Structure & Development 57 (2020) 100951

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Arthropod Structure & Development

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First description of male genital sclerites and associated musculature for two members of (Insecta: Neuropterida: ) based on X-ray microCT imaging

* Stephan Handschuh a, Ulrike Aspock€ b, c, a VetCore Facility for Research, Imaging Unit, University of Veterinary Medicine Vienna, Veterinaerplatz 1, A-1210, Vienna, Austria b Natural History Museum, 2nd Zoological Department, Burgring 7, A-1010, Vienna, Austria c Department of Evolutionary Biology, University of Vienna, Althanstraße 14, A-1090, Vienna, Austria article info abstract

Article history: Coniopterygidae are the dwarfs among the Neuroptera. Despite their miniaturisation, the males are Received 12 September 2019 equipped with genital sclerites that are excessively heterogeneous. They function in copulation and Received in revised form sperm transfer and have been widely utilized for species identification, as well being considered of high 24 April 2020 phylogenetic relevance. The present study is the first to describe the musculature associated with the Accepted 24 April 2020 genital sclerites of two species of Coniopterygidae, Helicoconis lutea (Wallengren, 1871) (Aleuropter- Available online xxx yginae), and Coniopteryx pygmaea (Enderlein, 1906) (Coniopteryginae) based on X-ray microCT imaging. We found six pairs of muscles associated with the genital sclerites in H. lutea and seven in C. pygmaea. Keywords: Gonocoxite hypothesis The images depict other internal organs of the posterior abdominal segments, such as gonads and Terminalia alimentary canal. In both investigated species, the internal sclerites support the ductus ejaculatorius, Homologisation which e surprisingly e turned out to be a landmark for the identification of closely adjacent internal Copulation sclerites and associated musculature. The interpretation of these sclerites as gonocoxites and gona- Helicoconis pophyses of the tenth segment (traditionally denoted as parameres and penis) could be corroborated. Coniopteryx Thus it is no longer tenable to assert that possession of a “penis” is exclusive to Coniopterygidae, since this sclerite is part of the ground pattern in Neuroptera. Interactions of genital sclerites and corre- sponding musculature during copulation are discussed. © 2020 Elsevier Ltd. All rights reserved.

1. Introduction forewing length between 1.2 and 6 mm and by being equipped with diverse morphological characters that are not yet functionally un- The Coniopterygidae Burmeister, 1839, (Insecta: Neuropterida: derstood. Secondly, due to several disagreements between Neuroptera) include approximately 580 known species assigned to morphological and molecular analyses, their systematic position three subfamilies: Brucheiserinae Navas, 1927, with only four within Neuroptera is still unresolved. known species restricted to South America, and the species-rich Coniopterygidae are equipped with excessively heterogeneous subfamilies Coniopteryginae Burmeister, 1839, with 201 known male genital sclerites which function during copulation and sperm species and Enderlein, 1905, with 370 known transfer. They have been widely utilized for species identification species, which are distributed worldwide (Meinander, 1972, 1990; (Tjeder, 1957; Meinander, 1972; Sziraki, 2011; Martins and Sziraki, 2007, 2011; Martins and Amorim, 2016; Oswald, 2017; Amorim, 2016) and are of high phylogenetic relevance Oswald and Machado, 2018). Coniopterygidae have been a matter (Meinander, 1972, U. Aspock€ and H. Aspock,€ 2008). The first of permanent interest for two reasons: Firstly, as the dwarfs among “portraits” of genital sclerites of Coniopterygidae were published Neuroptera and Neuropterida they remain enigmatic with a in the early twentieth century by Enderlein (1906).Almostfifty years later, Tjeder (1954) created a general terminology for genital sclerites of Neuropterida, which still serves as a reference for many * Corresponding author. Natural History Museum, 2nd Zoological Department, neuropterologists (Meinander, 1972; Sziraki, 2011; Martins and Burgring 7, A-1010, Vienna, Austria. Amorim, 2016). Tjeders terminology for Coniopterygidae E-mail addresses: [email protected] (S. Handschuh), ulrike. included, among other characters, a pair of external sclerites [email protected], [email protected] (U. Aspock).€ https://doi.org/10.1016/j.asd.2020.100951 1467-8039/© 2020 Elsevier Ltd. All rights reserved. 2 S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951

(gonarcus), two pairs of internal sclerites (penis, parameres) and a segmental muscles, the alimentary canal, and the reproductive prominent external hypandrium. organs), may provide new insights in order to either corroborate or More recently, male genital sclerites in Neuropterida have been falsify current homology assumptions with respect to genital studied and interpreted in the context of an extended gonocoxite sclerites. Specific questions include: (i) Does musculature support hypothesis (U. Aspock€ and H. Aspock,€ 2008, Liu et al., 2016,U. the interpretation of the external genital sclerites as appendages of Aspock,€ 2019). Although not the first to do so, Matsuda (1957) segment nine? Furthermore, can the musculature of the external argued that abdominal segments eight, nine, and ten of female sclerites be homologised with muscles of other Hexapoda? (ii) Can Archaeognathan Machilidae are equipped with serially homolo- the musculature of the two pairs of internal sclerites be homolo- gous gonocoxites, gonostyli and gonapophyses. Lawrence et al. gised with muscles of other Hexapoda? If yes, does musculature (1991) extended the gonocoxite hypothesis of Matsuda to the corroborate interpretation of these sclerites as tenth segmental genital appendages of female Holometabola. U. Aspock€ and H. appendages, and does the muscle homologisation allow a homo- Aspock€ (2008) adopted the gonocoxite hypothesis to Neuro- logisation of the internal genital sclerites of Coniopterygidae to pterida, involving also male genital appendages by using the male sclerites of other Hexapoda? (iii) Do the presented data allow for a terminalia of Raphidioptera as key link. In this extended gonocoxite sound interpretation of the identity of the external hypandrium of hypothesis, they included also sclerites of abdominal segment 11. Coniopterygidae? (iv) Do the presented data allow for any new clue The extended gonocoxite hypothesis assumes that each of the male on specific and unique configurations and modifications of the genital sclerites of Coniopterygidae can be homologized with either genital sclerites found in the two investigated genera? (3) To gonocoxites, gonostyli or gonapophyses of the abdominal segments explore the informational value of genital sclerite associated mus- nine, ten or eleven. Within the extended gonocoxite hypothesis, cles for phylogenetic evidence. sclerites of Neuroptera have been homologised according to their position and structure. In the Coniopterygidae, several sclerites 2. Material and methods show large positional and structural similarities with other neu- ropterans providing reasonable support for homology assumptions 2.1. (U. Aspock€ and H. Aspock,€ 2008). These include a prominent pair of external sclerites (gonarcus sensu Tjeder (1957)), which has been For this study we used one male specimen of H. lutea interpreted as derived appendages of the ninth abdominal segment (Wallengren, 1871) (Aleuropteryginae Enderlein, 1905) from sam- (gonocoxites, or potentially fused gonocoxites þ gonostyli). Also, pling locality Allershausen, Bavaria, Germany, N48,42541 two pairs of internal sclerites are present in Coniopterygidae E11,61307, July 2017, leg. Axel Gruppe, and one male specimen of (parameres and penis sensu Tjeder (1957)), which have been C. pygmaea Enderlein (1906) (Coniopteryginae Burmeister, 1839), interpreted as genital sclerites of abdominal segment ten (U. from sampling locality Windischhütte near Klosterneuburg, Lower Aspock€ and H. Aspock,€ 2008). The wealth of data on con- Austria, N48,1719,9 E16,1620,5, May 2013, leg. Ulrike Aspock.€ iopterygid genitalia (Meinander, 1972, 1990) provides good support for homology assumptions for these three pairs of sclerites. 2.2. Sample preparation and microCT imaging Furthermore, their descent from segment nine and ten appendages receives reasonable support from comparison to other Neuro- One male of H. lutea and one male of C. pygmaea were fixed and pterida, and Hexapoda/Endopterygota in general (U. Aspock€ and H. stored in 70% ethanol. Prior to microCT imaging, they were dehy- Aspock,€ 2008). However, the exact identity of single sclerites drated with absolute ethanol and stained for 24h in 1% (w/v) (referring to the interpretation as gonocoxites, gonostyli, gona- elemental iodine in absolute ethanol (Metscher, 2009). After pophyses), the potential presence of amalgamated composite staining, specimens were washed in absolute ethanol and vertically sclerites (e.g., gonocoxite þ gonostylus), the identity of the external mounted in polypropylene pipette tips in absolute ethanol. hypandrium, as well as the presence of genital sclerites of segment Specimens were scanned using an XRadia MicroXCT-400 (Carl 11 are so far still largely subject to interpretation and hypothetical, Zeiss X-ray Microscopy, Pleasanton, CA, USA). Overview scans of the as no developmental and genetic data are available for these whole abdomen were acquired at 60kVp/133 mA using the 10X structures. detector assembly. Projections were recorded with 12s exposure While a vast amount of information has been published on the time (camera binning ¼ 2) and an angular increment of 0.2 be- morphology of male genital sclerites covering most species of tween projections over a 184 rotation. Tomographic slices were Coniopterygidae (Meinander, 1972, 1990), so far no data are avail- reconstructed with an isotropic voxel size of 2.33 mm for H. lutea able on the associated musculature and soft tissues of the posterior and 2.16 mm for C. pygmaea using the XMReconstructer software abdominal segments for any member of the family. Thus the cur- provided with the microCT system. Subsequently, high-resolution rent study has three main aims: (1) To provide a first detailed tomographies of the posterior abdominal segments were acquired description of the muscles associated with the genital sclerites at 40kVp/75 mA using the 20 X detector assembly. Projections were using microCT imaging, a technique which is highly suited for non- recorded with 120s exposure time (camera binning ¼ 2) and an destructive 3D imaging of morphology (Friedrich et al., angular increment of 0.2 between projections over a 182 rotation. 2013), for two divergent model species: Helicoconis lutea For both species, the isotropic voxel resolution was 0.79 mm. (Wallengren, 1871) (Aleuropteryginae) and Coniopteryx pygmaea Reconstructed image volumes were exported in DICOM format. Enderlein (1906) (Coniopteryginae). Along with this description we discuss the possible functions of the muscles associated with male 2.3. Image processing, image segmentation, and 3D visualization genital sclerites during copulation. Future studies on the function of this apparatus would be especially intriguing, both because of the Image volumes were imported into the 3D software package miniaturisation of Coniopterygidae and the morphological di- Amira 6.5 (FEI SAS, Merignac, France (part of Thermo Fisher Sci- versity of genital sclerites within the group. (2) To explore the entific™)) and filtered using a 3D bilateral filter followed by a 3D informational value of these muscles for the homologisation of Gaussian filter to reduce image noise. Overview 10X scans were male genital sclerites within the general scope of Hexapoda/ used for general analysis of abdominal organs (e.g. position of Endopterygota. Data on genitalic muscles, together with a general reproductive organs) and for counting abdominal segments. High- re-investigation of the terminal abdominal segments (including resolution 20X scans were used for analysis of genital sclerites and S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951 3 associated muscles. Both genital sclerites and muscles of the pos- (Fig. 1A). All longitudinal muscles of the eighth abdominal segment terior abdominal segments were manually segmented using the insert at the T9 ring, including the dorsal medial (Mdm) and ventral Amira 6.5. Segmentation Editor. Furthermore we segmented the medial muscles (Mvm), as well as the dorsal lateral (Mdl) and reproductive organs (seminal vesicle and ejaculatory duct) and the ventral lateral (Mvl) muscles (Fig. 1E). digestive tract as they provide relevant anatomical landmarks for the interpretation of genital sclerites. Image segmentation of gen- 3.1.2. Male genital sclerites ital sclerites was cross-checked and validated by stereomicroscopic Five sclerotized structures are associated with the posterior examination. After segmentation, respective structures were visu- abdominal segments, three of which are paired and two unpaired. alized again in Amira 6.5. using a combined volume and surface The paired gonocoxites nine (gx9) are situated externally, articu- rendering approach. lating with the T9 ring at its most latero-posterior part (Figs. 1C, 2C and 3C) ventral to the ectoproct (Fig. 1A). The paired gx9 are heavily 2.4. Terminology and the extended gonocoxite hypothesis as related sclerotized and show three to four finger-shaped extensions that to Neuropterida are hollow and pointed (possibly representing gonostyli). Ventral to gx9, the paired sclerotized gonapophyses nine (gp9) articulate with Coniopterygidae (dustywings) have been treated in a general the gx9 dorso-laterally (Fig. 3C) and are fused to the S9 ventrally context by H. Aspock€ et al. (1980), U. Aspock€ and H. Aspock€ (2007), (Fig. 3A,C). On each side of the plate, a wing-like extension runs and H. Aspock€ and U. Aspock€ (2009), and the relevance of the anteriorly fusing along the midsagittal plane just ventral to the genital sclerites has recently been summarized by U. Aspock€ (2019). alimentary canal (Fig. 2E). This part is interpreted as gonocoxites 11 The extended gonocoxite hypothesis of Matsuda was described for (gx11). The S9 supports the genital sclerites of the tenth abdominal Neuropterida in a first approach by U. Aspock€ (2002), and in detail segment, which lie just dorsal to the S9 plate (Fig. 3A,C) and ventral by U. Aspock€ and H. Aspock€ (2008) and U. Aspock€ (2019). A com- to the antero-medial fusion of the gx11 (Figs. 1E,2E). Thus, S9 parison of traditionally used terminology for male genital sclerites together with gp9 and gx11 form a continuous ring around the (Tjeder, 1954, 1957; Meinander, 1972; Martins and Amorim, 2016; internal genital sclerites of the tenth segment, which include the Monserrat, 2016) with the terminology used according to the paired gonocoxites ten (gx10) and the paired, however, apically extended gonocoxite hypothesis is consistently applied throughout (terminally) fused gonapophyses ten (gp10). The gx10 are hollow the course of the present paper. Our colour code for the concerned and pointed, and they lie laterally to the gp10 (Figs. 2E and 3C). The sclerites of the genital segments is blue (segment 9), orange/red gp10 comprise two latero-anterior parts that fuse medio- (segment 10) and green (segment 11). The light colour refers to the posteriorly (Fig. 2E). The ejaculatory duct of the male reproduc- gonocoxites and dark colour to the gonapophyses. Present in- tive organs runs along the midsagittal plane. It originates from the terpretations on sclerite identity including the colour coding unpaired seminal vesicle at the height of the seventh abdominal (Figs. 1e3) constitutes a working hypothesis which is still under segment in a rather ventral position and then makes a sharp turn development. This means that the present 3D models illustrating dorsally, finally running into the fused gp10 (Fig. 1C). The opening the morphology of male genital sclerites of the two investigated of the ejaculatory duct is slightly anterior to the posterior tip of species were based on examination of the microCT scans; however, gp10 (Fig. 2E). For a summary of terminal sclerites of H. lutea see the colour coding scheme and terminology must be seen largely as Table 1. interpretation of the authors. Concerning muscle terminology, we decided to use a descriptive functional terminology, e.g. M.pro.gx10.dor for the dorsal protractor muscle of gonocoxite 10. 3.1.3. Musculature associated with male genital sclerites This musculature terminology, however, is inherently coupled to In total, eight paired muscles could be observed within or pos- the interpretation of genital sclerite identity, which also means that terior to abdominal segment nine. Six of these muscles are asso- the used muscle terminology must be seen as a working hypothesis. ciated with the genital sclerites (Table 2). The most prominent muscle is the adductor of gx9 (M.add.gx9), which originates from a 3. Results large lateral portion of the T9 ring and inserts at the gx9 close to its articulation (Fig. 4A). Its fibres run antero-posteriorly, indicating 3.1. Helicoconis lutea that contraction leads to a fast and powerful adduction. Two muscles are associated with each of the latero-anterior parts of the 3.1.1. Terminal abdominal segments terminally fused gp10. Both of them function as protractor muscles, Abdominal segments six, seven and eight have weakly sclero- moving gp10 posteriorly. The dorsal protractor of gp10 tized tergites and sternites, and each of them shows a distinct (M.pro.gp10.dor) originates from the antero-lateral rim of the T9 spiraculum as well as dorsal and ventral fields of wax glands, while ring inserting at the dorsal surface of the respective latero-anterior plicaturae, paired sacks of the abdominal segments (for more in- process of gp10 (Fig. 4B). The ventral protractor of gp10 formation see Zimmermann et al., 2009), can be found up to (M.pro.gp10.ven) originates from the antero-medio-ventral rim of segment six (Fig. 1A). The segmental musculature in these seg- the T9 ring and inserts at the ventral surface of the respective ments is well developed. For each of the segments six through latero-anterior process of gp10 (Fig. 4B). In a similar way, three eight, several prominent segmental muscles are found, (i) the muscles are associated with each of the paired gx10, again all of longitudinal dorsal medial muscles (Mdm), (ii) longitudinal dorsal them functioning as protractors. The dorsal protractor of gx10 lateral muscles (Mdl), (iii) longitudinal ventral medial muscles (M.pro.gx10.dor) originates from the medio-dorsal part of the T9 (Mvm), (iv) ventral lateral muscles (Mvl) and (v) lateral muscles ring and inserts at the dorso-lateral surface of the most anterior (Ml) (Fig. 1E). Tergite nine (T9) forms a strongly sclerotized ring part of the respective gx10 (Fig. 4C). The ventral protractor muscle lacking any wax glands (Fig. 1A). Sternite nine (S9) is a vertically of gx10 (M.pro.gx10.ven) originates from the anterior surface of S9 oriented plate that articulates with T9 ventrally (Figs. 1C, 3A and plate and inserts at the ventral surface of the most anterior part of 3C). The posterior portion of the alimentary canal (ad), i.e. the the respective gx10 (Fig. 4C). Finally, a small medial protractor rectum, reveals six rectal pads (rp). The rectum runs through the T9 muscle (M.pro.gx10.med) originates from gx11 and inserts at the ring just slightly ventral to its most dorsal portion (Fig. 1C). The dorso-medial surface of the most anterior part of the respective weakly sclerotized ectoproct (ec) lies posterior to the T9 ring gx10 (Fig. 4C). 4 S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951

Fig. 1. Comparison of lateral views of Helicoconis lutea and Coniopteryx pygmaea based on microCT scans. (A) Terminal abdominal segments and their wax glands, spiracles, plicaturae and genital sclerites in Helicoconis lutea and (B) Coniopteryx pygmaea. (C) Alimentary duct, reproductive organs and genital sclerites in Helicoconis lutea and (D) Con- iopteryx pygmaea. (E) Segmental muscles and genital sclerites in Helicoconis lutea and (F) Coniopteryx pygmaea. Abbreviations: ad alimentary duct, de ejaculatory duct, ec ectoproct, Mdm dorsal medial muscle (tergo-tergal), Mdl dorsal lateral muscle, Ml lateral muscle (tergo-sternal), Mvm ventral medial muscle (sterno-sternal), Mvl ventral lateral muscle, gp gonapophysis, gx gonocoxite, pl plicatura, Proc processus, rp rectal pad, S sternite, sp spiracle, sv seminal vesicle, T tergite, wg wax gland. S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951 5

Fig. 2. Comparison of dorsal views of Helicoconis lutea and Coniopteryx pygmaea based on microCT scans. (A) Terminal abdominal segments and their wax glands and genital sclerites in Helicoconis lutea and (B) Coniopteryx pygmaea. (C) Alimentary duct and genital sclerites in Helicoconis lutea and (D) Coniopteryx pygmaea. (E) Reproductive organs and genital sclerites in Helicoconis lutea and (F) Coniopteryx pygmaea. Abbreviations: ad alimentary duct, an anus, de ejaculatory duct, ec ectoproct, gp gonapophysis, gx gonocoxite, Mt Malpighi tubule, rp rectal pad, S sternite, sv seminal vesicle, T tergite, wg wax gland. 6 S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951

Fig. 3. Comparison of posterior views of Helicoconis lutea and Coniopteryx pygmaea based on microCT scans. (A) Terminal abdominal segments, their wax glands and genital sclerites in Helicoconis lutea and (B) Coniopteryx pygmaea. (C) Alimentary duct and genital sclerites in Helicoconis lutea and (D) Coniopteryx pygmaea. Abbreviations: ad alimentary duct, an anus, ec ectoproct, gp gonapophysis, gx gonocoxite, S sternite, T tergite, wg wax gland.

In addition to the muscles associated with genital sclerites, two Fields of wax glands are observable up to segments six and seven. more pairs of muscles were observed. One of them serves as a There are no plicaturae (as generally in Coniopteryginae). Seg- dilator for the rectum (M.dil.rec), originating from the posterior- ments six and seven possess a full set of segmental muscles dorsal rim of the T9 ring and inserting at the lateral wall of the (Fig. 1F). The eighth abdominal segment shows several signs of rectum. The second muscle also originates from the posterior- reduction. As such, it lacks wax glands (Fig. 1B) and the vertical dorsal rim of the T9 ring and inserts at the weakly sclerotized lateral (tergo-sternal) muscles (Ml) (Fig. 1F). The tergite of the cuticle of the ectoproct close to the articulation of gx9. We desig- ninth segment is part of the weakly sclerotized ectoproct. In nated it the ectoproct muscle (M.ect). contrast, sternite nine (S9) is a strongly sclerotized broad half- ring shaped plate (Fig. 1B, D, 1F, 3D). The rectum has six rectal 3.2. Coniopteryx pygmaea pads. The ventral medial (Mvm) and ventral lateral (Mvl) muscles of the eighth segment insert at the sclerotized S9 (Fig. 1F). The 3.2.1. Terminal abdominal segments dorsal medial muscles (Mdm) of the eighth segment insert at the In this species, the seventh abdominal segment is the last that ectoproct (ec). shows a distinct spiraculum. The spiraculum on the eighth abdominal segment is reduced or at least vestigial, so it could not 3.2.2. Male genital sclerites be observed in our microCT images (Fig. 1B). Tergites and ster- The paired external gonocoxites nine (gx9) articulate with the nites of segments six, seven and eight are weakly sclerotized. dorsal edge of S9. The gx9 have a plate-like dorsal part and a hollow S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951 7

Table 1 Summary of terminal sclerites observed in Helicoconis lutea and Coniopteryx pygmaea.

sclerite Helicoconis lutea Coniopteryx pygmaea S9 (sternum segment 9) Vertically oriented plate that Strongly sclerotized broad ventrally articulates to the T9 half-ring shaped plate that ring and dorsally is fused to the dorsally articulates with the paired gp9. S9, gp9 and gx11 gx9. are fused to a ring around the internal genitalia (gx10 and gp10).

gx 9 (gonocoxites segment 9) Paired and multifurcate, Paired, articulate with S9. articulate with the T9 ring. Not Ventrally fused to the gp9. fused to any other sclerites.

gp 9 (gonapophyses segment 9) Paired, articulate with gx9. The Paired and fused ventro- gp9 are ventrally fused to S9 medially. Dorsally fused with and dorsally fused to gx 11. the gx9. Connected to gx10.

gx 10 (gonocoxites segment 10) Paired. Ventrally supported by Paired and fused posteriorly. the S9, and lateral to gp10. Not Connected to gp9. Support parts fused to any other sclerites. of the ejaculatory duct.

gp 10 (gonapophyses segment 10) Paired but apico-medially Fused/tubular and adjacent to fused. Ventrally supported by gx10. Supports the most distal the S9 medial to the paired part of the ejaculatory duct and gx10. Supports the ejaculatory bears the genital pore. duct and bears the genital pore.

gx 11 (gonocoxites segment 11) Unpaired/fused, forming a absent small bridge dorsal to the gp10. Fused to the paired gp9. Need validation by histology or confocal microscopy.

and pointed ventral part (which may represent the gonostylus). The plate-shaped part of gx9. Fibres of the M.add.gx9 run vertically gx9 are fused with the paired gonapophyses nine (gp9), which are (i.e. dorso-ventrally). Paired dorsal and ventral protractors of the ventrally connected (Figs. 1D and 3D). This bridge supports the gx10 are present. The dorsal protractor (M.pro.gx10.dor) origi- genital sclerites of the tenth segment, which are tightly integrated nates from the plate-shaped portion of gx9 (Fig. 5C)andinserts into a single functional unit. The paired gonocoxites ten (gx10) are on the dorsal surface of the anterior part of the gx10. The ventral long and slender and each of them possesses a ventrally directed protractor (M.pro.gx10.ven) originates from the medio-ventral tooth, which is connected to gp9. The gx10 are fused posteriorly in a portion of S9 and inserts at the ventral surface of the anterior V-shape (Fig. 2D). Posterior to the fusion site, a slender process part of gx10. Two muscles serve as retractors for the internal extends downward supporting the gonapophyses ten (gp10) genitalia. A dorsal retractor muscle of the gx10 (M.ret.gx10) laterally on each side (Fig. 3B,D). The gp10 are highly reduced in originates from the plate-shape portion of gx9 and inserts mainly size but form long slender elements that are fused to a hollow tube at the ventral tooth of gx10, while few fibres may also insert at with a distal opening (Fig. 3B). In total, the functional tube con- gp9. Contraction of M.ret.gx10.dor will pull the gx10/gp10 com- sisting of fused gp10 medially and gx10 laterally supports the plex anteriorly. A ventral retractor muscle originates from the ejaculatory duct, which enters the tube near the fusion site of the V- lateral portion of S9 and inserts at the ventral fusion of gp9, thus shaped gx10 (Figs. 1D and 2F) and runs through the whole tube to serving as a retractor of gp9 (M.ret.gp9) and as a consequence, its opening at the distal tip (Fig. 3D). For a summary of terminal most likely as a retractor for the whole internal sclerite appa- sclerites of C. pygmaea see Table 1. ratus. Further, a set of two pairs of dorso-medial muscles is associated with the genital sclerites. The larger of these muscles originates from the cuticle of the ectoproct and inserts at the 3.2.3. Musculature associated with male genital sclerites dorsal surface of gx10 at their fusion site, potentially serving as a In total, ten paired muscles were observed within or posterior levator muscle of the gx10 (M.lev.gx10). The smaller muscle to abdominal segment nine, and seven of those were directly connects the lateral surface of the gx10 with the gp9 associated with genital sclerites (Table 3). The most voluminous (M.inter.gp9.gx10) and may be a functional extension of the muscle is M.add.gx9. It originates from S9 and inserts to the 8 S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951

Table 2 Summary of genitalic muscles observed in Helicoconis lutea. * muscles that are unique to H. lutea in comparison to C. pygmaea.

muscle origin insertion hypothesised function comment

This is a very strong M.add.gx9 T9 gx9 adductor of gx9 muscle

M.pro.gx10.dor T9 gx10 protractor of gx10 -

M.pro.gx10.ven S9 gx10 protractor of gx10 -

Due to the small size of the gx11, the M.pro.gx10.med* gx11 gx10 protractor of gx10 muscle origin needs validation from histology.

M.pro.gp10.dor* T9 gp10 protractor of gp10 -

M.pro.gp10.ven* T9 gp10 protractor of gp10 -

Fig. 4. Musculature associated with the genital-sclerites of Helicoconis lutea. (A) Single adductor muscle of gx9. (B) Paired protractor muscles of gp10. (C) Three protractor muscles of gx10. (D) Miscellaneous muscles in abdominal segment nine. Abbreviations: ad alimentary duct, M.add.gx9 adductor muscle of gx9, M.dil.rec rectum dilator muscle, M.ect ectoproct-associated muscle, M.pro.gp10.dor dorsal protractor muscle of gp10, M.pro.gp10.ven ventral protractor muscle of gp10, M.pro.gx10.dor dorsal protractor muscle of gx10, M.pro.gx10.med medial protractor muscle of gx10, M.pro.gx10.ven ventral protractor muscle of gx10, S9 sternite nine, T9 tergite nine. S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951 9

Table 3 Summary of genitalic muscles observed in Coniopteryx pygmaea. ** muscles that are unique to C. pygmaea in comparison to H. lutea.

muscle origin insertion hypothesised function comment

gx9 (plate- This is a very strong M.add.gx9 S9 adductor of gx9 shaped part) muscle

gx9 (plate- M.pro.gx10.dor gx10 protractor of gx10 - shaped part)

M.pro.gx10.ven S9 gx10 protractor of gx10 -

gx9 (plate- M.ret.gx10** gx10 retractor of genitalia - shaped part)

gp 9 (ventr.- M.ret.gp9** S9 retractor of genitalia - med. fusion)

The function of this muscle needs further evaluation. Some M.lev.gx10** ectoproct gx10 levator of genitalia? fibres potentially continuous with M.inter.gp9.gp10.

This minute muscle needs validation by M.inter.gp9.gx10** gp 9 gx10 unknown histology or confocal microscopy.

M.lev.gx10. In fact, some fibres of the M.lev.gx10 may extend into spiracles, plicaturae, wax glands, as well as internal organs, such as M.inter.gp9.gx10. However, due to its minute size the male reproductive organs and the alimentary tract. M.inter.gp9.gx10 needs to be validated by histology. Three pairs of muscles are not related to the genital sclerites. 4.1.1. Terminal abdominal segments and male genital sclerites One is the rectum dilator (M.dil.rec) (Fig. 5E), and the other two are One of the key findings in the present study concerns the interpreted as continuations of segmental muscles. Dorsally, a number and identity of abdominal segments in the two genera. muscle originates at the boundary of tergite eight and the ectoproct Going back to the works of Tjeder (1954, 1957), the last complete as a continuation of the dorsal medial muscle of segment eight. abdominal segment of Coniopteryx was commonly interpreted as Thus, this muscle is interpreted as the dorsal medial muscle of segment nine, a convention continued by many later authors (H. segment nine (Mdm 9) (Fig. 5E). Laterally, a muscle originates at the Aspock€ et al., 1980; Monserrat, 2016). However, the present work lateral rim of the sclerotized sternite nine as a continuation of unambiguously shows that the last complete abdominal segment ventral lateral muscles. This muscle inserts posteriorly at the nar- of C. pygmaea is segment eight, and it lacks wax glands and paired row cuticle and is interpreted as a ventral lateral muscle of segment spiracles (Fig. 1B), as previously pointed out and discussed by nine (Mvl 9) (Fig. 5D). Meinander (1972) for the genus Coniopteryx and the subfamily Coniopteryginae, respectively. This has relevance for the inter- pretation of the identity of male genital sclerites, providing a 4. Discussion strong argument for the interpretation of the unpaired ventral sclerite (external hypandrium sensu Tjeder (1954, 1957)) as ster- 4.1. Findings of the present study nite of the ninth abdominal segment (Fig. 1B, D, F). In H. lutea the eighth abdominal segment shows both wax glands and a pair of In the present study we provide the first three-dimensional spiracles, furthermore the ninth segment is represented by a investigation of male genital sclerites and associated muscles for heavily sclerotized ring as pictured in many previous works two members of the family Coniopterygidae based on X-ray (Tjeder, 1957; Meinander, 1972,H.Aspock€ et al., 1980). The identity microCT imaging. In addition, we depict several other morpho- of this sclerotized ring remains somewhat speculative at this point, logical traits of the posterior abdominal segments, including leaving two possible scenarios: (i) the ring is formed by 10 S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951

Fig. 5. Musculature associated with the genital-sclerites of Coniopteryx pygmaea. (A) Single adductor muscle of gx9. (B) Paired retractor muscles of the internal genital sclerite complex. (C) Paired protractor muscles of gx10. (D) and (E) miscellaneous muscles associated with genital sclerites or segment nine. Abbreviations: ad alimentary duct, M.add.gx9 adductor muscle of gx9, M.dil.rec rectum dilator muscle, M.inter.gp9.gx10 muscle connecting gp9 and gx10, M.lev.gx10 levator muscle of gx10, Mvl9 ventral lateral muscle of segment nine, Mdm9 dorsal medial muscle of segment nine, M.pro.gx10.dor dorsal protractor muscle of gx10, M.pro.gx10.ven ventral protractor muscle of gx10, M.ret.gp9 retractor muscle of gp9, M.ret.gx10 retractor muscle of gp10, S9 sternite nine. amalgamation of tergite nine and sternite nine, or (ii) the ring is Next to the internal sclerites is a prominent pair of external formed only by tergite nine, which extends and fuses ventrally. We sclerites (gonarcus sensu Tjeder (1954, 1957); Meinander (1972))in currently favour the latter explanation, interpreting the sclerotized C. pygmaea and H. lutea,(Figs. 1e3). These sclerites are interpreted ring as tergite nine and the unpaired, vertically oriented sclerite as gonocoxites of the ninth abdominal segment. In H. lutea they that articulates with the ring as sternite nine (Fig. 3C). This hy- articulate with the sclerotized tergite nine ring, while in C. pygmaea pothesis is supported both by topological (ventral plate associated they articulate with sternite nine. In both species, they might fulfila with abdominal segment nine) and structural (large plate carrying prehensile function during copulation (see discussion of muscles bristles) similarities between H. lutea and C. pygmaea and other below). The pointed and hollow tips of these sclerites (one single, members of the Coniopterygidae. downwards directed tip in C. pygmaea (entoprocessus sensu Tjeder When unravelling the homologies in the other genital sclerites, (1957))(Fig. 1D); multiple tips in H. lutea (Fig. 2C)) may have been the internal sclerites associated with the ejaculatory duct provide derived from the gonostyli of the ninth abdominal segment, but an excellent starting point. In most Neuropteran lineages, two pairs this remains speculative at the current point. Hypothetically, the of internal sclerites are present (Tjeder, 1954, 1966, Meinander, gonocoxites nine of C. pygmaea even represent a composite sclerite 1972,H.Aspock€ et al., 1980, 1991, U. Aspock€ and H. Aspock,€ 2008), of tergite nine, gonocoxite nine and gonostylus nine. This inter- both of which may fuse. Due to the wealth of data concerning the pretation would be supported by the fact that the dorsal protractor structure and position of these two pairs of internal sclerites among of gonocoxites ten (M.pro.gx10.dor) originates of the plate-shaped different genera of coniopterygids, the homology of these two part of gx9 in C. pygmaea and from T9 in H. lutea.InC. pygmaea the sclerite pairs is comparatively well supported. The outer pair gonocoxites nine furthermore carry an anteroventrally oriented (parameres sensu Tjeder (1954, 1957)) is prominent and robust in processus that fuses medially (this condition represents an evolu- C. pygmaea and H. lutea,(Figs. 1e3), but only in C. pygmaea is it tionary novelty) and is connected to a tooth of the gonocoxites ten. fused (Fig. 2D). These paired sclerites are interpreted as gonocoxites This processus is interpreted as gonapophysis nine (Fig. 1D). of the tenth abdominal segment. The inner pair of sclerites (penis However, this interpretation remains at this point highly specula- sensu Tjeder (1954, 1957)) is fused in both species. It is V-shaped tive. Similarly, the paired dorsal extensions, which arise from and comparatively robust in H. lutea (Fig. 2E), while it is distinctly sternite nine in H. lutea (Fig. 3C), articulate with the gonocoxites slender in C. pygmaea (Fig. 3B). In both species it accommodates the nine (Fig. 1C) and extend further dorsomedially (Fig. 2E), are ejaculatory duct (Figs. 1 and 2), representing the terminal tip of the interpreted as gonapophyses nine. As with gonostyli nine, inter- copulatory apparatus bearing the genital pore, thus homology of pretation of gonapophyses nine is purely speculative at this point. these parts is comparatively safe to establish. These sclerites are Finally, we hypothesise that the small bridge connecting the interpreted as gonapophyses of the tenth abdominal segment. gonapophyses nine in H. lutea, which is situated dorsal to the S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951 11 internal genital sclerites of segment ten, may be the fused gon- 4.2. Functional considerations ocoxites of the eleventh abdominal segment (Fig. 2E, gx11). In a similar position, sclerites were reported for the conioperygid The morphology of the genital sclerites shows similarities and genera Conwentzia Enderlein, 1905, Flintoconis Sziraki, 2007, and clear differences between the two investigated species, likewise Brucheiser Navas, 1927 (U. Aspock,€ 2019) as well as for other neu- regarding the functional interpretation of the sclerites. The two ropterids (U. Aspock€ and H. Aspock,€ 2008). These sclerites have internal paired sclerites (gx10 and gp10) clearly support the ejac- been previously interpreted as gonocoxites 11, and thus we adopted ulatory duct and thus very likely fulfil an important function in this interpretation also for Helicoconis. copulation. In H. lutea both the gx10 and gp10 are equipped with a set of prominent protractor muscles which moves these structures posteriorly, and possibly also independent from each other. Inter- 4.1.2. Musculature estingly, no retractor muscles are found, thus the retraction In both examined species, the largest observable muscle is mechanism is obscure. In C. pygmaea, the condition is different. associated with the external sclerites, which we interpret as Most importantly, the gp10 are very thin, lie between gx10 and are gonocoxites nine (gx9). In H. lutea, the course of the muscle's fi- incapable of independent movements, due to lack of muscles bres is horizontal, while in C. pygmaea it is vertical (Figs. 4A and attaching to the gp10. As such, protractors of the copulatory 5A). In both species, fibre course indicates that the muscle func- apparatus insert only at the gx10, and no muscles are associated tions as a strong adductor of the gx9 (M.add.gx9). The origins of with gp10. Notably, two large muscles insert at the connection of the muscle are either at tergite nine ring (H. lutea, Fig. 4A)or gp9 and gx10, potentially providing a mechanism for muscular sternite nine (C. pygmaea, Fig. 5A), indicating that the muscle is retraction of the internal genitalia. Broad similarities are found in most likely of extrinsic origin. If this hypothesis is correct and the the large muscles associated with gx9 between the two species. M.add.gx9 is an extrinsic muscle, this would provide support for These are powerful muscles adducting the gx9, which may function the hypothesis that these sclerites are actually derived from the as grasping tools ensuring prehension during copulation. While the gonocoxites of the ninth abdominal segment. Regarding the in- gx9 in H. lutea has multiple tips and is adducted in a horizontal ternal sclerites, which we interpreted as gonocoxites of segment fashion, the large single apex of gx9 in C. pygmaea is downwardly ten (gx10), three associated muscles were found in H. lutea and oriented and adducted in a vertical movement. In both species, an two in C. pygmaea. In both species these muscles function as antagonist is lacking. Presumably, abduction/extension of gx9 oc- protractors which convey the gx10 posteriorly. In H. lutea, a dorsal curs by changes in hemolymph pressure. Ultimately, behavioural protractor originates from the tergite nine ring and a ventral observations based on high-speed cinematography would be protractor from the sternite nine (Fig. 4C), thus indicating that needed to clarify the exact movements of male genital sclerites these may also be derived from extrinsic muscles and at least during copulation. partly corroborate the hypothesis that these sclerites were derived from gx10. The third, medial, protractor originates from the 4.3. Miniaturisation sclerotized bridge, which we interpret as gx11 (Fig. 4C). In C. pygmaea, a dorsal and a ventral protractor of the gx10 are The phenomenon of miniaturisation in Coniopterygidae is present (Fig. 5C). While the ventral protractor originates from substantial and has been discussed recently based on the adult sternite nine, the dorsal protractor originates from gx9. This may head structure in C. pygmaea (Randolf et al., 2017) and two species reveal a derived condition, since a sclerotized tergite nine is not of Aleuropteryx (Randolf and Zimmermann, 2019). These studies present and thus insufficient mechanical support would be pro- concluded that miniaturisation caused modifications in some or- vided for protraction of the copulatory apparatus. As an alternative gans of the head, while no modifications in number and complexity explanation, as hypothesised in 4.1.1., tergite nine may be amal- of head muscles were observed. Coniopterygids show no striking gamated with gx9 (and potentially also with gonostylus 9) to signs of miniaturisation with respect to the genital sclerites. On the form the dorsal plate-shaped part of this sclerite where the contrary, the complexity and diversity of these sclerites is signifi- M.pro.gx10.dor originates (Fig. 5C). cant and extraordinary compared to other Neuroptera, e.g. Hem- While the muscles associated with gx9 and gx10 are similar, erobiidae (U. Aspock€ and H. Aspock,€ 2008). Similarly, no obvious there are enormous differences between the remaining muscles reduction due to miniaturisation can be found in genitalic muscles. associated with the genital sclerites, reflecting the highly diver- In a recent study, Boudinot (2018) described nine muscles as gent evolutionary scenarios that took place in the two species. In belonging to the groundplan of Neuropterida (three extrinsic, six H. lutea, two more muscles are observed, both associated with the intrinsic) and five muscles for a member of the Neuroptera (Myr- internal sclerites, which we interpret as gonapophyses ten (gp10). meleontidae). The present study concluded that six muscles are Both of them originate from the tergite nine ring and serve as associated with the genital sclerites in H. lutea and seven in protractors of the fused and V-shaped gp10 (Fig. 4B). Interpreta- C. pygmaea. tion of this condition is intricate, since in C. pygmaea the gp10 are markedly reduced in size and have no associated muscles. In 4.4. Homology of the genitalic muscles and genital sclerites within C. pygmaea, four more muscles are observed. Two comparatively the general scope of Hexapoda/Endopterygota large muscles originate from the sternite nine and gx9 and insert at the connection between gp9 and gx10. Together, these two Concerning genitalic muscles, we found clear differences be- muscles may serve as comparatively strong retractors for the tween the two studied species, which mainly relates to the fact that copulatory apparatus (Fig. 5B). However, our present data do not the two species studied in the present paper are highly diverse allow us to interpret these muscles as originally extrinsic or concerning their genital sclerites. As such, it was not a big surprise intrinsic. Additionally, two small muscles are found, one con- to find clear distinctions in genitalic muscles as well. These differ- necting gp9 with gx10, and the other extends from gx10 dorsally, ences can be mostly explained by the reduction of the gp10 (penis potentially serving as a levator for the copulatory apparatus sensu Tjeder (1954, 1957))ofC. pygmaea to form a minute, fused (Fig. 5D). Again, the current state of data prohibits any interpre- sclerite. This element is functionally integrated into the gx10 tube tation of these muscles, whether they are of extrinsic or intrinsic and lacks muscle attachments, a situation dramatically different to origin. H. lutea, where the terminally fused gp10 are large and equipped 12 S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951 with a set of prominent dorsal and ventral protractor muscles. Due classification within the family. In the past, one of the most popular to this highly derived condition of internal genitalia in C. pygmaea, complaints/excuses has been that KOH macerated specimens have we hypothesise that the musculature of H. lutea is much closer to no muscles left which would have been the crucial elements for the groundplan of Coniopterygidae. solving problematic homologisation. In the present study we pro- Compared to data recently presented for Neuropterida and vided first data on musculoskeletal system in the male genitalia in Neuroptera by Boudinot (2018), some cautious interpretations on two representative members of the Coniopterygidae. As such, the the homology of H. lutea muscles within the general scope of present study should be seen as proof that microCT is suitable for Hexapoda can be formulated. First, the M.add.gx.9 reported here unravelling complex morphology of the muscles associated with may be homologous to the M. adductor exopodalis/stylalis genital sclerites even for these minute specimens. Genitalic mus- (IXAxad)ofBoudinot (2018). Second, it seems likely that the two cles may provide valuable data for phylogenetic comparisons once protractors of the gp10 (M.pro.gp10.dor, M.pro.gp10.ven) are ho- more representatives have been studied. Forthcoming microCT mologous to the M. promotor penialis dorsalis (IXAppd) and M. studies on Coniopterygidae including also Brucheiserinae may shed promotor penialis ventralis (IXAppv) of Boudinot. Since the IXAppv light on both inter- and intra-coniopterygid relationships. was not part of Boudinot's inferred groundplan of Neuropterida, this groundplan may be updated to include this muscle. Third, the 5. Conclusions and outlook dorsal protractor of gx10 (M.pro.gx10.dor) may be homologous to Boudinot's M. lateropenitis magnus (IXAlm), while the ventral One of the main aims of the present study was exploring the protractor of gx10 (M.pro.gx10.ven) may be homologous to the M. informational value of genitalic muscles of H. lutea and C. pygmaea lateropenitis parvus (IXAlp). If this interpretation is correct, this for the homologisation of male genital sclerites in Coniopterygidae. would imply retention of these muscles in Coniopterygidae, while We found that genitalic muscles in these two species are highly parallel losses of these muscles may have occurred in Megaloptera diverse, mirroring their different genital sclerite morphologies. and the remainder of Neuroptera (Boudinot, 2018). Finally, a ho- Muscles of H. lutea could be cautiously integrated within the gen- mology between our M.pro.gx10.med and Boudinot's M. later- eral scope of genitalic muscles of Hexapoda/Endopterygota openitis (IXAl) seems possible, but rather speculative at this point. (Boudinot, 2018), while C. pygmaea shows a highly derived muscle Due to the highly derived situation in C. pygmaea, no attempts are configuration. Relating to the specific questions formulated in the made here to draw any conclusions on muscle homologies within introduction, we conclude that our data on muscles (i) corroborate the general scope of Hexapoda. It will require more data on geni- an interpretation of the external sclerites as gx9 (or composite talic muscles based on broader sampling of coniopterygid genera to sclerites from T9 þ gx9 þ gonostylus 9) and suggest a homology get a clearer picture of the variability of genitalic muscles in this between the M.add.gx.9 reported here and the IXAxad reported by group in order to draw sound conclusions on their homologies. Boudinot, (ii) corroborate the interpretation of internal genital Suggested muscle homologies corroborate the interpretation of sclerites as appendages of the tenth segment, showing that muscles the external sclerites as appendages of abdominal segment nine of the internal genitalia of H. lutea are similar to that of other (either gonocoxite 9 or a composite sclerite from tergite Hexapoda and thus supporting a homologisation of the gp10 and 9 þ gonocoxite 9 þ gonostylus 9). Furthermore, they suggest that gx10 of Coniopterygidae with penis and lateropenites of other the internal gx10 (parameres sensu Tjeder (1954, 1957)) are ho- Hexapoda (Boudinot, 2018), and (iii) provide strong support that mologous with the lateropenites sensu Boudinot (2018), and that the external hypandrium (Tjeder, 1954, 1957) represents the ster- the internal gp10 are homologous with the penis sensu Boudinot nite of abdominal segment 9. However, our data (iv) provide no (2018). The interpretations of the internal genitalia as tenth new evidence on highly modified sclerites (e.g., gp9, gx11) leaving segmental appendages also receive considerable support. Klass and their interpretation at this point speculative. We conclude that a Matushkina (2018) recently presented their comprehensive hy- broader taxon sampling adding data on genitalic muscles of a larger potheses on the early evolution of the genitalia in based on a number of coniopterygid genera will be necessary to improve our thorough analysis of the exoskeleton of the male genital region in understating of evolutionary modifications in coniopterygid geni- Archaeognatha. They concluded that the penis sclerite is formed by talia and their muscles. Future studies may further elucidate the parts of the limbs of segment ten and considered that the gona- groundplan of coniopterygid genitalic muscles, thus potentially pophyses ten would be a plausible candidate. Boudinot (2018) also clarifying even intricate questions concerning genital sclerite explicitly accepted that the penis is derived from tenth segmental homologies. appendages. Previous interpretations of the large external sclerite (hypan- Author statement drium) of Coniopterygidae, which traditionally has been inter- preted as fused gonocoxites/coxopodites (Tjeder, 1954; Meinander, Stephan Handschuh: Investigation; Methodology; Visualiza- 1972), should be revised in consideration of the present findings tion; Writing - original draft, Ulrike Aspock€ : Conceptualization, (see discussion in 4.1.1.). The traditional interpretation is severely Investigation, Project administration, Writing - original draft. questioned by the fact that the last complete abdominal segment in Coniopteryx is segment eight. Thus the data presented here clearly Acknowledgements favour interpreting the external hypandrium as the sternite of the ninth segment (Fig. 1B). Grateful thanks are due to Dr. Axel Gruppe, Freising, Germany, for providing the specimen of Helicoconis lutea, to Dr John Plant 4.5. Phylogenetic implications (Madison, Connecticut) for linguistically checking and critically reading the manuscript, and to Univ.-Prof. Dr Horst Aspock€ To date, the genital sclerites of almost all described species of (Vienna) for constructive review of the manuscript and many Coniopterygidae, estimated at approximately 580, have been fruitful discussions. Finally, we thank two anonymous reviewers for depicted based on KOH macerated specimens. Irrespective of this their substantial contributions, which significantly helped to tremendous knowledge and the utilisation of this trait for phylo- improve this manuscript. This research was supported using re- genetic comparisons, severe conflicts arise regarding the position of sources of the VetCore Facility (Imaging) of the University of Vet- the Coniopterygidae within the order Neuroptera, and the erinary Medicine Vienna. S. Handschuh, U. Aspock€ / Arthropod Structure & Development 57 (2020) 100951 13

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