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Evolution of Flight Morphology in Stick Insects Supplementary A tale of winglets: evolution of flight morphology in stick insects Supplementary Materials Text section Notes on ancestral state reconstruction for relative wing size in stick insects Figure S1, Summary of morphological sampling S2, Polytomous tree with mapped traits S3, Ancestral state reconstruction for relative wing size S4, Ancestral state reconstruction for body size S4, Ancestral state reconstruction for sexual size dimorphism (SSD) and sexual wing dimorphism (SWD) S5, Phylogenetic mapping of hindwing state for sampled tribes S6, Summary of phylogenetic correlations for flight morphology among winged stick insects Table S1, Information on nuclear and mitochondrial loci S2, Summary of PGLS results for all winged stick insects S3, Summary of derived wing functions for stick insects with miniaturized wings Datasets 1-4 1. Morphometrics (taxon, wing length, body length) 2. Scaling of wing loading (body mass, wing length, wing area) 3. Molecular sampling (list of species), including accession numbers for new sequences on GenBank 4. Nexus input files Script MatLab script for organizing taxonomic data from Phasmida Species Files Supplementary Text Notes on ancestral state reconstruction for wing size evolution in stick insects Our maximum-likelihood ancestral state reconstruction showed that a short relative wing size (Q < 0.4) preceded multiple gains and losses observed in extant phasmids (Fig. S4,5). Nevertheless, this result offered only a preliminary perspective on wing size evolution in phasmids, as it reflects but a small portion of extant phasmid diversity (~ 10% documented species). A complete molecular phylogeny and comprehensive trait sampling are not currently available. Preliminary sampling for the presence of hindwings showed a highly variable pattern across phasmid clades, even below the tribe level (Fig. S7). In order to properly model evolutionary rates of change and to reconstruct ancestral states for relative wing size, future work should increase phylogenetic resolution and incorporate trait sampling across extant tips. Properly modeling gain and loss of wings in phasmids will require complex models to account for (1) variation in the rate of wing size evolution across the tree (Beaulieu et al. 2013), and (2) variation in rates of species diversification across the tree (Maddison 2006). In particular, Goldberg and Igić (2008) suggested that the loss and gain of wings may affect diversification patterns. Our results demonstrating a bimodal distribution of wing size further indicated a more complex and wing size-dependent diversification scenario. Supplementary Figures Figure S1. Summary of taxa sampled for flight-related morphology. (a) All documented phasmid tribes, with the number of species based on Brock et al. (2019). Colors represent the hindwing condition within tribes; asterisks denote tribes sampled in this study. (b) Summary of the number of taxa sampled for hindwing size (see Supplementary Dataset 1). 1 Canachus_sp_CAN5 1 Canachus_sp_CAN3 0.93 0.93 Canachus_alligator Canachus_sp_TRB2009 0.92 Microcanachus_matileorum 1 Asprenas_impennis 1 0.81 Asprenas_sp_ASP5 Trapezaspis_sp_TRB2009 0.92 Carlius_fecundus Eurycanthini Labidiophasma_rouxi 1 0.96 Erinaceophasma_vepres 1 Neopromachus_doreyanus 10.96 Eurycantha_insularis 1 Eurycantha_calcarata Thaumatobactron_guentheri Dryococelus_australis 1 Dimorphodes_mancus 1 Dimorphodes_prostasis 0.96 Dimorphodes_sp_TRB2009 1 Xeroderus_sp_TRB2009 Xeroderini 1 Cotylosoma_amphibius 0.97 Leosthenes_sp_TRB2009 Cnipsus_rachis 1 Acanthoxyla_intermedia 1 Acanthoxyla_geisovii 0.92 Acanthoxyla_inermis 1 Argosarchus_horridus Acanthoxylini 1 Pseudoclitarchus_sentus 1 Clitarchus_hookeri 1 Tectarchus_huttoni 1 Tectarchus_ovobessus 1 Hemipachymorphini 1 Tectarchus_salebrosus Spinotectarchus_acornutus 1 Asteliaphasma_jucundum 1 Niveaphasma_annulata 1 Micrarchus_hystriculeus Pachymorphini Pachymorpha_sp_TRB2009 1 Rhaphiderus_scabrosus Monandroptera_acanthomera Monandropterini Anchiale_sp_TRB2009 Phasma_gigas 0.95 0.97 Anchiale_briareus Anchiale_sp_STI17 Ctenomorpha_marginipennis 1 Acrophylla_titan Phasmatini 1 Acrophylla_wuelfingi 1 Acrophylla_thoon 1 Eurycnema_osiris Eurycnema_goliath 0.98 Ctenomorpha_sp_LAN3 1 Ctenomorpha_sp_LAN2 1 Tropidoderus_sp_LAN9 0.99 Tropidoderus_sp_LAN5 1 0.96 Tropidoderus_sp_LAN8 Tropidoderini 1 Tropidoderus_childrenii Podacanthus_wilkinsoni Malandania_pulchra Didymuria_sp_LAN10 1 Megacrania_alpheus 1 Megacrania_phelaus 1 1 Ophicrania_leveri Platycranini Graeffea_sp_GRA1 Graeffea_crouanii 1 Extatosoma_tiaratum_tiaratum 1 Extatosoma_tiaratum Extatosomatini Extatosoma_tiaratum_bufonium 1 Hermarchus_sp_HER2 Hermarchus_pythonius 1 Phasmotaenia_sp_TRB2009 Stephanacridini Macrophasma_biroi 1 Lamponius_guerini 1 Agamemnon_cornutus Hesperophasmatini 0.95 Pterinoxylus_crassus Rhynchacris_ornata Phobaeticus_heusii 1 Phobaeticus_serratipes Pharnaciini Pharnacia_ponderosa 1 Lopaphus_sphalerus 1 Lopaphus_sphalerus_YZ 1 Lopaphus_perakensis 1 Lopaphus_iolas 1 Asceles_tanarata_amplior 1 1 Asceles_tanarata_tanarata Asceles_tanarata_singapura Necrosciini 1 Orxines_semperi 1 1 Orxines_macklottii Sipyloidea_sipylus 1 Oxyartes_lamellatus 1 Oxyartes_yunnanus Phaenopharos_khaoyaiensis Baculofractum_insigne 1 Neohirasea_maerens Spinohirasea benaglensis Bactrododema_sp_TRB2009 Necrosciinae_TRB2009 1 Sceptrophasma_langkawicensis 1 Sceptrophasma_hispidulum 0.99 Zehntneria_mystica 0.98 Clonaria_conformans 1 Baculini_sp_WS22 Bacillus_rossius 0.95 1 Medaura_sp_WS34 Gratidiini Medauroidea_extradentata 1 Baculum_thaii Ramulus_thaii Gratidia_sp_TRB2009 1 Pylaemenes_pui Epidares_nolimetangere Datamini Oreophoetes_peruana 1 Diapheromera_femorata 1 Pseudosermyle_phalangiphora Oncotophasma_martini Diapheromerini Spinonemia_chilensis 1 Sungaya_inexpectata 1 Trachyaretaon_carmelae 1 Aretaon_asperrimus Obrimini Pterobrimus_depressus Haaniella_echinata 1 Heteropteryx_dilatata Heteropterygini Haaniella_dehaanii 1 Anisomorpha_ferruginea 1 Anisomorpha_buprestoides 1 Peruphasma_schultei Anisomorphini 0.9 1 Agathemera_crassa Agathemera_sp_TRB2009 Agathemerini 1 Pseudophasma_velutinum Pseudophasma_rufipes Achrioptera_punctipes Hyrtacus_sp_TRB2009 1 Carausius_morosus Lonchodini Chondrostethus_woodfordi Xylica_oedematosa 0.99 Chitoniscus_brachysoma 1 Phyllium_celebicum 0.93 Phyllium_siccifolium Phylliini 1 1 Phyllium_giganteum 1 Phyllium_bioculatum Chitoniscus_feejeeanus 1 Dinophasma_saginatum Aschiphasmatini 1 Abrosoma_festinatum Timema_californicum 1 Metoligotoma_pugionifer Metoligotoma_brevispina -150 -125 -100 -75 -50 -25 0 MY Figure S2. Bayesian species tree inferred with BEAST, with node labels showing posterior probabilities > 0.9. Figure S3. Phylogenetic correlations between wing and body size among winged species. Details are provided in Table S2. Figure S4. Ancestral state reconstruction for relative wing size (Q) of males (a) and females (b), respectively, featuring repeated gain and reduction. The last common ancestor of the main clades is characterized by short wings (Q < 0.4); however, this inference is liable to change with more complete sampling. Arrows on main branches highlight increases and reductions of wing size. In long-wing lineages (e.g., Tropidoderini and Necrosciini), relative wing size between females and males is correlated (see Fig. 7a). Carlius fecundus Carlius fecundus Eurycantha insularis Eurycantha insularis Neopromachus doreyanus Neopromachus doreyanus Erinaceophasma vepres Erinaceophasma vepres Dryococelus australis Dryococelus australis a Dimorphodes prostasis b Dimorphodes prostasis Dimorphodes mancus Dimorphodes mancus Cnipsus rachis Cotylosoma amphibius Acanthoxyla geisovii Cnipsus rachis Acanthoxyla geisovii Argosarchus horridus Argosarchus horridus Clitarchus hookeri Clitarchus hookeri Pseudoclitarchus sentus Pseudoclitarchus sentus Tectarchus huttoni Tectarchus huttoni Niveaphasma annulata Niveaphasma annulata Monandroptera acanthomera Monandroptera acanthomera Rhaphiderus scabrosus Rhaphiderus scabrosus Phasma gigas Phasma gigas Anchiale briareus Anchiale briareus Ctenomorpha marginipennis Ctenomorpha marginipennis Acrophylla wuelfingi Acrophylla wuelfingi Acrophylla titan Acrophylla titan Acrophylla thoon Acrophylla thoon Eurycnema goliath Eurycnema goliath Eurycnema osiris Eurycnema osiris Tropidoderus childrenii Tropidoderus childrenii Podacanthus wilkinsoni Podacanthus wilkinsoni Ophicrania leveri Malandania pulchra Graeffea crouanii Ophicrania leveri Graeffea crouanii Extatosoma tiaratumtiaratum Extatosoma tiaratumtiaratum Hermarchus pythonius Hermarchus pythonius Macrophasma biroi Macrophasma biroi Agamemnon cornutus Pterinoxylus crassus 1.18 2.19 Lamponius guerini 1.33 2.39 Agamemnon cornutus Phobaeticus serratipes Lamponius guerini Pharnacia ponderosa Phobaeticus serratipes Lopaphus iolas Pharnacia ponderosa Lopaphus perakensis Lopaphus iolas Asceles tanaratatanarata Lopaphus perakensis log (L ) Asceles tanarataamplior log (L ) Lopaphus sphalerus 10 M Asceles tanaratasingapura 10 F Asceles tanaratatanarata Sipyloidea sipylus Asceles tanarataamplior Oxyartes lamellatus Asceles tanaratasingapura Phaenopharos khaoyaiensis Sipyloidea sipylus Baculofractum insigne Oxyartes lamellatus Medauroidea extradentata Phaenopharos khaoyaiensis Bacillus rossius Baculofractum insigne Sceptrophasma hispidulum Medauroidea extradentata Bacillus rossius Sceptrophasma langkawicensis Sceptrophasma hispidulum Clonaria conformans Sceptrophasma langkawicensis Ramulus thaii Clonaria conformans Diapheromera femorata Ramulus thaii Epidares nolimetangere Diapheromera femorata
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