A tale of winglets: evolution of flight morphology in stick

Supplementary Materials

Text section Notes on ancestral state reconstruction for wing size in stick insects

Figure S1, Summary of morphology sampling S2, Polytomous tree with traits mapped S3, Ancestral state reconstruction for 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 hind wing state for sampled tribes S6, Summary of phylogenetic correlation of flight morphology among all winged stick insects

Table S1, Information on the nuclear and mitochondrial loci S2, Summary of PGLS results in all winged stick insects S3, Summary of derived wing utility in stick insects with miniaturized wings

Datasets 1-4 1. Morphometrics (taxa, wing length, body length) 2. Scaling of wing loading (mass, wing length, wing area) 3. Molecular sampling (list of ), including accession numbers for new sequences on GenBank 4. Nexus input files

Script MatLab script for organizing 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 a short wing size (Q < 0.4) preceded various levels of gains and losses observed in extant phasmids (Fig. S4,5). Nevertheless, this result only offered a preliminary perspective on wing size evolution in phasmids. This study reflects a small portion of the extant phasmid diversity (~ 10% documented species), and a complete molecular phylogeny and a complete trait sampling are not available. Our preliminary sampling for the presence of hind wings showed a highly variable pattern across phasmid , even below the tribe level (Fig. S7). In to properly model evolutionary rates of change and ancestral states for wing size, future work may increase the phylogenetic resolution and incorporate trait sampling across extant tips. Properly modeling the gain and loss of wings in phasmids will require complex models to account for (1) variation in rate of wing size evolution across the tree (Beaulieu et al. 2013), (2) variation in rate 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 on bimodal distribution of wing size further indicated a potentially more complex, 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 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 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. In long-wing lineages, wing size between females and males is correlated (see Fig. 7a).

Carlius fecundus Carlius fecundus 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 gigas Phasma gigas Anchiale briareus Anchiale briareus Ctenomorpha marginipennis Ctenomorpha marginipennis wuelfingi Acrophylla wuelfingi Acrophylla titan Acrophylla thoon Acrophylla thoon goliath Eurycnema osiris Eurycnema osiris Tropidoderus childrenii Podacanthus wilkinsoni Podacanthus wilkinsoni Ophicrania leveri Malandania pulchra Graeffea crouanii Ophicrania leveri Graeffea crouanii Extatosoma tiaratumtiaratum Extatosoma tiaratumtiaratum pythonius Hermarchus pythonius Macrophasma biroi Macrophasma biroi Agamemnon cornutus Pterinoxylus crassus 1.18 2.19 Lamponius guerini 1.33 2.39 Agamemnon cornutus serratipes Lamponius guerini ponderosa 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 sipylus Asceles tanarataamplior Oxyartes lamellatus Asceles tanaratasingapura Phaenopharos khaoyaiensis Sipyloidea sipylus Baculofractum insigne Oxyartes lamellatus extradentata Phaenopharos khaoyaiensis Bacillus rossius Baculofractum insigne Sceptrophasma hispidulum Bacillus rossius Sceptrophasma langkawicensis Sceptrophasma hispidulum Clonaria conformans Sceptrophasma langkawicensis thaii Clonaria conformans Diapheromera femorata Ramulus thaii nolimetangere Diapheromera femorata Oreophoetes peruana Epidares nolimetangere Spinonemia chilensis Oreophoetes peruana Trachyaretaon brueckneri Spinonemia chilensis Aretaon asperrimus Trachyaretaon brueckneri Pterobrimus depressus Aretaon asperrimus dilatata Pterobrimus depressus echinata Heteropteryx dilatata buprestoides Haaniella echinata Anisomorpha ferruginea Anisomorpha buprestoides Peruphasma schultei Anisomorpha ferruginea Paraphasma rufipes Peruphasma schultei Pseudophasma velutinum Paraphasma rufipes Achrioptera punctipes Pseudophasma velutinum Carausius morosus Achrioptera punctipes Carausius morosus Chondrostethus woodfordi Chondrostethus woodfordi Xylica oedematosa Xylica oedematosa Phyllium celebicum Phyllium celebicum Chitoniscus brachysoma Chitoniscus brachysoma Phyllium siccifolium Phyllium siccifolium Phyllium bioculatum Phyllium bioculatum Phyllium giganteum Phyllium giganteum Chitoniscus feejeeanus Chitoniscus feejeeanus Abrosoma festinatum Abrosoma festinatum Dinophasma saginatum Dinophasma saginatum californicum Timema californicum

Figure S5. Ancestral state reconstruction for body size (L) of males (a) and females (b), respectively. Body size evolution is coupled between two sexes but independent of wing size evolution, corresponding with the correlational pattern summarized in Fig. 7a.

Carlius fecundus Eurycantha insularis Carlius fecundus a Neopromachus doreyanus b Eurycantha insularis Erinaceophasma vepres Neopromachus doreyanus Dryococelus australis Erinaceophasma vepres Dimorphodes prostasis Dryococelus australis Dimorphodes mancus Dimorphodes prostasis Cnipsus rachis Dimorphodes mancus Female- Acanthoxyla geisovii Cnipsus rachis Male-biased Argosarchus horridus Acanthoxyla geisovii biased Clitarchus hookeri Argosarchus horridus Pseudoclitarchus sentus Clitarchus hookeri Tectarchus huttoni Pseudoclitarchus sentus −0.21 0 0.5 1 Niveaphasma annulata Tectarchus huttoni Monandroptera acanthomera −0.35 -0.3 -0.2 -0.1 0 Niveaphasma annulata Rhaphiderus scabrosus Monandroptera acanthomera Phasma gigas Rhaphiderus scabrosus Anchiale briareus Phasma gigas Ctenomorpha marginipennis Anchiale briareus Acrophylla wuelfingi Ctenomorpha marginipennis ΔQ Acrophylla titan ΔL Acrophylla wuelfingi Acrophylla thoon Acrophylla titan Eurycnema goliath Acrophylla thoon Eurycnema osiris Eurycnema goliath Tropidoderus childrenii Eurycnema osiris Podacanthus wilkinsoni Tropidoderus childrenii Ophicrania leveri Podacanthus wilkinsoni Graeffea crouanii Ophicrania leveri Extatosoma tiaratumtiaratum Graeffea crouanii Hermarchus pythonius Extatosoma tiaratumtiaratum Macrophasma biroi Hermarchus pythonius Agamemnon cornutus Macrophasma biroi Lamponius guerini Agamemnon cornutus Phobaeticus serratipes Lamponius guerini Pharnacia ponderosa Phobaeticus serratipes Lopaphus iolas Pharnacia ponderosa Lopaphus perakensis Lopaphus iolas Asceles tanaratatanarata Lopaphus perakensis Asceles tanarataamplior Asceles tanaratatanarata Asceles tanaratasingapura Asceles tanarataamplior Sipyloidea sipylus Asceles tanaratasingapura Oxyartes lamellatus Sipyloidea sipylus Phaenopharos khaoyaiensis Oxyartes lamellatus Baculofractum insigne Phaenopharos khaoyaiensis Medauroidea extradentata Baculofractum insigne Bacillus rossius Medauroidea extradentata Sceptrophasma hispidulum Bacillus rossius Sceptrophasma langkawicensis Sceptrophasma hispidulum Clonaria conformans Sceptrophasma langkawicensis Ramulus thaii Clonaria conformans Diapheromera femorata Ramulus thaii Epidares nolimetangere Diapheromera femorata Oreophoetes peruana Epidares nolimetangere Spinonemia chilensis Oreophoetes peruana Trachyaretaon brueckneri Spinonemia chilensis Aretaon asperrimus Trachyaretaon brueckneri Pterobrimus depressus Aretaon asperrimus Heteropteryx dilatata Pterobrimus depressus Haaniella echinata Heteropteryx dilatata Anisomorpha buprestoides Haaniella echinata Anisomorpha ferruginea Anisomorpha buprestoides Peruphasma schultei Anisomorpha ferruginea Paraphasma rufipes Peruphasma schultei Pseudophasma velutinum Paraphasma rufipes Achrioptera punctipes Pseudophasma velutinum Carausius morosus Achrioptera punctipes Chondrostethus woodfordi Carausius morosus Xylica oedematosa Chondrostethus woodfordi Phyllium celebicum Xylica oedematosa Chitoniscus brachysoma Phyllium celebicum Phyllium siccifolium Chitoniscus brachysoma Phyllium bioculatum Phyllium siccifolium Phyllium giganteum Phyllium bioculatum Chitoniscus feejeeanus Phyllium giganteum Abrosoma festinatum Chitoniscus feejeeanus Dinophasma saginatum Abrosoma festinatum Timema californicum Dinophasma saginatum Timema californicum Figure S6. Ancestral state reconstruction for sexual wing dimorphism (SWD) index (ΔQ; a) and sexual size dimorphism (SSD) index (ΔL; b). (a) The ancestral state reconstruction of ΔQ shows repeated increases and reductions, with an intermediate level of male-biased SWD (0 < ΔQ < 0.5, as represented by yellow color) as the ancestral state for most clades. (b) Ancestral state reconstruction of ΔL, showing an intermediate level of female-biased SSD (-0.2 < ΔL < -0.1) as the ancestral state preceding repeated increase and reduction. The lack of evolutionary correlation between ΔL and ΔQ, as summarized in Fig. 7c, is evident by comparing (a) and (b).

a b Varieganecroscia Acanthoxylini Thrasyllus Tagesoidea Hemipachymorphini Wingless Syringodes Sumatranius Pachymorphini Spinosipyloidea Spinohirasea Sosibia Sipyloidea Monandropterini Winged Sinophasma Singaporoidea Eurycanthini Septopenna Scionecra Sabahphasma Rhamphosipyloidea Xeroderini Qiongphasma Pterohirasea Phasmatini Pseudososibia Pseudoparamenexenus Pseudoneoclides Tropidoderini Pseudodiacantha Pseudocentema Pomposa Platycranini Platysosibia Planososibia Extatosomatini Phamartes Phaenopharos Paroxyartes Parastheneboea Stephanacridini Parasosibia Parasipyloidea Hesperophasmatini Parasinophasma Paraprosceles Necrosciini Paranecroscia Pharnaciini Paramenexenus Paraloxopsis Paradiacantha Necrosciini Pachyscia Oxyartes Medaurini Ovacephala Otraleus Orxines Orthostheneboea Bacillini Orthonecroscia Nuichua Gratidiini Notaspinius Neososibia us Neoqiongphasma Neooxyartes Clitumnini n Neonescicroa Neohirasea Cladoxerini Neoclides Ge Necroscia Neoasceles Datamini Micadina Moritasgus Oreophoetini Meionecroscia Mesaner Marmessoidea Diapheromerini Malandella Macrocercius Maculonecroscia Heteronemiini Lopaphus Loxopsis Obrimini Lamachodes Lobonecroscia Labanphasma Laevediacantha Heteropterygini Hennemannia Huananphasma Anisomorphini Hemiplasta Hemisosibia Galactea Gargantuoidea Agathemerini Diesbachia Eurynecroscia Stratocleini Dianphasma Diardia Pseudophasmatini Diacanthoidea Diangelus Conogalactea Cornicandovia Achriopterini Channia Cheniphasma Lonchodini Caudasceles Centrophasma Candovia Capuyanus Xylicini Brockphasma Calvisia Phylliini Austrosipyloidea Brevinecroscia Aschiphasmatini Aschiphasmodes Asystata Andropromachus Asceles Timematini Anarchodes Anasceles Acacus Acanthophasma 0 200 400 600 0 20 40 60 0 20 40 60 0 50 100 150 Number of species Number of species Number of species MY

Figure S7. Summary of species richness for tribes represented by the molecular phylogeny. (a) The number of known species mapped on a phylogeny of tribes sampled in this study, with colored bars showing the number of species with and without hind wings. (b) A more detailed summary of the number of known species and the presence of wings for Necrosciini, the most diversified tribe. Taxonomic organization followed Phasmida Species File (Brock, 2019).

Supplementary Table

Locus Forward primer Reverse primer Ta (˚C) Length (bp) Reference Buckley et al., 28s AGAACTTTGAAGAGAGAGTTCAAGA TCAAGACGGGTCGGGAGA 50 495 2008

COI TTGATTTTTTGGTCATCCAGAAGT TCCAATGCACTAATCTGCCATATTA 50 814 Simon et al., 1994

COII AATATGCAGATTAGTGCA GTTTAAGAGACCAGTACTTG 50 736 Simon et al., 1994 Buckley et al., H3 ATGGCTCGTACCAAGCAGAC ATATCCTTRGGCATRATRGTGAC 50 358 2008

Table S1. Information on the nuclear and mitochondrial loci used in this study.

PGLS correlations N slope slope.SE P

Male QM ~ Log10(LM) ‡ 273 -0.271 ( 0.005 ) 0.072 ( 0.001 ) < 0.001

Female QF ~ Log10(LF) ‡ 262 -0.107 ( 0.02 ) 0.1 ( 0.001 ) 0.296 ( 0.096 )

QF ~ QM ‡ 158 0.876 ( 0.01 ) 0.063 ( 0.001 ) < 0.001

Wing size, QM ~ ΔQ 183 -0.029 ( 0.003 ) 0.038 ( 0 ) 0.438 ( 0.04 ) species- Q ~ ΔQ ‡ 158 -0.534 ( 0.007 ) 0.068 ( 0 ) < 0.001 wise F comparison ΔQ ~ ΔL ‡ 158 -0.637 ( 0.033 ) 0.22 ( 0.003 ) 0.005 ( 0.003 )

ΔQ ~ Lmean ‡ 158 0.001 ( 0 ) 0.001 0.332 ( 0.11 )

LM ~ LF 367 0.61 ( 0.001 ) 0.014 ( 0 ) < 0.001 Body size, species- LF ~ ΔL 367 -118.893 ( 5.122 ) 18.92 ( 0.38 ) < 0.001 wise LM ~ ΔL 367 31.48 ( 3.534 ) 13.999 ( 0.276 ) 0.03 ( 0.02 ) comparison ΔL ~ Lmean 158 0 0 0.03 ( 0.026 )

Table S2. Summary of pairwise correlational analyses applied to all winged species using PGLS models. Values represent means from analyses using 100 randomly resolved trees, with 1 s.d. in brackets. The symbol ‡ indicates that equivalent significance was found for analyses using the original data with variables converted to pseudo-continuous ordinal numbers in analyses (see Methods).

Taxa Startle display Reference Pterinoxylus spinulosus female and male x x Robinson, 1968 Hanniella spp. female and male x Pers. Observ. Oxyartes spp. female and male x Pers. Observ. Phaenopharus spp. female and male x Pers. Observ.

Diapherodes spp. female x Pers. Observ. Parectatosoma spp. female and male x Pers. Observ.

Diesbachia hellotis female x Pers. Observ. Achrioptera spp. female and male x Pers. Observ. Peruphasma spp. female and male x Pers. Observ.

Table S3. A summary of known cases of derived wing utility in stick insects with miniaturized wings.

References

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