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Title Origin and transformation of the in-flight wing-coupling structure in (Insecta: )

Author(s) Ogawa, Naoki; Yoshizawa, Kazunori

Journal of Morphology, 279(4), 517-530 Citation https://doi.org/10.1002/jmor.20785

Issue Date 2018-04

Doc URL http://hdl.handle.net/2115/75534

This is the peer reviewed version of the following article: "Origin and transformation of the in-flight wing-coupling structure in Psocodea (Insecta: Paraneoptera)."Journal of Morphology; 279(4) pp517-530 Apr 2018, which has been Rights published in final form at DOI:10.1002/jmor.20785. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

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Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Page 1 of 45 Journal of Morphology

1 1 OGAWA 2 3 4 5 Origin and transformation of the in-flight wing-coupling structure in 6 7 8 Psocodea (Insecta: Paraneoptera) 9 10 11 12 Naoki OGAWA*, Kazunori YOSHIZAWA 13 14 15 Systematic Entomology, School of Agriculture, Hokkaido University, 16 17 Sapporo 060-8589, Japan 18 19 TEL: +81-11-706-2486 20 21 22 Fax: +81-11-706-2424 23 24 [email protected] 25 26 27 28 Short title: Wing coupling structure in ‘’ 29 30 31 32 33 34 35 ABSTRACT 36 37 38 Many four-winged have mechanisms that unite the fore- and 39 40 hindwings in a single plane. Such an in-flight wing coupling apparatus may 41 42 improve flight performance in four-winged insects, but its structure is 43 44 45 variable among different groups. The wings of bark lice (Insecta: 46 47 Psocodea: ‘Psocoptera’) also have an in-flight wing coupling apparatus, but 48 49 to date, its morphology has not been studied in detail. In this study, we 50 51 52 examined the wing-coupling structure in representative species of the three 53 54 suborders of bark lice (, and ) and 55 56 inferred its origin and transformation. We conclude that the main 57 58 59 60 John Wiley & Sons Journal of Morphology Page 2 of 45

2 1 OGAWA 2 3 4 5 component of the psocodean wing coupling apparatus evolved once in the 6 7 common ancestor via modification of cuticular structures at the apex of the 8 9 10 forewing CuP vein. Morphological differences in components of the coupling 11 12 structures are phylogenetically informative at the intraorder level and 13 14 include an autapomorphy that characterizes Troctomorpha and a 15 16 17 synapomorphy that supports a sister relationship between Troctomorpha 18 19 and Psocomorpha. 20 21 22 23 24 INTRODUCTION 25 26 The evolution of insect wings and powered flight are cited as epochal events 27 28 in their history (Brodsky, 1994), and insect flight mechanisms have 29 30 31 attracted significant interest from functional and comparative biologists 32 33 (Wootton, 1992; Brodsky, 1994; Grodnitsky, 1995, 1999). Winged insects 34 35 usually have two pairs of (i.e., four) wings. Some four-winged insects, such 36 37 38 as dragonflies (Odonata) and locusts (Orthoptera), flap the fore- and 39 40 hindwings independently (Chapman, 2013), but this condition is rather 41 42 exceptional among insects, possibly because this mode is less efficient and 43 44 45 less stable (Pope, 1994). Loss of the thrust-generating function in either the 46 47 fore- or hindwing pair (i.e., dipterous flight) is more frequent; this occurs, for 48 49 example, in Diptera (true flies) and Strepsiptera (the twisted wing 50 51 52 parasites). The forewings of Coleoptera (beetles), the elytra, function to 53 54 protect the wings from external damage (Linz et al., 2016). The elytra are 55 56 sclerotized and held away from the body during flight, and they do not 57 58 59 60 John Wiley & Sons Page 3 of 45 Journal of Morphology

3 1 OGAWA 2 3 4 5 generate thrust. Complete loss of the capacity to generate thrust in either 6 7 the fore- or hindwing pair occurs in mayflies (Ephemeroptera), earwigs 8 9 10 (Dermaptera), crickets (Orthoptera), stick insects (Phasmida), scale insects 11 12 (), wasps (Hymenoptera), lacewings (Neuroptera), and moths 13 14 (Lepidoptera) (Grodnitsky, 1995). 15 16 17 The predominant flight mode in four-winged insects is functionally 18 19 two-winged flight (Grodnitsky, 1999). In such insects, the fore- and 20 21 hindwings are connected by an in-flight coupling apparatus. Grodnitsky 22 23 24 (1999) defined two coupling modes in the functionally two-winged insects: 25 26 wings that are coupled during the downstroke only (part-time coupling) and 27 28 wings that are mechanically connected and fully synchronized during flight 29 30 31 (full-time coupling). The wing coupling apparatus of “full-time” coupled 32 33 flyers has been reported in Paraneoptera [bark lice (Psocodea: Weber, 1936), 34 35 thrips (Thysanoptera: Pesson, 1951a; Moritz, 1997), aphids, cicadas and 36 37 38 true bugs (Hemiptera: Pesson, 1951b; Bohne and Schneider, 1979; D’Urso 39 40 and Ippolito, 1994; Ni et al., 2002)] and Holometabola [wasps 41 42 (Hymenoptera: Basibuyuk and Quicke, 1997), moths and butterflies 43 44 45 (Lepidoptera: Tillyard, 1918; Braun, 1924), and caddisflies (Trichoptera: 46 47 Tillyard, 1918; Stocks, 2010a)]. Such full-time wing coupling apparatuses 48 49 are one of two primary types: 1) setae of one wing that interact with setae or 50 51 52 other structures on the other wing, such as in Thysanoptera (Pesson, 1951a; 53 54 Moritz, 1997), Hymenoptera (Basibuyuk and Quicke, 1997), and Trichoptera 55 56 (Tillyard, 1918; Stocks, 2008, 2010a,b); 2) non-setal cuticular structures, 57 58 59 60 John Wiley & Sons Journal of Morphology Page 4 of 45

4 1 OGAWA 2 3 4 5 often in the form of grooves, such as in various Hemiptera (Pesson, 1951b; 6 7 D’Urso and Ippolito, 1994) and the jugum in Lepidoptera and Trichoptera 8 9 10 (Tillyard, 1918; Stocks, 2010ab). 11 12 The in-flight wing coupling apparatus of the free-living Psocodea, or 13 14 “Psocoptera”, is located on the distal end of the CuP vein (Fig. 2A, “fli”) and 15 16 17 clasps the hindwing at all times during wing flapping. The psocopterans 18 19 also have a repose-coupling apparatus located on the R or Sc vein that 20 21 engages the costa of the hindwing at rest (New, 1974; Mockford, 1967) (Fig. 22 23 24 2A, “rep”), but this structure is not further examined here. The psocopteran 25 26 in-flight wing coupling structure is formed from non-setal cuticles and 27 28 consists of a “hook” that engages the anterior margin of the hindwing 29 30 31 (Weber, 1936; Günther, 1974). Although there are some SEM studies on the 32 33 psocopteran in-flight coupling structures (New, 1974; Lawson and Chu, 34 35 1974), the structures have not been studied comprehensively. Therefore, 36 37 38 their evolutionary pattern and phylogenetic value are as yet unclear. 39 40 Furthermore, there is also uncertainty about the homology of the structures 41 42 with those of other paraneopteran orders (i.e., Thysanoptera and 43 44 45 Hemiptera: Lawson and Chu, 1974) 46 47 Currently, three suborders of ‘Psocoptera’ are recognized: 48 49 Trogiomorpha, Troctomorpha (including the parasitic lice) and Psocomorpha 50 51 52 (Yoshizawa et al., 2006). Mockford (1967) and Yoshizawa (2002, 2005) 53 54 recognized that in-flight wing coupling structures in psocids are 55 56 phylogenetically informative characters, having a ‘hook’ composed of 57 58 59 60 John Wiley & Sons Page 5 of 45 Journal of Morphology

5 1 OGAWA 2 3 4 5 truncated ‘spines’ fused at their bases as either a synapomorphy supporting 6 7 the clade Troctomorpha + Psocomorpha (Mockford, 1967) or an 8 9 10 autapomorphy of Psocomorpha (Yoshizawa, 2002; 2005). Molecular 11 12 phylogenetic approaches support Troctomorpha and Psocomorpha as sister 13 14 taxa (Yoshizawa et al., 2006; Yoshizawa and Johnson, 2014), but few 15 16 17 morphological characters that might support this relationship are known. 18 19 We expanded on the previous wing characters surveys that were mostly 20 21 based on light microscopy by including additional taxa and using SEM to 22 23 24 examine structures at higher magnification and resolution. We evaluated 25 26 wing coupling characters based on the phylogenetic hypotheses presented by 27 28 Yoshizawa & Johnson (2010, 2014), Friedemann et al. (2014) and Yoshizawa 29 30 31 & Lienhard (2016). We examined the homology and character state 32 33 transformations of the wing coupling apparatus. 34 35 36 37 38 MATERIALS & METHODS 39 40 Taxon selection (Table 1) 41 42 The taxa examined were as follows: Trogiomorpha, 5 species 43 44 45 representing 4 families; Troctomorpha, 7 species representing 9 families; 46 47 and 16 species representing the 23 families of Psocomorpha. We included a 48 49 species of Psocomorpha: Calopsocidae, recently synonymized with 50 51 52 (Yoshizawa and Johnson, 2014), because of the highly 53 54 modified, elytra-like wings. Aeolothrips kurosawai (Thysanoptera: 55 56 Aeolothripidae) and Cinara sp. (Hemiptera: Aphididae) were selected as 57 58 59 60 John Wiley & Sons Journal of Morphology Page 6 of 45

6 1 OGAWA 2 3 4 5 outgroups. 6 7 8 9 10 Treatment of Specimens 11 12 Specimens examined were stored in 80% or 99% ethanol. Forewings 13 14 were removed and dehydrated in 100% ethanol for 1 hour. Wing cuticle was 15 16 17 hardened by soaking with 1,1,1,3,3,3-hexamethyldisilazane for 1 hour and 18 19 air drying prior to mounting on 10mm aluminum stubs. Wings were held in 20 21 place with sticky urethane sheets (Kokuyo Hittsuki Sheet, Kokuyo Co. Ltd., 22 23 24 Tokyo) and coated with Au-Pd in a Hitachi E101 ion sputter-coater (Hitachi 25 26 High Technologies Corp., Tokyo) for 120 sec. We used a Jeol JSM-5310LV 27 28 scanning electron microscope (Jeol Ltd., Tokyo) and photographed screen 29 30 31 images with Jeol Digi Capture SUP-7707 Version 1.0.11. Figures were 32 33 arranged with Adobe Photoshop CC 2014 and Adobe Illustrator CC 2014. 34 35 36 37 38 Character Coding 39 40 Character state changes were reconstructed on the composite 41 42 phylogenetic tree (Fig. 1 in Yoshizawa and Johnson 2014), which included 43 44 45 almost all psocid families and was used as a backbone tree. Placement of 46 47 and was based on Yoshizawa & Johnson 48 49 (2010); those families were not sampled in Yoshizawa & Johnson (2014). The 50 51 52 outgroup taxa were based on Friedemann et al. (2014) and Yoshizawa & 53 54 Lienhard (2016). Unsampled families were trimmed from the tree, and a 55 56 coded character matrix was reconstructed by the parsimony criterion using 57 58 59 60 John Wiley & Sons Page 7 of 45 Journal of Morphology

7 1 OGAWA 2 3 4 5 Mesquite version 3.04 (Maddison and Maddison, 2015). 6 7 8 9 10 Terminology 11 12 Different terms have been used for the wing structures in 13 14 Psocoptera, but the terms used here are based on Weber (1936) and Günther 15 16 17 (1974). 18 19 20 21 22 RESULTS 23 24 25 26 Summary of general morphology (Fig. 2) 27 28 Structures forming the forewing in-flight coupling apparatus are near the 29 30 31 apical-most region of the CuP vein and are termed the re t i n a c u l u m , Cu P --- 32 33 titipp , and re t a i n e r (Figs. 2A, B). The term “nodulus” indicates the region 34 35 where the CuP and A1 veins join near the posterior wing margin (Fig. 2A, B, 36 37 38 junction of two green lines). Ri b ---li k e structures (Fig. 2B, r) are arranged at 39 40 almost equal intervals along the CuP vein (Günther, 1974) (Fig. 2B) 41 42 generally with each structure arranged transverse to the vein. The rib-like 43 44 45 structures occur on other veins, but their presence is taxonomically variable. 46 47 The “re t i n a cu l u m ” (Fig. 2B, Rc) is composed of many spine-like 48 49 cuticles (“re t i n a cu l a r s p i n e s ”) that as a unit form a hook-like structure. 50 51 52 The “Cu P ---ti p ” (Fig. 2B, Ct) refers to the apical-most section of the 53 54 CuP vein between the Rc and the posterior wing margin that bears modified 55 56 rrii b ---li k e s t r u c tu r e s . 57 58 59 60 John Wiley & Sons Journal of Morphology Page 8 of 45

8 1 OGAWA 2 3 4 5 The “re t a i n e rr” (Fig. 2B, Rtr) is a highly thickened and bent 6 7 structure on the posterior wing margin that bears a large number of fine 8 9 10 stud-like projections. The retainer is taxonomically variable in the degree of 11 12 swelling and the morphology of the projections. The retinaculum and 13 14 retainer together form a ‘clip-like’ functional unit that engages the costal 15 16 17 margin of the hindwing. 18 19 The counterpart of the in-flight wing coupling apparatus in 20 21 hindwings (i.e., the anterior margin) is not specialized among the taxa 22 23 24 examined, although the wing coupling apparatus of forewings is diversified. 25 26 The anterior margin of the hindwing engages in both types of wing coupling 27 28 (repose and in-flight) and is bent and rolled inwardly (Fig. 2C). At rest, the 29 30 31 repose-coupling apparatus (Fig. 2A, “rep”) fits into the bend (white arrow, 32 33 Fig. 2C) and supports the hindwing. During flight, the ‘clip-like’ unit formed 34 35 by the retinaculum and retainer catches the hindwing margin. 36 37 38 39 40 Trogiomorpha 41 42 43 44 45 Venation and the rib-like structure (Fig. 3) 46 47 The A1 and CuP veins do not join (i.e., the nodulus is absent), 48 49 although they terminate closely. Psoquilla sp. () lacks A1. The 50 51 52 CuP is thickened as in other veins. The rib-like structure is distributed on 53 54 all veins, but the ribs on the CuP are more prominent. 55 56 57 58 59 60 John Wiley & Sons Page 9 of 45 Journal of Morphology

9 1 OGAWA 2 3 4 5 Morphology of the retinaculum (Fig. 4) 6 7 The retinaculum is present in all examined trogiomorphan taxa, 8 9 10 and all are composed of several separated spines (Fig. 4; Character 3:1; 4:0) 11 12 that are continuous with the row of ribs on the CuP. In Prionoglaris 13 14 () (Fig. 4A), the retinaculum is composed of approximately 15 16 17 nine nearly straight spines. () also possesses a 18 19 simple retinaculum composed of approximately 10 straight spines (Fig. 4E). 20 21 In all other species, the retinaculum is composed of curled spines (Fig. 4C, 22 23 24 D). Psyllipsocus () has a simple retinaculum composed of only 25 26 four bent spines (Fig. 4C). The retinaculum of Psoquilla consists of 27 28 approximately 10 densely arranged and strongly curled and twisted spines 29 30 31 (Fig. 4D). The spines of Psyllipsocus, Echmepteryx and Psoquilla are 32 33 apically fringed (Fig. 4A, C, D; Character 5:1). 34 35 The retinaculum of Neotrogla (Prionoglarididae) (Fig. 4B) shows an 36 37 38 extremely different structure from that of the other trogiomorphan species, 39 40 including a species of the family (Prionoglaris). The rib-like structure on the 41 42 CuP vein becomes gradually recumbent and merges into the vein toward the 43 44 45 retinaculum (Character 6:1). The retinaculum is composed of one short and 46 47 three long, sharp spines projecting posteriorly. 48 49 50 51 52 Morphology of the CuP-tip (Fig. 4) 53 54 The CuP-tips in Prionoglaris, Psyllipsocus, Psoquilla sp. and 55 56 Echmepteryx are continuous from the basal CuP without remarkable 57 58 59 60 John Wiley & Sons Journal of Morphology Page 10 of 45

10 1 OGAWA 2 3 4 5 modification (Fig. 4A, C–E). The ribs on the CuP-tips are transversely 6 7 8 arranged without detectable modifications from those on the CuP vein 9 10 (Character 8:0). The CuP-tip of Neotrogla retains only one transversely 11 12 arranged rib near the base of the retinaculum (Fig. 4B). 13 14 15 16 17 Morphology of the retainer (Fig. 4) 18 19 The retainer of Trogiomorpha tends to be thickened and bent 20 21 22 (Character 11:1), but no further remarkable deformation compared to the 23 24 unmodified posterior wing margin was detected. The surface is uniformly 25 26 covered with scaly studs (Characters 14:0; 15:0), but the studs of Neotrogla 27 28 are recumbent and mostly merge into the vein (Fig. 4B). In Echmepteryx, 29 30 31 the surface studs distal to the retainer are gradually reduced (Fig. 3E; 32 33 Character 15:2). 34 35 36 37 38 Troctomorpha 39 40 41 42 Venation and rib-like structure (Fig. 5) 43 44 45 The A1 and CuP veins closely approximate at the distal ends. 46 47 They are clearly joined (= nodulus: Fig. 5C, D, E), separated (Fig. 5B), or 48 49 are intermediate in condition (Fig. 5A). The rib-like structures are 50 51 52 distributed on all veins, but they are more prominent on the CuP. 53 54 The species in two families of have somewhat 55 56 simplified forewings. The forewing of Embidopsocus (Liposcelididae) 57 58 59 60 John Wiley & Sons Page 11 of 45 Journal of Morphology

11 1 OGAWA 2 3 4 5 lacks a rib, a coupling structure and most veins (Character 2:1) (Fig. 6 7 9A). The forewing of Tapinella (Pachytroctidae) lacks the coupling 8 9 10 structure and nodulus (Fig. 9C), but all principal veins and the reduced 11 12 ribs (Character 1:1) are retained (Fig. 9B). 13 14 15 16 17 Morphology of the retinaculum (Fig. 5) 18 19 The retinaculum is composed of curled, separated spines 20 21 (Character 3:1: 4:0). The number of spines and their condition are 22 23 24 variable: they are numerous and in contact with each other in 25 26 Stimulopalpus (Fig. 5A); they are 5–10 in number and closely 27 28 approximated in Gen. (Troctopsocidae) (Fig. 5B), 29 30 31 Selenopsocus (Troctopsocidae) (Fig. 6C) and Manicapsocus 32 33 (Electrentomidae) (Fig. 6E); and there are five well-separated spines in 34 35 Musapsocus (Musapsocidae) (Fig. 5D). The spines are apically fringed 36 37 38 (Fig. 6A–C, E) except for Musapsocus with simple spines (Fig. 6D). 39 40 41 42 Morphology of the CuP-tips (Fig. 6) 43 44 45 The ribs on the CuP-tip of troctomorphan species are arranged 46 47 diagonally or vertically against the CuP (Character 8:1) and are thinner 48 49 than those on the basal CuP vein (Fig. 6A–E). In particular, the ribs on 50 51 52 the CuP-tips of Musapsocus, Stimulopalpus, Selenopsocus and 53 54 Manicapsocus are highly modified; much smaller ribs are arranged 55 56 densely and vertically in multiple rows (Character 9:1; Fig. 6A, C–E), 57 58 59 60 John Wiley & Sons Journal of Morphology Page 12 of 45

12 1 OGAWA 2 3 4 5 although the ribs on the CuP-tips of Troctopsocidae Gen. are not reduced 6 7 in size and are arranged in a row (Character 9:0) (Fig. 6B). 8 9 10 11 12 Morphology of the retainer (Fig. 5) 13 14 The retainer is inwardly rolled (Character 10:1) and uniformly 15 16 17 ornamented with scale-like studs (Characters 14:0; 15:0). However, the 18 19 retainers of some troctomophan taxa are more modified. Musapsocus 20 21 has a slightly broadened retainer (Character 12:1), and the studs are 22 23 24 more densely arranged on the counterpart of the retinaculum (Fig. 5D). 25 26 The retainer of Selenopsocus is also swollen (Character 12:1; Fig. 5C). 27 28 Stimulopalpus has a wide swelling (Character 12:1) with a narrow dent 29 30 31 (Character 13:1) (Fig. 5A). The retainer of Manicapsocus is also swollen 32 33 (Character 12:1), and the retainer is fully expanded and rolled inwardly 34 35 (Character 10:1) (Fig. 5E). The retainer of Troctopsocidae Gen. is only 36 37 38 slightly bent (Character 11:1) (Fig. 5B) and lacks a swelling (Character 39 40 12:0). 41 42 43 44 45 Psocomorpha 46 47 48 49 Venation and the rib-like structure (Fig. 7) 50 51 52 The CuP and A1 veins are joined distally, consistently forming 53 54 the nodulus. The CuP vein has well developed ribs, but ribs are not 55 56 present on the other veins. 57 58 59 60 John Wiley & Sons Page 13 of 45 Journal of Morphology

13 1 OGAWA 2 3 4 5 6 7 8 Morphology of the retinaculum (Fig. 8) 9 10 The morphology of the retinaculum is quite stable throughout 11 12 the suborder; it consists of completely fused curled spines (Character 13 14 3:1; 4:1). Judging from the numbers and condition of the slits, the 15 16 17 retinaculum appears to be composed of many twisted spines in most 18 19 psocomorphans. In Archipsocus (), the retinaculum is 20 21 simplified and apically pointed, and it appears to be composed of three 22 23 24 spines (Fig. 8A). The retinacular spines are not fringed in the 25 26 Psocomorpha in general, but the retinacular spines of Matsumuraiella 27 28 () (Fig. 8B), Amphipsocus () (Fig. 8D), 29 30 31 () (Fig. 8E), () (Fig. 8I), 32 33 Goja () (Fig. 8M) and Psilopsocus () (Fig. 8O) 34 35 have apparent to obscure apical fringes. 36 37 38 39 40 Morphology of the CuP-tips (Fig. 8) 41 42 In Archipsocus, the CuP-tip becomes obscure with only a 43 44 45 reduced rib (Fig. 8A). In the other infraorders, the morphology of the 46 47 CuP-tip is stable. The ribs are arranged diagonally (Character 8:1), and 48 49 the anterior ribs are continuous with the retinaculum (Character 7:1) (cf. 50 51 52 Fig. 8B). The CuP-tip of Calopsocus (Calopsocidae) (Fig. 8K) lacks the 53 54 rib (Character 9:2), making the surface of the CuP-tip smooth. The ribs 55 56 of Heterocaecilius (Pseudocaeciliidae) are also reduced and are only 57 58 59 60 John Wiley & Sons Journal of Morphology Page 14 of 45

14 1 OGAWA 2 3 4 5 represented by some diagonal slits (Fig. 8L). 6 7 8 9 10 Morphology of the retainer (Fig. 7) 11 12 The retainer is generally bent and rolled inwardly (Characters 13 14 10:1, 11:1). Further deformation of its shape also occurs. In Archipsocus 15 16 17 (Fig. 7A), the spiny area is restricted to a position in front of the 18 19 retinaculum, and the other areas lack any ornamentation (Characters 20 21 14:2, 15:2). In Valenzuela (Caeciliusidae) (Fig. 7E), Peripsocus 22 23 24 () (Fig. 7F), Aaroniella (Philotarsidae) (Fig. 7I), 25 26 () (Fig. 7J) and Calopsocus (Calopsocidae) 27 28 (Fig. 7K), the retainer has a swelling (Character 12:1), which is 29 30 31 particularly prominent in Calopsocus and Heterocaecilius (Character 32 33 12:2). 34 35 The retainer surface is generally ornamented with fine scale- 36 37 38 like studs. However, the retainer decoration of some species (Valenzuela 39 40 (Fig. 7E), Aaroniella (Fig. 7I), Trichopsocus (Fig. 7J), Calopsocus (Fig. 41 42 7K) and Heterocaecilius (Fig. 7L)) is modified to fine spines (Characters 43 44 45 14:1, 15:1). The shapes of the surface studs of Amphipsocus 46 47 (Amphipsocidae) (Fig. 7D) and Matsumuraiella (Dasydemellidae) (Fig. 48 49 7B) differ significantly between the proximal and distal regions with the 50 51 52 retinaculum as the boundary; the proximal surface is covered with scale- 53 54 like studs (Character 14:0), but the distal surface is arranged with fine 55 56 spines (Character 15:0). Goja (Epipsocidae) (Fig. 7M) has scale-like 57 58 59 60 John Wiley & Sons Page 15 of 45 Journal of Morphology

15 1 OGAWA 2 3 4 5 studs only on the proximal surface, and the studs disappear distally 6 7 (Character 15:2). 8 9 10 11 12 Outgroups (Fig. 10) 13 14 The wing coupling apparatuses are found consistently in the 15 16 17 outgroups (Hemiptera and Thysanoptera), but their conditions are 18 19 completely different from those of the psocopterans. Aphids (Hemiptera) 20 21 possess hamuli (= hooked hairs) along the costal margin of the hindwing 22 23 24 (Ni et al., 2002). The coupling apparatus of thrips (Thysanoptera) 25 26 consists of the marginal setae of the fore- and hindwings (Ellington, 27 28 1980). Their vein surfaces are decorated occasionally similar to those of 29 30 31 Psocoptera (Fig. 10A, C). Cinara (Aphididae: Hemiptera) possesses ribs 32 33 on all of the veins (Character 1:1) (Fig. 10B). The veins of Aeolothrips 34 35 (Aeolothripidae: Thysanoptera) are covered with embossed tiles, each 36 37 38 bearing a microtrichium (Fig. 10D) (Character 1:0). In the outgroups, 39 40 the nodulus is not formed. 41 42 43 44 45 Character coding and phylogenetic reconstruction 46 47 Based on the observations, 16 characters that may be relevant 48 49 to psocid phylogeny were selected and coded from the wing coupling 50 51 52 structure (Table 2). Only qualitative or discontinuous quantitative 53 54 characters were coded, although some continuous characters were also 55 56 mentioned in the above morphological descriptions. The result of the 57 58 59 60 John Wiley & Sons Journal of Morphology Page 16 of 45

16 1 OGAWA 2 3 4 5 parsimonious reconstruction of these characters is shown in Fig. 11. The 6 7 8 character indices are as follows: Consistency Index = 0.41; Retention 9 10 Index = 0.65. 11 12 13 14 Character 1. Decoration of CuP: tile pattern with microtrichia (0); rib- 15 16 17 like structure (1). State 0 was only observed in Aeolothrips (Thysanoptera) 18 19 (Fig. 10C, D). State 1 was observed in Cinara sp. (Hemiptera) (Fig. 10A, B) 20 21 and all Psocoptera (Figs. 3A–E; 5A–E; 7A–P) except for the veinless 22 23 24 Embidopsocus (Liposcelididae) (Fig. 9A). 25 26 Character 2. Venation. Present (0); reduced (1). Almost all examined 27 28 taxa possessed venation (State 0), but the venation is reduced in 29 30 31 Embidopsocus (Liposcelididae: Troctomorpha) (Fig. 9A), which almost entirely 32 33 lacks veins (state 1). 34 35 Character 3. Retinaculum on CuP. Absent (0); present (1). This is 36 37 38 identified as an autapomorphy of Psocodea (state 1), but its secondary absence 39 40 was detected in Embidopsocus and Tapinella (state 0). 41 42 Character 4. Arrangement of retinacular spines. Clearly, separated 43 44 45 from each other (0); fused with each other (1). State 1 was identified as an 46 47 autapomorphy of Psocomorpha (Fig. 7A–P) (State 1). 48 49 Character 5. Tip of the retinacular spines. Not divided (0); fringed (1). 50 51 52 State 1 was observed in some taxa of all three suborders (Fig. 6B–E) and was 53 54 identified as a highly homoplasious condition. 55 56 57 58 59 60 John Wiley & Sons Page 17 of 45 Journal of Morphology

17 1 OGAWA 2 3 4 5 Character 6. Retinacular spines. Standing (0); laying (1). State 1 was 6 7 only observed in Neotrogla (Prionoglarididae) (Fig. 4B). 8 9 10 Character 7. Retinaculum and ribs on CuP-tip. Separated (0); 11 12 anterior rib merged into the retinaculum (1). State 1 was identified as an 13 14 autapomorphy of Psocomorpha (Fig. 8A–P). 15 16 17 Character 8. Angle of the ribs against the CuP-tip. Transversal (0); 18 19 diagonal to vertical (1). State 1 was identified as a synapomorphy of 20 21 Troctomorpha and Psocomorpha. 22 23 24 Character 9. Number of rows of ribs on CuP-tip. 1 (0); 2 or more (1); 25 26 absent (2). State 1 was observed in almost all troctomorpha except for 27 28 Troctopsocidae Gen. (Fig. 6A, B, D, E). The CuP-tip of Calopsocus completely 29 30 31 lacks ribs, and this was coded as state 2. 32 33 Character 10. Retainer. No inward rolling (0); rolling inwardly (1). 34 35 State 1 was detected in all Psocoptera (Fig. 3A–E; 5A–E; 7A–P) except for the 36 37 38 species lacking the wing coupling structure (Tapinella and Embidopsocus). 39 40 Although the outgroups lack the retainer, state 0 was adopted due to it having 41 42 a non-rolling forewing hind margin (Figs. 9A, B; 10A, C) 43 44 45 Character 11. Posterior margin of the retainer. Not bent (0); bent (1). 46 47 Almost all examined taxa have a bent retainer (State 1), but the retainer of 48 49 Stimulopalpus (), Tapinella (Pachytroctidae), 50 51 52 Embidopsocus (Liposcelididae), Heterocaecilius (Pseudocaeciliidae), 53 54 Hemipsocus (), and Psilopsocus (Psilopsocidae) is not bent (Figs. 55 56 5B; 7L, N, O; 9A, B) (State 0). 57 58 59 60 John Wiley & Sons Journal of Morphology Page 18 of 45

18 1 OGAWA 2 3 4 5 Character 12. Retainer. No swelling (0); with swelling (1); strongly 6 7 8 swelling and forming a protrusion (2). State 1 was observed in several 9 10 scattered taxa (Figs. 5A, B, D; 7E–G, I, J, M, O), and Heterocaecilius 11 12 (Pseudocaeciliidae) and Calopsocus (Calopsocidae) have a huge protrusion on 13 14 15 the retainer (State 2) (Fig. 7K, L). 16 17 Character 13. Surface of the retainer swelling. No dent (0); with dent 18 19 (1). State 1 was only observed in Stimulopalpus (Amphientomidae) (Fig. 5B). 20 21 22 Character 14. Surface of the proximal region of the retainer. Covered 23 24 with scale-like studs (0); with trichomes (1); bare (2). The proximal region of 25 26 the retainer surface of Cinara sp. and almost all psocopterans is covered with 27 28 tiny scale-like studs (State 0). The surface of several psocopteran and 29 30 31 thysanopteran taxa is covered by fine trichomes (Figs. 7E, I–L). The proximal 32 33 retainer surface of Archipsocus sp. is bare (Fig. 7A) (State 2). 34 35 Character 15. The surface of the distal region of the retainer. Covered 36 37 38 with scale-like studs (0); with trichomes (1); bare (2). The distal retainer 39 40 surface of Cinara sp. and almost all psocopterans is also covered with tiny 41 42 scale-like studs (State 0). The distal surface ornamentations of Thysanoptera 43 44 45 and psocopteran taxa are trichomes (Fig. 7 B, D, E, I–L) (State 1). This region 46 47 is bare in Echmepteryx (Lepidopsocidae), Archipsocus (Archipsocidae) and 48 49 50 Goja (Epipsocidae) (Figs. 3E; 7A, M) (State 2). 51 52 53 54 55 56 57 DISCUSSION 58 59 60 John Wiley & Sons Page 19 of 45 Journal of Morphology

19 1 OGAWA 2 3 4 5 6 7 8 Origin and homology 9 10 The psocopteran wing coupling system is composed of three functional 11 12 units: two on the forewing— the retinaculum and retainer (Fig. 2B)— 13 14 15 and the costal margin of the hindwing, in which the retinaculum and 16 17 retainer engage the anterior margin of the hindwing during flight. 18 19 SEM observations clearly suggest that the retinaculum is 20 21 22 composed of highly modified rib-like structures. Their homology is most 23 24 clearly indicated in Prionoglaris (Trogiomorpha: Prionoglarididae; Fig. 25 26 3A), in which the retinacular spines and normal ribs differ only by the 27 28 degree to which their apexes are extended. 29 30 31 Wing veins in other insects are often arrayed with rows of 32 33 microtrichia (Fig. 10), and the microtrichia are probably homologous 34 35 with the ribs. The rib-like structures are thin and semi-circular 36 37 38 projections that give the CuP vein a rasp-like appearance. Similar rib- 39 40 like structures occur in Cinara (Hemiptera: Aphididae) (Fig. 10B) but 41 42 are not part of a wing-coupling system. Therefore, the presence of the 43 44 45 retinaculum is apparently an autapomorphic condition for Psocodea. 46 47 The retainer is formed by various but relatively simple 48 49 modifications of the cuticle on the surface of the posterior wing margin. The 50 51 52 retainers in all specimens examined curved inward to some degree (Fig. 2) 53 54 with additional bends and/or protrusions in some species of Troctomorpha 55 56 and Psocomorpha. 57 58 59 60 John Wiley & Sons Journal of Morphology Page 20 of 45

20 1 OGAWA 2 3 4 5 We conclude that the psocopteran wing coupling system is 6 7 8 unique. The morphology of the components is distinct from those of 9 10 outgroup taxa, although Lawson and Chu (1974) suggested the 11 12 homology of the structure between Psocoptera and Hemiptera. The wing 13 14 15 coupling system in Thysanoptera is formed by setae located on the fore- 16 17 and hindwing margins (Ellington, 1980). In Hemiptera: Sternorrhyncha 18 19 it is composed of ‘hamuli-like’ projections on the hindwing margin (Ni et 20 21 22 al., 2002), and in Hemiptera: Auchenorrhyncha the coupling system is 23 24 composed of grooves on the fore- and hindwing margins (D’Urso and 25 26 Ippolito, 1994). The wing-coupling system of some Heteroptera is 27 28 morphologically and functionally similar to that of Psocoptera (Bohne 29 30 31 and Schneider, 1979; Stocks, 2008), although the forewing components 32 33 are on the A vein. 34 35 36 37 38 Phylogenetic significance 39 40 The retinacular spines are clearly separated in Trogiomorpha and 41 42 Troctomorpha, although they are more closely set in the latter (cf. Fig. 5E) 43 44 45 and are fused in Psocomorpha (cf. Fig. 7P). Since outgroup taxa lack this 46 47 structure, there is no basis on which to assess the polarity of the 48 49 transformation series. However, homology of the retinacular spines and the 50 51 52 ribs on the CuP vein permit estimation of character polarity; we might for 53 54 example consider distinctly separated retinacular spines as a plesiomorphy. 55 56 The surface structure of the CuP-tip is identical to the that of the ribs on the 57 58 59 60 John Wiley & Sons Page 21 of 45 Journal of Morphology

21 1 OGAWA 2 3 4 5 basal CuP vein in the Trogiomorpha (cf. Fig. 3A) and is also considered a 6 7 plesiomorphy with gradual modification throughout Troctomorpha and 8 9 10 Psocomorpha taxa. The troctomorphan CuP-tip has multiple rows of 11 12 diagonally to vertically arranged ribs (cf. Fig. 6A), but the ribs are clearly 13 14 separated from the retinaculum, whereas the psocomorphan CuP-tip is 15 16 17 arranged diagonally (cf. Fig. 7P) with anterior ribs integrated into the 18 19 retinaculum (cf. Fig. 2B). 20 21 The wing coupling apparatus in Trogiomorpha and Troctomorpha is 22 23 24 less modified. However, the multiple rows of ribs on the CuP-tip (Character 25 26 9:1), which have not been reported previously, is an autapomorphy 27 28 supporting the monophyly of Troctomorpha. Troctopsocidae Gen. (Fig. 5B) 29 30 31 has a single row of diagonal ribs, as observed in Trogiomorpha and 32 33 Psocomorpha (Character 9:0), but this trait is a reversal in the most 34 35 parsimonious reconstruction (Fig. 11). However, this condition may be 36 37 38 plesiomorphic, since the higher-level relationships among troctomorphan 39 40 families are poorly understood (Yoshizawa and Johnson, 2014). 41 42 A close relationship between Troctomorpha and Psocomorpha is 43 44 45 supported by molecular data (Yoshizawa and Johnson, 2014), but there is 46 47 little support based on morphology. The ribs arranged diagonally to 48 49 vertically (Character 8:1) was identified here as a synapomorphy supporting 50 51 52 a close relationship. Based on light microscopy, Mockford (1967) suggested 53 54 that a retinaculum composed of truncated spines fused at their bases is a 55 56 potential synapomorphy of Troctomorpha and Psocomorpha. However, the 57 58 59 60 John Wiley & Sons Journal of Morphology Page 22 of 45

22 1 OGAWA 2 3 4 5 SEM images reveal more detail, and we could not discern such fusion in 6 7 Troctomorpha. The following character states were recovered as 8 9 10 autapomorphies of Psocomorpha: retinacular spines fused (Character 4:1) 11 12 (Yoshizawa, 2002, 2005) and anterior ribs on the CuP-tip merged into the 13 14 retinaculum (Character 7:1) (Fig. 11). 15 16 17 The retainer is less variable throughout Psocoptera. The posterior 18 19 forewing margin covered by scale-like studs and curved inward (Character 20 21 10:1) is considered an autapomorphy of Psocoptera, and its absence in 22 23 24 Nanopsocetae (Pachytroctidae and Liposcelididae: Fig. 9A, B) is a secondary 25 26 loss. A bent retainer (Character 11:1) may be an autapomorphy of the order, 27 28 but several taxa of Troctomorpha and Psocomorpha (Figs. 3A, 7N, O, 9A, B) 29 30 31 indicate secondary reversal. The shapes of the surface studs on the posterior 32 33 wing margin (Characters 14:0; 15:0) are highly variable. Trichome-like studs 34 35 were observed only in Caeciliusetae and Philotarsetae (Characters 14:1; 36 37 38 15:1) (Fig. 7B, D, E, I–L), but the distant phylogenetic relationship of the 39 40 families suggests independent origins (Fig. 11). Trichome-like studs occur in 41 42 Matsumuraiella and Amphipsocus (Caeciliusetae) but are restricted to the 43 44 45 distal region with scale-like studs proximally (Character 14:0). The retainer 46 47 of Archipsocus is almost completely devoid of surface decorations (Fig. 7A), 48 49 and those of Echmepteryx (Lepidopsocidae) and Goja (Epipsocidae) diminish 50 51 52 gradually distally from the junction of the CuP (Character 14:2; 15:2) (Fig. 53 54 11). Modifications of the bent retainer, retinaculum and CuP-tip occurred 55 56 independently in different taxa and overall appear to be highly 57 58 59 60 John Wiley & Sons Page 23 of 45 Journal of Morphology

23 1 OGAWA 2 3 4 5 homoplasious. If variation in these character systems contains a 6 7 phylogenetic signal, a much denser taxon sampling is needed. 8 9 10 The modifications observed in the retainer displayed by some taxa 11 12 may be phylogenetically informative at a lower level of phylogeny. For 13 14 example, the strongly developed, thumb-like protrusion in Calopsocus 15 16 17 (Calopsocidae) (Figs. 7K, 8K, L) and Heterocaecilius (Pseudocaeciliidae) 18 19 (Figs. 7L, 8L) (Character 12:2) may be a synapomorphy that supports a close 20 21 relationship; this is supported by other morphological (Yoshizawa, 2002) and 22 23 24 molecular (Yoshizawa and Johnson, 2014) data. 25 26 Among Psocoptera, there are two different cases of reduction of the 27 28 wing coupling apparatus. Neotrogla (Prionoglarididae) has a simplified wing 29 30 31 coupling structure in which the retinaculum consists of a few recumbent 32 33 spines (cf. Fig. 4B), and the hindwings are largely diminished. In most 34 35 analyses, taxa of Prionoglarididae are considered to be the most plesiotypic 36 37 38 overall, and the simplified retinaculum may also represent a plesiomorphy. 39 40 However, the most parsimonious reconstruction (MPR) of the retinacular 41 42 character (Character 6) implies that the simplified retinaculum of Neotrogla 43 44 45 is an autapomorphy (Fig. 11). Neotrogla species inhabit caves and exhibit 46 47 many specialized behaviors (Yoshizawa et al., 2014), and the simplified 48 49 retinaculum is probably associated with diminution of the hindwing 50 51 52 (Lienhard and Ferreira, 2013). 53 54 Liposcelididae and Pachytroctidae also have simplified wings, and 55 56 they completely lack a wing coupling apparatus. However, their hindwings 57 58 59 60 John Wiley & Sons Journal of Morphology Page 24 of 45

24 1 OGAWA 2 3 4 5 keep their size, unlike Neotrogla. Based on phylogenetic analysis and MPR, 6 7 loss of wing coupling structures and the nodulus in Liposcelididae (Fig. 9A) 8 9 10 and Pachytroctidae (Fig. 9B) (Troctomorpha: Nanopsocetae) is considered as 11 12 a secondary loss (Characters 10:0: 11:0) (Fig. 11). The insects can flap the 13 14 fore- and hindwings independently during flight, and the reduction of their 15 16 17 wing coupling apparatus may be involved with the different ecology of 18 19 Neotrogla in its functional aspect. 20 21 In summary, the common ancestor of Psocodea gained a unique wing 22 23 24 coupling apparatus composed of the retainer, CuP-tip and a retinaculum at 25 26 the end of the CuP vein. These structures are consistently retained 27 28 throughout the Psocodea, and some modifications reflect their deep 29 30 31 phylogenetic relationships, including the first potential autapomorphy of 32 33 Troctomorpha (Character 9:1) and synapomorphy of Troctomorpha and 34 35 Psocomorpha (Character 8:1). Independent origins of the wing coupling 36 37 38 apparatus among the paraneopteran orders were suggested by the 39 40 morphological analysis. The factors driving the independent evolution of the 41 42 wing coupling apparatus are still unknown. To answer this question, 43 44 45 comprehensive observations of the morphology and flight behavior must be 46 47 conducted throughout the paraneopteran orders. 48 49 50 51 52 ACKNOWLEDGEMENT 53 54 We are grateful to Charles Lienhard, Rodrigo Ferreira, Tadaaki Tsutsumi, 55 56 and Toshifumi Nonaka for providing identified sample and Shin-ichi 57 58 59 60 John Wiley & Sons Page 25 of 45 Journal of Morphology

25 1 OGAWA 2 3 4 5 Akimoto for assistance with specimen identification, and Masanori Yasui 6 7 and the Electron Microscope Laboratory, Research Faculty of Agriculture, 8 9 10 Hokkaido University for the support of the Electron Microscope observation. 11 12 This study was supported by the JSPS pre-doctoral fellowship (DC1) and 13 14 JSPS research grant No. 15J03697 to NO, and JSPS grant No. 15H04409 to 15 16 17 KY. 18 19 20 21 22 REFERENCES 23 24 25 26 27 28 Basibuyuk HH, Quicke DLJ. 1997. Hamuli in the Hymenoptera (Insecta) 29 30 and their phylogenetic implications. J Nat Hist 31 :1563–1585. 31 32 33 Bohne VJ, Schneider P. 1979. Morphologische gestaltung des 34 35 gleitkoppelmechanismus bei wanzenflügeln (Heteroptera). Zool 36 37 Anzeiger Jena 20 22:307–316. 38 39 40 Braun AF. 1924. The frenulum and its retinaculum in the Lepidoptera. Ann 41 42 Entomol Soc Am 17 :234–257. 43 44 Brodsky AK. 1994. The Evolution of Insect Flight. Oxford: Oxford 45 46 47 University Press,. 1-229 p. 48 49 Chapman RF. 2013. The Insects. Structure and Function. 5th ed. 50 51 Cambridge: Cambridge University Press. 929 p. 52 53 54 D’Urso V, Ippolito S. 1994. Wing-coupling apparatus of Auchenorrhyncha 55 56 (insecta: Homoptera). Int J Insect Morphol Embryol 23 :211–224. 57 58 59 60 John Wiley & Sons Journal of Morphology Page 26 of 45

26 1 OGAWA 2 3 4 5 Ellington CP. 1980. Wing Mechanics and Take-Off Preparation of Thrips 6 7 8 (Thysanoptera). J Exp Biol 858585:129–136.85 9 10 Friedemann K, Spangenberg R, Yoshizawa K, Beutel RG. 2014. Evolution of 11 12 attachment structures in the highly diverse Acercaria (Hexapoda). 13 14 15 Cladistics 303030:170–201.30 16 17 Grodnitsky DL. 1995. Evolution and classification of insect flight 18 19 kinematics. Evolution 494949:1158–1162.49 20 21 22 Grodnitsky DL. 1999. Form and Function of Insect Wings. Baltimore: The 23 24 Johns Hopkins University Press. 1-261 p. 25 26 Günther KK. 1974. Staubläuse, Psocoptera. Jena: Gustav Fischer Verlag. 1- 27 28 314 p. 29 30 31 Lawson FA, Chu J. 1974. Wing coupling in a bark : A light and SEM 32 33 study (Psocoptera: ). J Kans Entomol Soc 474747(1):47 136-140. 34 35 Lienhard C, Ferreira R. 2013. A new species of Neotrogla from Brazilian 36 37 38 caves ( Psocodea: “ Psocoptera ”: Prionoglarididae ). Rev Suisse Zool 39 40 120120:3–12. 41 42 Linz DM, Hu AW, Sitvarin MI, Tomoyasu Y. 2016. Functional value of 43 44 45 elytra under various stresses in the red flour beetle, Tribolium 46 47 castaneum. Sci Rep 666:34813.6 48 49 Maddison WP, Maddison DR. 2015. Mesquite: a modular system for 50 51 52 evolutionary analysis. Version 3.03 [WWW Document]. URL 53 54 http://mesquiteproject.org 55 56 Mockford EL. 1967. The electrentomoid psocids (Psocoptera). Psyche 57 58 59 60 John Wiley & Sons Page 27 of 45 Journal of Morphology

27 1 OGAWA 2 3 4 5 774:118–165.4 6 7 Moritz G. 1997. Structure, Growth and Development. In: Lewis T, editor. 8 9 10 Thrips as crop pests: CAB International. p. 15–63. 11 12 Ni X, Johnson GD, Quisenberry SS. 2002. Comparison of hindwing hamuli 13 14 from five species of cereal aphids (Hemiptera: Aphididae). Ann Entomol 15 16 17 Soc Am 9955 : 109–114. 18 19 New TR. 1974. Structural variation in psocopteran wing-coupling 20 21 mechanisms. Int J Insect Morphol Embryol 333(2):3 193-201. 22 23 24 Pesson P. 1951a. Ordre des Thysanoptera. In: Grassé PP, editor. Traité de 25 26 Zoologie Vol. 10 Paris: Masson. p. 1805–1869. 27 28 Pesson P. 1951b. Ordre des Homoptères (1). In: Grassé PP, editor. Traité de 29 30 31 Zoologie Vol. 10 Paris: Masson. p. 1390–1656. 32 33 Pope A. 1994. From Functionally four-winged flight to functionally two- 34 35 winged flight. In: The Evolution of Insect Flight p. 132–151. 36 37 38 Stocks IC. 2008. Wing Coupling. In: Capinera JL, Editor. Encyclopedia of 39 40 Entomology, Second Edition: Springer . p. 4266-4271. 41 42 Stocks IC. 2010a. Comparative and functional morphology of wing coupling 43 44 45 structures in Trichoptera: Annulipalpia. J Morphol 272 711 :152–168. 46 47 Stocks IC. 2010b. Comparative and functional morphology of wing coupling 48 49 structures in Trichoptera: Integripalpia. Ann Zool Fenn 47 : 351-386. 50 51 52 Tillyard RJ. 1918. The panorpoif complex. Part i.-The Wing-coupling 53 54 Apparatus, with special refference to the Lepidoptera. Proc Linn Soc 55 56 New South Wales 434343:169–172.43 57 58 59 60 John Wiley & Sons Journal of Morphology Page 28 of 45

28 1 OGAWA 2 3 4 5 Weber H. 1936. Copeognatha. Biol der Tiere Deutschlands 393939 (27): 1-50. 6 7 8 Wootton RJ. 1992. Functional Morphology of Insect Wings. Annu Rev 9 10 Entomol 373737:113–140.37 11 12 Yoshizawa K. 2002. Phylogeny and higher classification of suborder 13 14 15 Psocomorpha (Insecta: Psocodea: “Psocoptera”). Zool J Linn Soc 16 17 136136136:371–400.136 18 19 Yoshizawa K. 2005. Morphology of Psocomorpha (Psocodea: “Psocoptera”). 20 21 22 Insecta Matsumurana, New Ser 626262:1–44.62 23 24 Yoshizawa K, Ferreira RL, Kamimura Y, Lienhard C. 2014. Female penis, 25 26 male vagina, and their correlated evolution in a cave insect. Curr Biol 27 28 24:1006–1010.24 29 2424 30 31 Yoshizawa K, Johnson KP. 2010. How stable is the “Polyphyly of Lice” 32 33 hypothesis (Insecta: Psocodea)?: a comparison of phylogenetic signal in 34 35 multiple genes. Mol Phylogenet Evol 555555:939–951.55 36 37 38 Yoshizawa K, Johnson KP. 2014. Phylogeny of the suborder Psocomorpha: 39 40 congruence and incongruence between morphology and molecular data 41 42 (Insecta: Psocodea: “Psocoptera”). Zool J Linn Soc 171171171:716–731.171 43 44 45 Yoshizawa K, Lienhard C. 2016. Bridging the gap between chewing and 46 47 sucking in the hemipteroid insects: new insights from 48 49 amber. Zootaxa 407940794079:229–245.4079 50 51 52 Yoshizawa K, Lienhard C, Johnson KP. 2006. Molecular systematics of the 53 54 suborder Trogiomorpha (Insecta: Psocodea: “Psocoptera”). Zool J Linn 55 56 Soc 146146146:287–299.146 57 58 59 60 John Wiley & Sons Page 29 of 45 Journal of Morphology

29 1 OGAWA 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Morphology Page 30 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 1. Phylogeny of ‘Psocoptera’ and relatives adopted in this study. This tree was constructed based on Yoshizawa & Johnson (2010, 2014) (for ‘Psocoptera’) and Friedmann et al. (2014) and Yoshizawa & Lienhard 48 (2016) (for the relationship with the outgroups). 49 50 212x324mm (300 x 300 DPI) 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 31 of 45 Journal of Morphology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 3. In-flight wing coupling structures in Trogiomorpha, ventral view. A. Prionoglaris stygia (Prionoglarididae). B. Neotrogla curvata (Prionoglarididae). C. Psyllipsocus yucatan (Psyllipsocidae). D. 48 Psoquilla sp. (Psoquillidae). E. (Lepidopsocidae). 49 50 211x357mm (300 x 300 DPI) 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Morphology Page 32 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 2. Right fore- and hindwing of Metylophorus sp. (). A. Ventral view of the forewing, with names of relevant veins. Red rectangle indicates the in-flight wing coupling region (fli). Abbreviations: rep, repose- 48 coupling apparatus; fli, in-flight coupling apparatus. B. Enlarged in-flight wing coupling structure. 49 Abbreviations: Rc, retinaculum; Ct, CuP tip; Rtr, retainer; r, rib-like structure; A1, First Anal vein; CuP, 50 Posterior Cubital vein. C. Dorsal view of the hindwing. White and black arrows indicate engagement point 51 with repose-coupling appatarus and in-flight coupling apparatus, respectively. 52 53 211x298mm (300 x 300 DPI) 54 55 56 57 58 59 60 John Wiley & Sons Page 33 of 45 Journal of Morphology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Fig. 4. In-flight wing coupling structures in Trogiomorpha, ventrolateral view. A. Prionoglaris stygia 30 (Prionoglarididae). B. Neotrogla curvata (Prionoglarididae). C. Psyllipsocus yucatan (Psyllipsocidae). D. 31 Psoquilla sp. (Psoquillidae). E. Echmepteryx hageni (Lepidopsocidae). 32 33 211x140mm (300 x 300 DPI) 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Morphology Page 34 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 5. In-flight wing coupling structures in Troctomorpha, ventral view. A. Stimulopalpus japonicus (Amphientomidae). B. Gen. sp. (Troctopsocidae). C. Selenopsocus sp. (Troctopsocidae). D. Musapsocus sp. 48 (Musapsocidae). E. Manicapsocus alettae (Electrentomidae). 49 50 211x357mm (300 x 300 DPI) 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 35 of 45 Journal of Morphology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Fig. 6. In-flight wing coupling structures in Troctomorpha, ventrolateral view. A. Stimulopalpus japonicus 30 (Amphientomidae). B. Gen. sp. (Troctopsocidae). C. Selenopsocus sp. (Troctopsocidae). D. Musapsocus sp. 31 (Musapsocidae). E. Manicapsocus alettae (Electrentomidae). 32 33 211x140mm (300 x 300 DPI) 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Morphology Page 36 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 7. In-flight wing coupling structures in Psocomorpha, ventral view. A. Archipsocus sp. (Archipsocidae). B. Matsumuraiella radiopicta (Dasydemellidae). C. Stenopsocus nigricellus (). D. Amphipsocus 48 japonicus (Amphipsocidae). E. (Caeciliusidae). F. Peripsocus quercicola (Peripsocidae). 49 G. Ectopsocus briggsi (). H. Idatenopsocus orientalis (Mesopsocidae). I. Aaroniella badonneli 50 (Philotarsidae). J. (Trichopsocidae). K. Calopsocus furcatus (Calopsocidae syn: 51 Pseudocaeciliidae). L. Heterocaecilius solocipennis (Pseudocaeciliidae). M. Goja sp. (Epipsocidae). N. 52 Hemipsocus chloroticus (Hemipsocidae). O. Psilopsocus malayensis (Psilopsocidae). P. Metylophorus sp. 53 (Psocidae). 54 55 254x342mm (300 x 300 DPI) 56 57 58 59 60 John Wiley & Sons Page 37 of 45 Journal of Morphology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Fig. 8. In-flight wing coupling structures in Psocomorpha, ventrolateral view. A. Archipsocus sp. 41 (Archipsocidae). B. Matsumuraiella radiopicta (Dasydemellidae). C. Stenopsocus nigricellus (Stenopsocidae). 42 D. Amphipsocus japonicus (Amphipsocidae). E. Valenzuela flavidus (Caeciliusidae). F. Peripsocus quercicola 43 (Peripsocidae). G. Ectopsocus briggsi (Ectopsocidae). H. Idatenopsocus orientalis (Mesopsocidae). I. 44 Aaroniella badonneli (Philotarsidae). J.Trichopsocus clarus (Trichopsocidae). K. Calopsocus furcatus 45 (Calopsocidae syn: Pseudocaeciliidae). L. Heterocaecilius solocipennis (Pseudocaeciliidae). M. Goja sp. (Epipsocidae). N. Hemipsocus chloroticus (Hemipsocidae). O. Psilopsocus malayensis (Psilopsocidae). P. 46 Metylophorus sp. (Psocidae). 47 48 282x282mm (300 x 300 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Morphology Page 38 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Fig. 9. Forewings of Nanopsocetae species (Troctomorpha) lacking the in-flight wing coupling structure, 25 ventral view. A. Embidopsocus sp. (Liposcelididae). B. Tapinella sp. (Pachytroctidae). C. ditto, enlarged view 26 of the end of the CuP and A1 in B. 27 28 223x111mm (300 x 300 DPI) 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 39 of 45 Journal of Morphology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig. 10. Right forewing and vein decorations of the outgroups. A. Cinara sp. (Hemiptera: Sternorrhyncha: 29 Aphididae), ventral view. B. ditto, enlarged view of the vein indicated by red rectangle in A. C. Aeolothrips 30 kurosawai (Thysanoptera: Aeolothripidae), ventral view. D. ditto, enlarged view of the vein indicated by red 31 rectangle in C. 32 211x132mm (300 x 300 DPI) 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Morphology Page 40 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Fig. 11. The parsimonious reconstruction of selected 16 characters. Character and character state changes 44 reconstructed on the branches are indicated by black (non-homoplasious) and gray bars (homoplasious). 45 46 446x491mm (300 x 300 DPI) 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 41 of 45 Journal of Morphology

1 2 3 4 5 Table 1. Taxa examined for this study. 6 7 8 9 10 ORDER HEMIPTERA 11 12 Cinara sp. (Aphididae) 13 14 15 16 17 ORDER THYSANOPTERA 18 19 Aeolothrips kurosawai Bhatti, 1971 (Aeolothripidae) 20 21 22 23 24 ORDER PSOCODEA 25 26 SUBORDER TROGIOMORPHA 27 28 Prionoglaris stygia Enderlein, 1909 (Prionoglarididae) 29 30 31 Neotrogla curvata Lienhard & Ferreira, 2013 (Prionoglarididae) 32 33 Psyllipsocus yucatan Gurney, 1943 (Psyllipsocidae) 34 35 Psoquilla sp. (Psoquillidae) 36 37 38 Echmepteryx hageni (Packard, 1870) (Lepidopsocidae) 39 40 41 42 SUBORDER TROCTOMORPHA 43 44 45 Stimulopalpus japonicus Enderlein, 1906 (Amphientomidae) 46 47 Gen. sp. (Troctopsocidae) 48 49 Selenopsocus sp. (Troctopsocidae) 50 51 52 Musapsocus sp. (Musapsocidae) 53 54 Manicapsocus alettae Smithers, 1966 (Electrentomidae) 55 56 Embidopsocus sp. (Liposcelididae) 57 58 59 60 John Wiley & Sons Journal of Morphology Page 42 of 45

1 2 3 4 5 Tapinella sp. (Pachytroctidae) 6 7 8 9 10 SUBORDER PSOCOMORPHA 11 12 Archipsocus sp. (Archipsocidae) 13 14 15 Matsumuraiella radiopicta Enderlein, 1906 (Dasydemellidae) 16 17 Stenopsocus nigricellus Okamoto, 1907 (Stenopsocidae) 18 19 Amphipsocus japonicus (Enderlein, 1906) (Amphipsocidae) 20 21 22 Valenzuela flavidus (Stephens, 1836) (Caeciliusidae) 23 24 Peripsocus quercicola Enderlein, 1906 (Peripsocidae) 25 26 Ectopsocus briggsi McLachlan, 1899 (Ectopsocidae) 27 28 Idatenopsocus orientalis (Vishnyakova, 1986) (Mesopsocidae) 29 30 31 Aaroniella badonneli (Danks, 1950) (Philotarsidae) 32 33 Trichopsocus clarus (Banks, 1908) (Trichopsocidae) 34 35 Calopsocus furcatus (New, 1978) (Calopsocidae syn: 36 37 38 Pseudocaeciliidae) 39 40 Heterocaecilius solocipennis (Enderlein, 1907) (Pseudocaeciliidae) 41 42 Goja sp. (Epipsocidae) 43 44 45 Hemipsocus chloroticus (Hagen, 1958) (Hemipsocidae) 46 47 Psilopsocus malayensis New & Lee, 1991 (Psilopsocidae) 48 49 Metylophorus sp. (Psocidae) 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 43 of 45 Journal of Morphology

1 1 OGAWA 2 3 4 5 Table 2. Data matrix used for the parsimonious reconstruction. See text for 6 7 8 characters and their states. 9 10 11 5 10 15 12 Cinara sp. (Aphididae) 1 0 0 ? ? ? ? 0 ? 0 0 0 ? 0 0 13 Aeolothrips kurosawai (Aeolothripidae) 0 0 0 ? 0 0 0 0 0 0 0 0 ? 1 1 14 Prionoglaris stygia (Prionoglarididae) 1 0 1 0 1 0 0 0 0 1 1 0 ? 0 0 15 Neotrogla curvata (Prionoglarididae) 1 0 1 0 0 1 ? 0 0 1 1 0 ? 0 0 16 Psyllipsocus yucatan (Psyllipsocidae) 1 0 1 0 0 0 0 0 0 1 1 0 ? 0 0 17 Psoquilla sp. (Psoquillidae) 1 0 1 0 1 0 ? 0 0 1 1 0 ? 0 0 18 Echmepteryx hageni (Lepidopsocidae) 1 0 1 0 1 0 0 0 0 1 1 0 ? 0 2 19 Stimulopalpus japonicus (Amphientomidae) 1 0 1 0 2 0 0 1 1 1 0 1 1 0 0 20 Gen. sp. (Troctopsocidae) 1 0 1 0 2 0 0 1 0 1 1 0 ? 0 0 21 22 Selenopsocus sp. (Troctopsocidae) 1 0 1 0 2 0 0 1 1 1 1 1 0 0 0 23 Musapsocus sp. (Musapsocidae) 1 0 1 0 0 0 0 1 1 1 1 1 ? 0 0 24 Manicapsocus alettae (Electrentomidae) 1 0 1 0 2 0 0 1 1 1 1 1 ? 0 0 25 Embidopsocus sp. (Liposcelididae) 1 1 0 ? ? 0 ? ? ? 0 0 ? ? ? ? 26 Tapinella sp. (Pachytroctidae) 1 0 0 ? ? 0 ? ? ? 0 0 ? ? ? ? 27 Archipsocus sp. (Archipsocidae) 1 0 1 1 0 0 1 1 0 1 1 0 0 2 2 28 Matsumuraiella radiopicta (Dasydemellidae) 1 0 1 1 2 0 1 1 0 1 1 0 ? 0 1 29 Stenopsocus nigricellus (Stenopsocidae) 1 0 1 1 0 0 1 1 0 1 1 0 ? 0 0 30 Amphipsocus japonicus (Amphipsocidae) 1 0 1 1 2 0 1 1 0 1 1 0 ? 0 1 31 Valenzuela flavidus (Caeciliusidae) 1 0 1 1 1 0 1 1 0 1 1 1 0 1 1 32 Peripsocus quercicola (Peripsocidae) 1 0 1 1 0 0 1 1 0 1 1 1 0 0 0 33 Ectopsocus briggsi (Ectopsocidae) 1 0 1 1 0 0 1 1 0 1 1 1 ? 0 0 34 Idatenopsocus orientalis (Mesopsocidae) 1 0 1 1 0 0 1 1 0 1 1 0 ? 0 0 35 Aaroniella badonneli (Philotarsidae) 1 0 1 1 2 0 1 1 0 1 1 1 0 1 1 36 Trichopsocus clarus (Trichopsocidae) 1 0 1 1 0 0 1 1 0 1 1 1 0 1 1 37 Calopsocus furcatus (Calopsocidae) 1 0 1 1 0 0 1 1 2 1 1 2 0 1 1 38 Heterocaecilius solocipennis (Pseudocaeciliidae) 1 0 1 1 0 0 1 1 0 1 0 2 0 1 1 39 Goja sp. (Epipsocidae) 1 0 1 1 2 0 1 1 0 1 1 0 ? 0 2 40 Hemipsocus chloroticus (Hemipsocidae) 1 0 1 1 0 0 1 1 0 1 0 1 0 0 0 41 Psilopsocus malayensis(Psilopsocidae) 1 0 1 1 1 ? 1 1 0 1 0 1 ? 0 0 42 Metylophorus sp. (Psocidae) 1 0 1 1 0 0 1 1 0 1 1 0 ? 0 0 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Journal of Morphology Page 44 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Graphical Abstract Image 33 141x103mm (300 x 300 DPI) 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 45 of 45 Journal of Morphology

1 2 3 4 5 Wing-coupling structures of insects mechanically unite the fore- and hindwings. The 6 7 structure in different “Psocoptera” (barklice) groups consists of three functional units, 8 termed retinaculum, CuP tip and retainer, which originated in the common ancestor, 9 10 and are phylogenetically informative at different levels in the order. 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons