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Supporting Information Supporting Information Marek and Moore 10.1073/pnas.1500014112 Oxidus gracilis Oxidus gracilis Oxidus gracilis Nearctodesmus salix Melaphe vestita Riukiaria holstii Melaphe vestita Sigmocheir furcata Xystodesmus sp Dicellariusatlanta Dicellarius atlanta Ochthocelata adynata Stenodesmus tuobitus * Dynoria medialis Dicellarius atlanta Brachoria cumberlandmountainensis * Apheloria montana Parafontaria tonominea * Apheloria montana Melaphe vestita * Dynoria medialis Brachoria cumberland. Nearctodesmus salix Sigmocheir furcata Ochthocelata adynata Brachoria cumberlandmountainensis Ochthocelata adynata Parafontaria tonominea * Apheloria montana * Xystocheir modestior modestior * Riukiaria holstii * Dynoria medialis Xystocheir nsp Xystodesmus sp Stenodesmus tuobitus * Xystocheir modestior haerens Isaphe convexa Montaphe elrodi Xystocheir stenomacris Metaxycheir prolata Chonaphe armata Parcipromus cooki Orophe unicus Semionellus placidus Xystocheir reducta * Isaphe convexa Parcipromus gigantoarboricolus Chonaphe armata Orophe unicus Parcipromus tiemanni Montaphe elrodi Metaxycheir prolata Xystocheir stolonifera * Harpaphe haydeniana Harpaphe haydeniana Xystocheir prolixorama Selenocheir sinuata Selenocheir sinuata Xystocheir dissecta dissecta Xystocheir bistipita Sigmocheir furcata Xystocheir brachymacris Xystocheir solenofurcata Motyxia monica Xystocheir bistipita Anombrocheir bifurcata * Motyxia porrecta Motyxia monica 28S Motyxia tiemanni Motyxia pior Anombrocheir spinosa EF1a * Motyxia tularea tularea COI Wamokia sierrae Motyxia kerna Motyxia porrecta *Wamokia dentata 0.05 site substitutions Motyxia sequoiae 0.05 site substitutions * Motyxia tiemanni 1.0 site substitutions Wamokia hoffmani Motyxia pior Motyxia sequoiae * Wamokia falcata Motyxia tularea tularea Wamokia remota * Motyxia kerna Wamokia discordis * * Motyxia sequoia sequoia * Motyxia sequoia sequoia Wamokia placera Motyxia sequoia alia * Motyxia sequoia alia Xystocheir bistipita Motyxia tularea olla Motyxia tularea olla Parcipromus cooki Xystocheir solenofurcata * Motyxia monica Xystocheir dissecta * Motyxia sequoia sequoia * Parcipromus tiemanni * Xystocheir prolixorama Motyxia sequoia alia Parcipromus gigantoarboricolus Wamokia placera * Motyxia tularea ollae Xystocheir stolonifera * Motyxia pior * * Wamokia sierrae Motyxia porrecta * Xystocheir reducta Wamokia falcata Motyxia sequoiae * Xystocheir modestior haerens Wamokia hoffmani * Motyxia tularea tularea Xystocheir modestior modestior Wamokia dentata Motyxia kerna Xystocheir nsp Wamokia discordis Motyxia tiemanni Xystocheir dissecta Xystocheir brachymacris Parafontaria tonominea Xystocheir brachymacris * Anombrocheir bifurcata Riukiaria holstii Xystocheir solenofurcata Anombrocheir spinosa * Xystodesmus sp Parcipromus cooki * Metaxycheir prolata Wamokia falcata * Parcipromus gigantoarboricolus * Selenocheir sinuata Anombrocheir bifurcata * Parcipromus tiemanni Harpaphe haydeniana * Anombrocheir spinosa Xystocheir stenomacris Orophe unicus Wamokia discordis Xystocheir stolonifera Isaphe convexa Wamokia dentata Xystocheir modestior haerens Chonaphe armata Wamokia hoffmani Xystocheir reducta ** Montaphe elrodi Wamokia placera * Xystocheir modestior modestior Semionellus placidus Wamokia remota Xystocheir nsp Wamokia sierrae Nearctodesmus salix Oxidus gracilis Melaphe vestita Nearctodesmus salix Oxidus gracilis Melaphe vestita Stenodesmus tuobitus * Stenodesmus tuobitus * Dicellarius atlanta * Dicellarius atlanta Parcipromus gigantoarboricolus * Apheloria montana * Apheloria montana * Brachoria cumberlandmountainensis Brachoria cumberlandmountainensis * Dynoria medialis Dynoria medialis Xystocheir bistipita Ochthocelata adynata * Ochthocelata adynata Parafontaria tonominea Parafontaria tonominea * Riukiaria holstii * * Riukiaria holstii * Xystodesmus sp Xystodesmus sp * Motyxia monica Metaxycheir prolata * Isaphe convexa * Harpaphe haydeniana ** Metaxycheir prolata * Selenocheir sinuata * Harpaphe haydeniana Motyxia pior Isaphe convexa Selenocheir sinuata Orophe unicus Orophe unicus * Semionellus placidus Semionellus placidus * * * Chonaphe armata Motyxia tularea tularea Chonaphe armata * Montaphe elrodi Montaphe elrodi Sigmocheir furcata Sigmocheir furcata Xystocheir bistipita Xystocheir bistipita Motyxia tiemanni Motyxia monica * Motyxia monica * * Motyxia pior * Motyxia pior * * * Motyxia tularea tularea ** Motyxia tularea tularea ** Motyxia porrecta * * Motyxia porrecta Motyxia porrecta * Motyxia tiemanni * Motyxia tiemanni * Motyxia sequoiae Motyxia sequoiae * Motyxia kerna * Motyxia kerna * Motyxia sequoia sequoia ** Motyxia sequoia sequoia Motyxia sequoiae * Motyxia sequoia alia * * Motyxia sequoia alia * Motyxia tularea olla Motyxia tularea olla Xystocheir solenofurcata Parcipromus gigantoarboricolus Motyxia kerna Parcipromus gigantoarboricolus * Parcipromus cooki * Parcipromus cooki * Parcipromus tiemanni Parcipromus tiemanni * Xystocheir stenomacris * *** Xystocheir stenomacris * Xystocheir stolonifera Motyxia sequoia sequoia Xystocheir stolonifera * Xystocheir reducta * Xystocheir reducta * * Xystocheir modestior haerens * Xystocheir modestior haerens * Xystocheir modestior modestior Motyxia sequoia alia * Xystocheir modestior modestior Xystocheir nsp * Xystocheir nsp * Xystocheir dissecta * Xystocheir brachymacris Xystocheir prolixorama Xystocheir dissecta Xystocheir brachymacris Motyxia tularea ollae * Xystocheir prolixorama Xystocheir solenofurcata Anombrocheir bifurcata * Anombrocheir bifurcata * Anombrocheir spinosa Anombrocheir spinosa Wamokia sierrae Wamokia dentata * Wamokia discordis 16S/12S/tRNA-Val * Wamokia falcata concatenated coalescent * Wamokia remota * Wamokia hoffmani Wamokia sierrae −6 Wamokia placera 0.5 site substitutions 1.0 site substitutions 8.0 x 10 coalescent units * Wamokia dentata Wamokia placera * Wamokia falcata * Wamokia discordis * Wamokia hoffmani Wamokia remota Fig. S1. Separate gene histories for the loci concatenated in Fig. S4 analyzed in RAxML and the Motyxia species phylogeny from the multispecies coalescent model in *BEAST. (Scale bar, expected substitutions per site.) Branch lengths of *BEAST tree are shown in coalescent units. Asterisks denote bootstrap/posterior probability support values ≥ 70%. [Nuclear: elongation factor-1 alpha (EF1-a); 28S ribosomal RNA (28S); mitochondrial: small subunit ribosomal RNA (16S/12S/ tRNA-Val); and cytochrome c oxidase subunit I (COI)]. Marek and Moore www.pnas.org/cgi/content/short/1500014112 1of4 OH O E HC N + N Mandelonitrile Catalytic enzyme Benzaldehyde Hydrogen cyanide OH precursor reservoir N reaction chamber E O HC N Fig. S2. Bicompartmental cyanide glands of polydesmidan millipedes, including Motyxia and relatives. Through a cyanohydrin reaction, mandelonitrile (a biologically stable chemical housed in the precursor reservoir) is disassociated by a catalytic enzyme (E) present in the reaction chamber to produce cyanide and benzaldehyde. Data from ref. 1. (Scale bar: 1.0 mm.) 1. Eisner T, Meinwald J (1966) Defensive secretions of arthropods. Science 153(3742):1341–1350. Fig. S3. Live habitus photographs of Motyxia and related xystodesmid species showing colors in life. Motyxia taxa photographed are to the right of the tree. Xystodesmid species: Sigmocheir furcata (A), Xystocheir new species (on dead oak leaf from habitat) (B), Stenodesmus tuobitus (C), and Xystocheir stenomacris (D). Character history of bioluminescent intensity are as in Fig. 2. All millipede images are calibrated to the scale bar shown in gray. Marek and Moore www.pnas.org/cgi/content/short/1500014112 2of4 Wamokia remota Wamokia discordis Wamokia placera Wamokia sierrae Wamokia hoffmani Wamokia falcata Wamokia dentata Anombrocheir spinosa Anombrocheir bifurcata Xystocheir solenofurcata Xystocheir brachymacris Xystocheir prolixorama Xystocheir dissecta dissecta Xystocheir n. sp. “Sierra” Xystocheir modestior modestior Xystocheir modestior haerens Xystocheir reducta Xystocheir stolonifera Xystocheir stenomacris Parcipromus tiemanni Parcipromus cooki Parcipromus gigantoarboricolus Motyxia tularea ollae Motyxia sequoia alia Motyxia sequoia sequoia Motyxia kerna Motyxia sequoiae Motyxia tiemanni Motyxia porrecta Motyxia tularea tularea Motyxia pior Motyxia monica Xystocheir bistipita Sigmocheir furcata Selenocheir sinuata Harpaphe haydeniana haydeniana Metaxycheir prolata Orophe unicus Isaphe convexa Montaphe elrodi Chonaphe armata Semionellus placidus Xystodesmus sp. Riukiaria holstii Parafontaria tonominea Ochthocelata adynata Dynoria medialis Brachoria cumberlandmountainensis Apheloria montana Dicellarius atlanta Stenodesmus tuobitus Melaphe vestita Nearctodesmus salix Oxidus gracilis Fig. S4. Species phylogeny of the millipede tribe Xystocheirini including the bioluminescent genus Motyxia and a broad taxonomic sample of representatives from 9 of the 12 tribes of the family Xystodesmidae. Tree estimated using five single-copy mitochondrial and nuclear genes under a mixed-model partitioned Bayesian analysis. Branches in gray indicate <0.95 posterior probability for the clade, and black branches are ≥0.95. Color key to taxa: Xystocheirini (light blue), Sigmocheirini (dark blue), Chonaphini (light green), Xystodesmini (orange), Orophini (magenta), Parafontariinae (yellow), Apheloriini (red), Pachydesmini (dark green), Rhysodesmini (gray), and Melaphini (purple). Outgroups without color: Nearctodesmus salix (Trichopolydesmidae) and Oxidus gracilis (Para- doxosomatidae). (Scale bar: 0.5 expected substitutions per site.) Marek and Moore www.pnas.org/cgi/content/short/1500014112
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