005312-RA Euscelidius variegatus GFTU01003387 Graminella nigrifrons GAQX01006206 Bemisia tabaci XP_018897134 Diaphorina citri XP_008479213 Pachypsylla venusta GAOP01019620 Phenacoccus solenopsis GGIT01015335 Adelges tsugae GBJX01018946 Daktulosphaira vitifoliae GDEB01025952 Sternorrhyncha Aphis citricidus ARD07922 Acyrthosiphon pisum XP_001942660 Diuraphis noxia XP_015363980 Myzus persicae XP_022180848 Perla marginata GATV02011223 Leuctra sp. GAUF01012297 Plecoptera Timema genevievae GFPR01021056 InR2_2 Aretaon asperrimus GAWC01082819 Phasmatodea Xu & Zhang Extatosoma tiaratum GAWG01064895 2017 Blattella germanica KN196784_1 Zootermopsis nevadensis KDR21366 Blattodea Cryptotermes secundus XP_023702527 InR2_1 Prorhinotermes simplex MH560589 (InR2) Xu & Zhang Tetrix subulata GASQ01015035 Orthoptera Zootermopsis nevadensis KDR21367 2017 Cryptotermes secundus XP_023702537 Prorhinotermes simplex MH560588 (InR3) Blattodea Blattella germanica KN196784_2 InR2-like Timema genevievae GFPR01028898 Armisén et al. Aretaon asperrimus GAZQ02010804_GAWC01050212 Phasmatodea 2018 Extatosoma tiaratum GAWG01048820 Tanzaniophasma sp. GAXB01155547 Mantophasmatodea Tetrix subulata GASQ02006861 Orthoptera Zorotypus caudelli GAYA02044618 Zoraptera InR3 Ecdyonurus insignis GCCL01030520 Kremer et al. Ephemera danica GAUK01018742 Ephemeroptera 2018 Eurylophella sp. GAZG01015571 InR2 Xu et al. 2015 Xu & Zhang 2017 Ding et al. 2017 Cluster I Lin et al. 2018 Supplementary Figure 2. Phylogenetic tree and nomenclature of insect insulin receptors and decoy of insulin receptors identified in the Cluster II in 98 species from 23 orders. The tree represents the full version of the simplified tree given in Figure 1 of the main text. Orange arrow marks the Cluster II duplication within Polyneoptera. Red arrow (DR2) marks the loss of the tyrosine kinase domain giving rise to the decoy of insulin receptor gene DR2 (red) in Mecopterida, a group of advanced Holometabola including Diptera, Lepidoptera, Trichoptera and Mecoptera. Asterisks mark the duplication identified in Tribolium castaneum and the multiple duplications in Auchenorrhyncha. The topology and branching supports were inferred using RAxML maximum likelihood algorithm with WAG + Γ model (-ln = 285839.374747) the bootstrap values calculated from 500 replicates are shown for nodes represented in more than 50% of trees. Black dots in the right part of the figure indicate the insect orders included in phylogenetic analyses in previous studies, the boxes give the nomenclature used by previous studies for the identified InR genes. Bold marking in brackets shows the InR nomenclature used in this study for P. simplex and P. apterus InRs. 2 Smýkal et al. Complex evolution of insect insulin receptors and two homologous decoy receptors SUPPLEMENTARY MATERIAL ONLINE Cluster II InR-2 InR1 Lu & Pietrantonio Armisén et al. 2011 2018 >96 Musca domestica XP_011290405 >90 bootstrap Sarcophaga peregrina GGEP01012125 InR2 >80 SDR Kremer et al. value (%) Ceratitis capitata XP_004519681 >65 Drosophila melanogaster NP_650408 2018 >50 Drosophila melanogaster AAF55903 Musca domestica XP_005178583 Sarcophaga peregrina GGEP01027089 Diptera 0.3 Ceratitis capitata XP_004518075 Clunio marinus CRL08130 Anopheles sinensis KFB49958 Aedes aegypti Q93105 Liogma simplicicornis GEMK01036784 Nyssomyia neivai JAV06640 Boreus hyemalis GAYK01004971 Mecoptera Ctenocephalides felis XP_026475386 Ceratophyllus gallinae GAWK02019316 Siphonaptera Plutella xylostella XP_011567916 Bombyx mori NP_001037011 Lepidoptera Danaus plexippus EHJ65074 Annulipalpia sp. GATX01009258_01085969_ Platycentropus radiatus