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1 Supporting Information Chen et al /pnas Fig. S1. Life cycle of the social amoeba D. discoideum. D. discoideum amoebae propagate vegetatively as a unicellular organism when food (naturally bacteria) is abundant. Upon starvation, social amoebae transition into multicellular development. During development, individual cells aggregate and differentiate, forming a multicellular slug that migrates and then forms a fruiting body in a highly coordinated process. The cartoons at the bottom half of the figure depict various stages during multicellular development: streaming, loose aggregate, tipped aggregate, slug, Mexican hat, and fruiting bodies. Chen et al. 1of1

2 Relative abundance (TIC x 1, ions) DdTPS DdTPS DdTPS DdTPS Retention time (min) Fig. S. Monoterpene synthase activity of D. discoideum terpene synthases. Genes were heterologously expressed in E. coli, and crude protein extracts were incubated with the substrate GPP. Enzyme products were collected by using solid-phase microextraction and analyzed by GC/MS. 1, (Z)-β-ocimene;, (E)-β-ocimene;3,allo-ocimene;,linalool;5,β-myrcene;, limonene; 7, α-terpineol. Chen et al. of1

3 DdTPS1 DdTPS DdTPS3 DdTPS DdTPS5 DdTPS9 Relative abundance (TIC x 1, ions) Relative abundance (TIC x 1, ions) I II III IV V III Relative abundance Relative abundance Relative abundance peak I peak II peak III peak IV peak V m/z Retention time (min) Fig. S3. Diterpene synthase activity of D. discoideum terpene synthases. Genes were heterologously expressed in E. coli, and crude protein extracts were incubated with the substrate GGPP. Enzyme products were extracted with hexane and analyzed by using GC/MS. GC traces (Left) and mass spectra of major products (Right) are shown. I, unidentified diterpene; II, unidentified diterpene; III, unidentified diterpene; IV, unidentified oxygenated diterpene; V, unidentified diterpene. Chen et al. 3of1

4 5 3 1 (E,E)-α-farnesene (peak#1) Uniden fied sesquiterpene (peak#) 3 1 Calarene (peak#5) 3 Uniden fied sesquiterpene (peak#) area) (peak Relative abundance 8 Uniden fied sesquiterpene (peak#7) 3 1 Uniden fied sesquiterpene (peak#8) (E)-nerolidol (peak#9) β-barbatene (peak#13) Uniden fied diterpene (peak#di) Uniden fied sesquiterpene (peak#1) Linalool (peak#li) Fig. S. Relative abundance of individual volatile terpenes at each time point during the h of multicellular development of D. discoideum. The emission rates were calculated based on the peak area of individual compound in GC chromatograms from three biological replicates with Fig. 5 as one of the replicates. Chen et al. of1

5 Table S1. this study Category Eukaryotes with well-annotated genomes analyzed in Species Homo sapiens Pan troglodytes Pan paniscus Gorilla gorilla gorilla Pongo abelii Nomascus leucogenys Macaca mulatta Macaca fascicularis Rhinopithecus roxellana Callithrix jacchus Mus musculus Rattus norvegicus Cricetulus griseus Nannospalax galili Heterocephalus glaber Oryctolagus cuniculus Tupaia chinensis Canis familiaris Ailuropoda melanoleuca Ursus maritimus Felis catus Panthera tigris altaica Bos taurus Bos mutus Pantholops hodgsonii Capra hircus Ovis aries Sus scrofa Camelus ferus Balaenoptera acutorostrata scammoni Lipotes vexillifer Equus caballus Myotis brandtii Myotis davidii Pteropus alecto Monodelphis domestica Sarcophilus harrisii Ornithorhynchus anatinus Gallus gallus Meleagris gallopavo Anas platyrhynchos Taeniopygia guttata Geospiza fortis Ficedula albicollis Pseudopodoces humilis Corvus cornix Falco peregrinus Falco cherrug Columba livia Alligator sinensis Alligator mississippiensis Pelodiscus sinensis Chelonia mydas Anolis carolinensis Python bivittatus Xenopus laevis Xenopus tropicalis Danio rerio Takifugu rubripes Maylandia zebra Oryzias latipes Xiphophorus maculatus Chen et al. 5of1

6 Table S1. Category Cont. Species Red algae Red algae Latimeria chalumnae Callorhinchus milii Branchiostoma floridae Ciona intestinalis Strongylocentrotus purpuratus Saccoglossus kowalevskii Drosophila melanogaster Drosophila pseudoobscura Drosophila ananassae Drosophila erecta Drosophila persimilis Drosophila sechellia Drosophila simulans Drosophila willistoni Drosophila yakuba Drosophila grimshawi Drosophila mojavensis Drosophila virilis Musca domestica Anopheles gambiae Aedes aegypti Culex quinquefasciatus Apis mellifera Solenopsis invicta Acromyrmex echinatior Harpegnathos saltator Camponotus floridanus Nasonia vitripennis Tribolium castaneum Bombyx mori Plutella xylostella Acyrthosiphon pisum Pediculus humanus corporis Ixodes scapularis Caenorhabditis elegans Caenorhabditis briggsae Brugia malayi Loa Trichinella spiralis Helobdella robusta Lottia gigantea Crassostrea gigas Octopus bimaculoides Schistosoma mansoni Nematostella vectensis Hydra vulgaris Trichoplax adhaerens Amphimedon queenslandica Monosiga brevicollis Salpingoeca rosetta Chlamydomonas reinhardtii Volvox carteri f. nagariensis Ostreococcus lucimarinus Ostreococcus tauri Bathycoccus prasinos Micromonas sp. RCC99 Micromonas pusilla Coccomyxa subellipsoidea Chlorella variabilis Auxenochlorella protothecoides Cyanidioschyzon merolae Galdieria sulphuraria Chen et al. of1

7 Table S1. Category Cont. Species Red algae Dictyosteliida Dictyosteliida Dictyosteliida Entamoeba Entamoeba Entamoeba Acanthamoeba Chondrus crispus Dictyostelium discoideum Dictyostelium purpureum Dictyostelium fasciculatum Entamoeba histolytica Entamoeba dispar Entamoeba invadens Acanthamoeba castellanii Plasmodium falciparum 3D7 Plasmodium falciparum Dd Plasmodium falciparum HB3 Plasmodium yoelii Plasmodium chabaudi Plasmodium berghei Plasmodium knowlesi Plasmodium vivax Plasmodium cynomolgi Theileria annulata Theileria parva Theileria orientalis Theileria equi Babesia bovis Cryptosporidium parvum Cryptosporidium hominis Toxoplasma gondii Tetrahymena thermophila Paramecium tetraurelia Phaeodactylum tricornutum Thalassiosira pseudonana Aureococcus anophagefferens Nannochloropsis gaditana Phytophthora infestans Phytophthora sojae Saprolegnia parasitica Trypanosoma brucei Trypanosoma cruzi Leishmania major Leishmania infantum Leishmania donovani Leishmania mexicana Leishmania braziliensis Naegleria gruberi Trichomonas vaginalis Giardia lamblia Chen et al. 7of1

8 Table S. Summary of the identified eukaryotic terpene synthase genes Gene name Gene ID Protein size (amino acids) Intron Note DdTPS1 DDB_G98 35 DdTPS DDB_G DdTPS3 DDB_G77 3 DdTPS DDB_G DdTPS5 DDB_G DdTPS DDB_G DdTPS7 DDB_G DdTPS8 DDB_G93 31 DdTPS9 DDB_G DdTPS1 DDB_G Pseudogene DdTPS11 DDB_G758 1 Partial DpTPS1 DPU_G DpTPS DPU_G DpTPS3 DPU_G DpTPS DPU_G751 3 DpTPS5 DPU_G751 3 DpTPS DPU_G DpTPS7 DPU_G DpTPS8 DPU_G DpTPS9 DPU_G DpTPS1 3 1 DpTPS11 35 DpTPS DpTPS13 DPU_G78 17 Partial PpTPS1 PPA_G PpTPS PPA_G PpTPS3 PPA_G PpTPS PPA_G PpTPS5 PPA_G PpTPS PPA_G PpTPS7 PPA_G PpTPS8 PPA_G PpTPS9 PPA_G PpTPS1 PPA_G PpTPS11 PPA_G PpTPS1 PPA_G PpTPS13 PPA_G PpTPS1 PPA_G PpTPS15 PPA_G PpTPS1 PPA_G PpTPS17 98 PpTPS18 PPA_G PpTPS19 PPA_G19 33 PpTPS PPA_G Partial PpTPS1 PPA_G Partial DfaTPS1 DFA_G DfaTPS DFA_G DfaTPS3 DFA_G AsTPS1 ADB NgTPS NgTPS NgTPS NgTPS NgTPS NgTPS 39 NgTPS As, Actyostelium subglobosum; Dd,Dictyostelium discoideum; Dfa,Dictyostelium fasciculatum;dp,dictyostelium purpureum;ng,naegleria gruberi;pp,polysphondylium pallidum. Chen et al. 8of1

9 Table S3. Conserved domains of the identified eukaryotic terpene synthases Domains Genes DDxx(x)D/E R NDxxSxxD/E AsTPS1 FDDGLDA LRK INDLVSYEKEV DdTPS1 IDDFLED IRK VNDCASYDKEY DdTPS FDDLFDG HRK VNDCVSYEKEI DdTPS3 LDDFLER IRT FNDCVSYAKEL DdTPS LDDYIFE KRS INDLYSFNREI DdTPS5 FDDPLDT IRK VNDAASFCKEI DdTPS FDDVLDA LRK INDLVSYEKEV DdTPS7 IDDFLES FKK FNDCASYAKEV DdTPS8 LDDYIYE SRH INDCGSFKMEI DdTPS9 VDDFLES FKR MNDCASYAKEI DfaTPS1 FDDALDS LRK INDLVSYEKEV DfaTPS YDEMHDY LRT LNDIVSWEVEK DpTPS1 FDDALDA LRK INDLVSYEKEV DpTPS FDDDLDT IRK YNDCVSYAKEV DpTPS3 VDDFYFE YRC INDMVSFERER DpTPS LDDFLER IRT VNDCVSYAKEI DpTPS5 LDDFLER IRT FNDCVSYAKEI DpTPS IDDFYFE SRY INDCYSFNKEK DpTPS7 IDDLYLE YRG INDIHSFNKEI DpTPS8 VDDFYLE SRG INDIYSFNKET DpTPS9 LDDILDS LRK VNDMASYCKEV DpTPS1 IDDYLDS IRT VNDAVSYAKEI DpTPS11 YDDEYLE TRS VNDIYSFVKES DpTPS1 TDDFFDG IRR INDLHSFEKEF NgTPS1 VDDACDC WRR INDLASFPKEL NgTPS VDDACDS LRE INDICSLPKDL NgTPS3 VDDRIDS RRI VNDLLSYHKDV NgTPS VDDACDE ERR LNDVASIAKDT NgTPS5 VDDACDE DRR LNDVASIAKDT NgTPS VDDACDE ERR FNDVASIAKDA NgTPS7 VDDACDE ERR FNDVASIAKDA PpTPS1 FDDLIDT MRR YNDIFSYEKEL PpTPS FDEKMDA IRI INDICSYEKEF PpTPS3 FDDLIDS IRR YNDIFSYEKEL PpTPS5 FDDNIDT LRR YNDVLSYEKEM PpTPS FDDLIDS MRR FNDLFSYEKEL PpTPS8 FDDLIDT MRR YNDVFSYEKEL PpTPS9 FDDFIDS LRI YNDVLSYEKEV PpTPS1 FDDLIDS LRR YNDVLSYEKEV PpTPS11 FDDLIDT MRN YNDVLSYEKEV PpTPS1 FDDFIDC LRI YNDVFSYEKEV PpTPS13 FDDLIDT MRI YNDVFSYEKEL PpTPS1 IDDIVIE FRS VNDLYSFVSEF PpTPS15 FDECIES LRR YNDLFSYEKEV PpTPS1 FDDFIET LRG FNDITSYEKEL PpTPS17 FDDYIEK IRL VNDVYSFRKEM PpTPS18 FDDGLDA LRK INDLVSYEKEV PpTPS19 TDDQLLE FRE VNDLYSFNKEI Bold characters denote conserved amino acids in respective domains. Chen et al. 9of1

10 Table S. Primers used for cloning of full-length cdnas of DdTPS genes Gene name Primers Sequences Forward 5 -ATGAATATTTTATTAAAAGATTTTAAATAC-3 DdTPS1 Reverse 5 -TTAAATGTATTTTGATTTTGTATTTTC-3 Forward 5 -ATGGATATTAAAAATCTATCATTAAAAG-3 DdTPS Reverse 5 -TTATTTATTTTTTAAATCATAAATTTTTG-3 Forward 5 -ATGGAAACTATTAATCTTGAAAAAC-3 DdTPS3 Reverse 5 -TTAATAAAGATTTGGATCTAAAATAG-3 Forward 5 -ATGGAAGAAAAAATAAAATTTTATTTTG-3 DdTPS Reverse 5 -TTAATTATATCTTTTACTTTTTTTATG-3 Forward 5 -ATGTTATGCTTTAATGATTTTAATTTTC-3 DdTPS5 Reverse 5 -TTAAATATAGTTTTTATTTGTTAATG-3 Forward 5-ATGTCTTTATCTTTGGGTGATATC-3 DdTPS Reverse 5 -TTAATTATTTAAATCAATTGGTGTTG-3 Forward 5 -ATGTATTCAATTTATGATTTTAAATAC-3 DdTPS7 Reverse 5 -TTATAATTTAATAATTTTATCAACAATATTTTC-3 Forward 5 -ATGGATTATGATATAAAATTTACTTG-3 DdTPS8 Reverse 5 -CTAATTATAACGATTTGCATGAC-3 Forward 5 -ATGTATTCTCTTCATGATTTCAAATTC-3 DdTPS9 Reverse 5 -TTATTTTAATAAAATTTTATCAACAATTTTTTC-3 Chen et al. 1 of 1

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Sheet1. Page 1. protein build 1 version 1 Ab initio model Ab initio GPIPE/6085/1.1/gnomon_prot build 1 version 1 Build GPIPE/6085/1.1/ Acyrthosiphon pisum build 2 version 1 Build GPIPE/7029/2.1/ Acyrthosiphon pisum build 2.1