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 3of4 Table S1. Taxa sampled Tribe Species Specimen no. 12S–16S EF1a 28S COI

Xystocheirini Anombrocheir bifurcata MTX0174 JN383854 JN383878 KR135885 KR135987 Anombrocheir spinosa MTX0180 JN383855 JN383879 KR135886 KR135988 Motyxia kerna MTX0129 JN383849 JN383874 KR135897 KR135999 Motyxia monica MTX0008 JN383871 JN383847 KR135898 KR136000 Motyxia pior MTX0022 JN383848 JN383872 KR135899 KR136001 Motyxia porrecta MTX0006/253 JN383851 JN383877 KR135938 KR136002 Motyxia sequoia alia MTX0242 KR135952 KR136052 KR135900 KR136003 Motyxia sequoia sequoia MTX0249 KR135954 KR136054 KR135902 KR136005 Motyxia sequoiae MTX0024 JN383850 JN383875 KR135901 KR136004 Motyxia tiemanni MTX0011 JN383852 JN383876 KR135903 KR136006 Motyxia tularea ollae MTX0228 KR135955 KR136055 KR135904 KR136007 Motyxia tularea tularea MTX0033 JN383853 JN383873 KR135905 KR136008 Parcipromus cooki MTX0431 KR135961 KR136060 KR135911 KR136014 Parcipromus gigantoarboricolus MTX0021 JN383856 JN383882 KR135912 KR136015 Parcipromus tiemanni MTX0040 JN383857 JN383883 KR135913 KR136016 Wamokia dentata MTX0334 KR135968 KR136065 KR135919 KR136022 Wamokia discordis MTX0335 KR135969 KR136066 KR135920 KR136023 Wamokia falcata MTX0329 KR135970 KR136067 KR135921 KR136024 Wamokia hoffmani MTX0183/324 JN383859 JN383880 KR135922 KR136025 Wamokia placera MTX0340 KR135971 KR136068 KR135923 KR136026 Wamokia remota MTX0313 KR135972 KR136069 KR135924 — Wamokia sierrae MTX0303 KR135973 KR136070 KR135925 KR136027 Xystocheir bistipita MTX0452 KR135976 KR136073 KR135926 KR136030 Xystocheir brachymacris MTX0182 JN383860 JN383881 KR135927 KR136031 Xystocheir dissecta dissecta MTX0437 KR135977 KR136074 KR135928 KR136032 Xystocheir modestior haerens MTX0381 KR135978 KR136075 KR135929 KR136033 Xystocheir modestior modestior MTX0371 KR135979 KR136076 KR135930 KR136034 Xystocheir Sierra MTX0395 KR135980 KR136077 KR135931 KR136035 Xystocheir prolixorama MTX0442 KR135981 — KR135932 KR136036 Xystocheir reducta MTX0407 KR135982 KR136078 KR135933 KR136037 Xystocheir solenofurcata MTX0308 KR135983 KR136079 KR135934 KR136038 Xystocheir stenomacris MTX0396 KR135984 — KR135935 KR136039 Xystocheir stolonifera MTX0383 KR135985 KR136080 KR135936 KR136040 Sigmocheirini Ochthocelata adynata MTX0152 JN383846 JN383885 KR135907 KR136010 Sigmocheir furcata MTX0367 KR135966 KR136064 KR135917 KR136020 Orophini Orophe unicus MTX0126 JN383865 JN383868 KR135908 KR136011 Xystodesmini Harpaphe haydeniana haydeniana MTX0137 JN383862 JN383870 KR135892 KR135994 Isaphe convexa MTX0106 JN383863 JN383869 KR135893 KR135995 Riukiaria holstii MTX0191 KR135963 KR136062 KR135914 KR136017 Semionellus placidus MTX0460 KR135965 — KR135916 KR136019 Xystodesmus sp. MTX0186 KR135986 KR136081 KR135937 KR136041 Chonaphini Chonaphe armata MTX0097 JN383861 JN383866 KR135889 KR135991 Metaxycheir prolata MTX0107 KR135949 KR136047 KR135895 KR135997 Montaphe elrodi MTX0083 KR135950 KR136048 KR135896 KR135998 Selenocheir sinuata MTX0319 KR135964 KR136063 KR135915 KR136018 Rhysodesmini Stenodesmus tuobitus MTX0199 KR135967 — KR135918 KR136021 Apheloriini Apheloria montana SPC000134 DQ490660 KR136042 KR135887 KR135989 Brachoria cumberlandmountainensis SPC000651 EU127864 KR136043 KR135888 KR135990 Dynoria medialis SPC000431 DQ490700 KR136045 KR135891 KR135993 Pachydesmini Dicellarius atlanta SPC000428 DQ490648 KR136044 KR135890 KR135992 Melaphinae Melaphe vestita MEL1 KR135948 KR136046 KR135894 KR135996 Parafontariinae Parafontaria tonominea MTX0189 KR135960 KR136059 KR135910 KR136013 Trichopolydesmidae Nearctodesmus salix MTX0215 KR135958 — KR135906 KR136009 Paradoxosomatidae Oxidus gracilis MTX0216 KR135959 KR136058 KR135909 KR136012

—, denotes missing data.

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