Arthropod Struct Dev 2014 Jaoszyski P

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ASD593_proof ■ 17 October 2014 ■ 1/22

Arthropod Structure & Development xxx (2014) 1e22

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Evolution of a giant intromittent organ in Scydmaeninae (Coleoptera: Staphylinidae): Functional morphology of the male postabdomen in Mastigini Q1

ski a, *, Yoko Matsumura b, Rolf G. Beutel b Paweł Jałoszyn a b

Museum of Natural History, Wrocław University, Wrocław, Poland Institut für Spezielle Zoologie and Evolutionsbiologie, FSU Jena, Jena, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 June 2014 Received in revised form 23 September 2014 Accepted 26 September 2014 Available online xxx

We compared the postabdominal architecture of Mastigini with extremely long (Stenomastigus) or short (Palaeostigus) aedeagus. A novel mode of copulation was discovered: males of Stenomastigus insert a paramere between the female's abdomen and elytra, and the intromission is stabilized by several structures in both sexes. The intrinsic aedeagal mechanism is indicated as responsible for inflating the endophallus, and the long flagellum does not penetrate the ductus spermathecae during copulation. The structure of the flagellum suggests that it is primarily responsible for the sperm transfer. Asymmetrical postabdominal rotators of the aedeagus were only found in Stenomastigus; they presumably facilitate the withdrawal of the genitalia. Their origin as bunches separated from larger muscles is postulated. We discuss a scenario in which the evolution of elongated genitalia was facilitated by the lack of structural constraints and existing preadaptations. Benefits of stabilizing the copulation and intromission are indicated as the driving force for the evolution of extremely long aedeagi, while the short aedeagi might have the advantage of freedom of movements facilitating the initiation of copulation by males. Disruptive selection is suggested as a working hypothesis to further investigate mechanisms that have played a role in the evolution of genital structures of Mastigini. © 2014 Published by Elsevier Ltd.

Keywords: Histology 3D reconstruction Anatomy Aedeagus Mastigini Copulation

1. Introduction The phenomenon of the 'hyper elongation' of insect genitalia is usually discussed in the context of specific intromittent structures of the male reproductive system, e.g., the flagellum in various beetle families (e.g., Gack and Peschke, 2005; Matsumura et al., 2013), the distiphallus in Tephritidae flies (Eberhard, 2005), the processus gonopori in Lygaeidae bugs (Gschwentner and Tadler, 2000), the virga of earwigs (Kamimura, 2000, 2005), the ‘flagellum’ or ‘elongated tube’ of Zoraptera (Mashimo et al., 2013; Matsumura et al., 2014), and others (summarized by Matsumura and Yoshizawa, 2012). Such elongated sclerotized structures have various functions, from sperm transfer to removal of a rival sperm from the female reproductive tract, and may play a role in cryptic female choice (e.g., Rodriguez, 1995; Gschwentner and Tadler, 2000; Kamimura, 2000; S. Naomi, pers. comm). Sexual selection

* Corresponding author. Museum of Natural History, Wrocław University, Sienkiewicza 21, 50-335 Wrocław, Poland. ski). E-mail address: [email protected] (P. Jałoszyn

mechanisms are broadly accepted to explain the genital diversification, and the evolution of enlarged and often complex intromittent organs can be attributed to post-insemination selection processes (e.g., Eberhard, 1985; Hosken and Stockley, 2004). A growing body of evidence shows correlations between the observed variation in male genital morphology and insemination/ fertilization success (e.g., House and Simmons, 2003; Hotzy and Arnqvist, 2009; Polak and Rashed, 2010). Most of the published studies deal with behavioral and anatomical specializations associated with the elongated genitalia. Recently questions related to storing the large genitalia in a limited space of abdomen and precise manipulations during the copulation (i.e., insertion and withdrawal) without tangling or breakage were discussed (e.g., Gack and Peschke, 2005; Matsumura et al., 2014). Anatomical constraints may have a profound effect on the available character change scenarios, and therefore preadaptations to evolve hyper elongated genitalia must play an important role in the diversification of genital traits. Despite the enormous diversity of male genital structures (especially, in the context of this study, the aedeagi in beetles), the function and basic mechanisms of insertion and withdrawal remain

http://dx.doi.org/10.1016/j.asd.2014.09.006 1467-8039/© 2014 Published by Elsevier Ltd.

ski, P., et al., Evolution of a giant intromittent organ in Scydmaeninae (Coleoptera: Staphylinidae): Please cite this article in press as: Jałoszyn Functional morphology of the male postabdomen in Mastigini, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.09.006

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ski et al. / Arthropod Structure & Development xxx (2014) 1e22 P. Jałoszyn

simple. The intromission and sperm transfer is a primary function of the intromittent organ, and only in exceptional cases the female genitalia take over a part of this task, as in the recently described female ‘penis’ and male ‘vagina’ of Neotrogla, a genus of Prionoglarididae barklice (Yoshizawa et al., 2014). The insertion and withdrawal seems to be universally based on an increased hemolymph pressure (insertion) and muscle contraction (withdrawal), independently of the length of genitalia (Verma and Kumar, 1972; Dallai et al., 1997; Matsumura and Yoshizawa, 2010; Matsumura et al., 2014). The architecture of postabdomen is crucial to understand the functional morphology of male genitalia. This problem, although frequently emphasized already since the classical work of Sharp and Muir (1912), is challenging, and in Coleoptera only a few authors attempted a detailed morphological analysis of the postabdomen. The most important and complete studies are those by Hieke (1966) dealing with Carabidae, Geotrupidae and Curculionidae; Verma and Kumar (1980) on Cerambycidae and Chrysomelidae; Krell (1996) on Scarabaeidae, and, most recently, Hünefeld et al. (2011), who characterized in detail the postabdomen of the ancestral archostematan beetle Tetraphalerus bruchi. A comprehensive study of postabdominal membranous structures in beetles by Wanat (2007) also contributed to the highly fragmentary knowledge of the evolution of male genitalia. Data on intrinsic aedeagal muscles (if present) can also be found in some of these studies. The most suitable subjects to study the diversification processes leading to extreme elongation of male genitalia are arguably lineages comprising closely related taxa differing significantly in the length of the intromittent organs. This approach yielded a wealth of interesting data in the case of criocerine leaf beetles that include forms with and without the flagellum and helped to explain how novel structures evolved from a simple groundplan (Matsumura and Yoshizawa, 2012). Within Coleoptera, another suitable group is the tribe Mastigini of the ant-like stone beetles (Staphylinidae: Scydmaeninae). In this case, however, the internal aedeagal structures seem similar throughout the tribe, while in two otherwise extremely similar and closely related genera, Stenomastigus Leleup and Palaeostigus Newton, the entire aedeagus differs strikingly in ski, length (Leleup, 1968; Bordoni and Castellini, 1973; Jałoszyn 2012a,b,c). Mastigini is one of the basal lineages within Mastigitae, a relatively small and distinct group within the large and diverse Scyd ski, 2012d). They are relatively maeninae (Staphylinidae) (Jałoszyn large beetles as compared to other Scydmaeninae, reaching nearly 1 cm in length. All species are wingless with the elytra fused along suture, and live in dense local populations in the Mediterranean subregion and in South Africa (Leleup, 1968; Bordoni and Castellini, ski, 2012d). Moreover, these are diurnal predacious 1973; Jałoszyn beetles actively patrolling not only loose layers of leaf litter, but also running on the soil or moss surface and climbing high grasses, bushes and trees (observed by the first author in South Africa). The monophyly of Mastigini is strongly supported by an exceptional ski, 2012d), number of 12 unique character changes (Jałoszyn among others a strong asymmetry of the aedeagus (Fig. 1), with the copulatory piece and the distal portion of the endophallus permanently everted and the flagellum coiled into several loops inside the median lobe (Fig. 1AeC). The aedeagi in Mastigitae are typically rather large in relation to the body length, and the parameres are long and largely fused with the dorsal wall of the median lobe, except for their free apical portions (Fig. 1AeH). In the majority of Mastigitae, as well as in Scydmaenini (the sister group of Mastigitae), the aedeagus is symmetrical or nearly symmetrical (Fig. 1DeH), while in Mastigini one paramere is always distinctly shorter than the other or even entirely reduced, and the median lobe is asymmetrical, with its capsular part containing an

extremely long flagellum (Fig. 1AeC). External structures of Stenomastigus, Mastigus and Palaeostigus, three components of the Mastigini lineage, are extremely similar and in recent phylogenetic ski (2012d) relationships between these reconstructions of Jałoszyn genera remained unresolved. Only Stenomastigus and Palaeostigus are well-defined and certainly monophyletic genera; Mastigus shows some intermediary characters (also in the aedeagus), which is problematic due to a large variability of the external and genital structures, and this genus requires a comprehensive revision (dis ski, 2012a,c). Stenomastigus and Palaeostigus cussed in Jałoszyn differ externally only in the shape of the mesoventral intercoxal process, the general body shape (Stenomastigus more slender and with longer appendages) and the dorsal microsculpture (Stenomastigus with distinct microgranulation), but their aedeagi are strikingly different. In Stenomastigus (Fig. 1A) the aedeagus is extremely elongated and slender, with one paramere rudimentary or missing, and the apex of the membranous inflatable endophallus located near the median region of the rigid long paramere. The entire aedeagus is as long or even longer than the abdomen and pterothorax together. Consequently, in some species the apical portion of the long paramere is permanently projecting outside from the male abdomen even in repose (Fig. 2A, arrow), while the aedeagal base rests near the anterior margin of the mesothorax ski, 2012a,b). In Palaeostigus (Fig. 1C), the (Leleup, 1968; Jałoszyn aedeagus is short and entirely contained within the abdomen posterior to segment VIII, the short paramere is always distinct, and the apex of endophallus is located near the apex of the long paramere or distinctly protrudes distally beyond the paramere (Leleup, 1968; Bordoni and Castellini, 1973). The significant difference in the length of the entire copulatory organ and the position of the permanently everted endophallus in relation to the rigid and immovable long paramere must have a profound effect on the mating and copulation. However, while studying these beetles in their natural environment and in photographs taken by participants of the 2013 South African expedition of the University of Wrocław, the first author surprisingly observed that the mating position in Palaeostigus and Stenomastigus appears identical, and the extremely long aedeagus of the latter genus during copulation seemed nearly entirely (except for a short basal portion) inserted into the female abdomen (Fig. 2B). Moreover, when collecting Stenomastigus using a beating net, males just uncoupled from females were observed with the entire aedeagus projecting from the abdomen (Fig. 2C). These observations prompted the present study. To understand the evolution of enlarged or variously armed male genitalia a multidisciplinary approach is required, with morphological, behavioral and genetic studies focused on adaptive functional anatomy, structural and behavioral factors modulating the reproductive success (male and female strategies to gain control over the mating process; cryptic female choice; sperm competition et cet.), the population spacial distribution, sex and genetic structure and phylogenetic reconstructions (e.g., Hosken and Stockley, 2004; Soulier-Perkins, 2002; Hotzy and Arnqvist, 2009; Richmond et al., 2012; Hotzy et al., 2012). The present study deals with the first of these aspects, the functional morphology of the short (Palaeostigus) and long (Stenomastigus) aedeagus in the context of the intromission, postabdominal architecture and endophallus mechanics in Mastigini. To our best knowledge, no other such closely related and morphologically similar genera so profoundly differing in the length of the entire aedeagus (and not only in its internal structures, as in some Chrysomelidae (Matsumura and Yoshizawa, 2010, 2012; Matsumura et al., 2013) or Zoraptera (Matsumura et al., 2014)) exist in the Coleoptera. Mastigini offer a unique opportunity of a deeper insight into the evolution of short vs. long male genitalia and closely associated problems of the internal architecture of the abdomen as a whole


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ski, 2012d). Above trees exemplars aedeagi in abparameral view are shown Fig. 1. Two alternative and equally supported hypotheses of the phylogeny of Mastigitae (after Jałoszyn ski (A), Mastigus spinicornis (Fabricius) (B), Palaeostigus micans Leleup (C); Papusus macer Casey (D); Leptomastax sp. (E), for each genus: Stenomastigus berlinafricanus Jałoszyn ski, Hlavac & Nomura (G); and Leptochromus agilis (Sharp) (H). Abbreviations: cp, copulatory piece; eph, permanently Ablepton treforti Frivaldszky (F); Clidicus aliquantulus Jałoszyn everted distal portion of endophallus; fg, flagellum; pm, paramere.

and specifically the postabdomen. We address four questions in this study: i) how works the copulation in Mastigini with the short and long aedeagus (e.g., what is the position of structures participating in the process, stabilizing or facilitating the intromission); ii) how is the extremely long aedeagus contained within the abdomen

and thorax of males (to clarify problems related to the available space for development of such a large internal organ and possible morphological constraints); iii) what is the postabdominal architecture of Mastigini, with special focus on the musculature related to the aedeagus movements; and iv) how does the intrinsic


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Fig. 2. Stenomastigus longicornis. A, male with permanently projecting tip of paramere (indicated by arrow); B, mating couple; C, male shortly after copulation in the process of retracting aedeagus.

mechanism of endophallus inflation and movements function in an aedeagus with extremely long and coiled flagellum. 2. Materials and methods 2.1. Taxa examined Adults of the following Mastigini (Staphylinidae, Scydmaeninae) were included in the study: Stenomastigus longicornis (Boheman, 1851) (Doreen Clark Nat. Res., KwaZulu-Natal, South Africa, leg. P. _ ski, R. Ruta, M. Wanat & K. Zuk); Jałoszyn Palaeostigus palpalis (Latreille, 1804) (Genilla Riv. valley, Cordoba, Spain, leg. A. Castro & ski); Palaeostigus micans Lelup, 1968 (Wilderness NaP. Jałoszyn tional Park, West Cape, South Africa, leg. R. Ruta & M. Wanat) (all preserved in the collection of Natural History Museum, University of Wrocław, Poland). 2.2. Methods Couples of P. micans found in copula were killed in hot 75% ethanol; dissections in ethanol were made to asses the size of the inflated endophallus and the degree of flagellum extension. The maximum inflation of the membranous endophallus was assessed in isolated aedeagi placed in glycerol for 15 min and then in hypotonic distilled water. The position of the aedeagus in copulae of S. longicornis was inferred from field observations and photographic documentation. Males of S. longicornis and P. palpalis with aedeagus in repose were preserved and stored in FAE (3.4 vol. 35% formalin, 1 vol. acetic acid, 6.7 vol. ethanol) or in 75% ethanol.

Female genitalia were dissected from 75% ethanol-preserved specimens (S. longicornis) or beetles freshly killed with ethyl acetate (P. palpalis). They were stained with a glycerol solution of chlorazole black. For scanning electron microscopy (SEM), specimens were macerated in a warm 10% NaOH solution until soft tissues were removed, washed thoroughly, disarticulated, dehydrated, sputter-coated with gold and observed under an EVO LS 15 scanning electron microscope (Carl Zeiss, Germany). Habitus images were taken with a Nikon Coolpix 4500 camera mounted on a Nikon Eclipse 1500 stereoscopic light microscope (Nikon, Tokyo, Japan); translucent structures in temporary (ethanol or water) or permanent (Canada balsam) transparent mounts were photographed with a KY-F75U (JVC, Japan) camera mounted on a Leica M205 C microscope. Image stacks were processed using COMBINE ZP (Hadley, 2010). For transmission electron microscopy (TEM), an ethanol-preserved male of S. longicornis was dissected and its aedeagus was dehydrated in an acetone series and embedded in Epon 812 (Serva, Heidelberg, Germany). Ultrathin (70e90 nm) cross-sections were cut with a Reichert Ultracut ultramicrotome, contrasted with uranyl acetate and lead citrate (Reynolds, 1963) and examined with a Zeiss EM 900 transmission electron microscope. For three-dimensional (3D) reconstructions of selected structures, one male of S. longicornis and one of P. palpalis (both preserved in FAE) were dehydrated, embedded in Araldite CY 212 (Agar Scientific, Stansted, England) and cross-sectioned at 1 mm using a microtome HM 360 (Microm, Walldorf, Germany) equipped with a diamond knife. Serial sections were stained with toluidine blue and pyronin G (WaldeckGmbH and Co.KG/Division Chroma, Münster, Germany). Sections were digitalized using a light


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microscope (Zeiss Axioplan, Germany) equipped with a camera (PixeLink Capture OEM). The images were aligned, outlined and transferred into surface objects with Amira 4.1.2 software (Visage Imaging, Berlin, Germany). The data files were then transferred to MAYA7 (Alias Wavefront, Toronto/Ontario, Canada) in order to refine the 3D images by using the smoothing functions and rendering options of this software. Final image adjustments were made in Corel PhotoPaint. Measurements of the flagellum and ductus spermathecae were carried out using photographs of partly fragmented flagella dissected from the aedeagus and intact ducti spermathecae using the curve measuring tool of Auto-Montage Pro v. 5.03.0061 software (Synoptics Ltd., U.K.). Muscle nomenclature follows that of Hünefeld et al. (2011), with novel muscles numbered with an asterisk. Names of muscles refer to their inferred results of contractions during retracting the aedeagus. The term ‘aedeagus’ is used to describe the entire complex composed of the median lobe, parameres, copulatory piece, endophallus with flagellum and internal muscles, delimited by the insertion site of the proximal connecting membrane to the rim surrounding the basal aedeagal foramen. As the aedeagus in Mastigini is twisted around its longitudinal axis in the abdomen the traditionally used terms “dorsal”, “ventral” and “lateral” may be misleading. For clarity, the following nomenclature was adopted: a parameral view shows the aedeagus with the basal foramen facing upward; an abparameral view shows the opposite side; left lateral view: aedeagus with long paramere facing upward; right lateral view: aedeagus with long paramere facing downward. 3. Results 3.1. Sex dimorphism in Palaeostigus and Stenomastigus (Figs. 3AeF and 13A) Palaeostigus (Fig. 3AeB) and Stenomastigus (Fig. 3CeF) are very similar in their external features. Males of Palaeostigus (Fig. 3A) are smaller and more slender than females and the distal portions of protibiae are slightly curved inwards. In females (Fig. 3B) the elytra are similar in shape to those of males and similarly evenly convex, with the apices separately rounded. The only noticeable difference is a pair of indistinct, shallow impressions in the anterior third of each elytron near the suture, which is present in females and absent in males. However, this character is scarcely recognizable in some females of P. palpalis (not noticeable in the specimen showed in Fig. 3B). In Stenomastigus (Fig. 3CeD), males (Fig. 3C) are also smaller and more slender than the females and they have longer antennae. They have distinctly emarginated and curved distal internal portions of the protibiae, enlarged protrochanters (Fig. 13A) and evenly convex elytra. In females (Fig. 3D) the elytra are distinctly differing from those of males. The sutural region is distinctly raised, especially in the subapical roof-shaped area, with a pair of elongate and often deep impressions on the anterior half. The elytra in posterior view (Fig. 3F) show the median convexity bordered at either side by an elongate anterior impression, and in lateral view (Fig. 3F) a weakly curved dorsal profile from the base to the subapical region, where the elytra are rapidly lowering toward their apex. In dorsal (Fig. 3D) and posterior (Fig. 3E) views, a long apical median gap between the elytra is visible. The height of the median raised area, the depth and length of lateral impressions, the lateral elytral profile and the width and length of the apical gap between each elytron show some degree of variation among females from the same population. Besides individuals with these features distinctly developed (as the one in Fig. 3DeF), females with weakly raised suture, indistinct impressions and a shallower gap between elytral apices can be found.

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3.2. Male abdomen and postabdomen e skeletal structures (Figs. 4AeJ and 5AeF) In males of both Palaeostigus and Stenomastigus the externally visible abdominal venter is composed of seven sternites (IIIeIX) (Figs. 4A and 5A). In both genera the abdominal sternite III (i.e., the first visible one) is broadly and firmly fused ventrally with the metaventrite between the metacoxae, and the metendosternite (metafurca) has broadly separated arms, each adjacent to the mesal margin of the metacoxa (Figs. 4B and 5B). Likewise in both genera tergite IX is composed of lateral hemitergites each with a long anteriorly directed tergal apodeme (Figs. 4C and 5C). In males of Palaeostigus (Fig. 4A) abdominal sternite VIII is distinctly emarginate posteriorly. The emargination does not reach half length of the sternite and forms a nearly equilateral triangle. The aedeagus (Fig. 4C) is entirely contained within segments VIIIeIX and twisted around its longitudinal axis so that the basal foramen is lateroventrally oriented. Hemitergites IX are not visible in ventral view, while sternite IX is largely exposed. The aedeagus (Fig. 4D) is suboval with a slightly asymmetrical median lobe, strongly asymmetrical parameres and an elongate copulatory piece with an asymmetrical and permanently everted distal portion of the endophallus. The abparameral wall of median lobe bears a large oval membranous area (diaphragm), while its remaining parts are heavily sclerotized and rigid. The copulatory piece (Fig. 4E) is also heavily sclerotized, equipped with a blunt and short subapical tooth and inserted eccentrically in the oval basal membrane. The apical portion of the copulatory piece is confluent with permanently everted endophallus (Fig. 4D), but in transparent mounts the border between the heavily sclerotized copulatory piece and membranous endophallus is sharply marked (Fig. 4I). In specimens preserved with the aedeagus in repose (Fig. 4F) the endophallus is folded along a complex system of lines, while in males preserved during copulation (Fig. 4I) and dissected thereafter the endophallus is inflated and forms an elongated sac with two finger-like projections. The same degree of inflation was obtained when isolated aedeagi were placed under hypotonic conditions. The flagellar opening (or gonopore) (Fig. 4IeJ) is located subapically on the inflated endophallus, on the apex of an elongate conical projection. The endophallic surface is irregularly covered with fine microtrichia locally forming dense patches (Fig. 4G). The median lobe contains the flagellar loops in its basal capsular part (Fig. 4IeJ). No noticeable variation in the general shape of aedeagus was found within the studied populations of Palaeostigus palpalis and P. micans. In Stenomastigus (Fig. 5AeF) skeletal abdominal and aedeagal structures are essentially similar to those observed in Palaeostigus. Differences can be seen in the emargination of sternite VIII, the length and shape of the aedeagus, and in the condition of the parameres. Abdominal sternite VIII (Fig. 5A) is deeply notched medially; the emargination is as long as 2/3 of the length of the sternite, narrow and its margins are distinctly raised (i.e., projecting ventrally). Segments IXeX are more elongate than those of Palaeostigus, and the extremely long and slender aedeagus is much longer than the abdomen and pterothorax combined. In the specimens shown in Fig. 5AeC the aedeagus and segments IXeX were shifted posteriorly during preparation; in living (Fig. 2A) males only the parameral apex is permanently projecting outside the abdomen and the aedeagal base rests between the mesofurcal arms near the anterior margin of the mesothorax. Postmortem (killed with ethyl acetate or liquid preservatives) the contraction of the abdomen usually causes a posterior shift of the aedeagus. The aedeagus (Fig. 5EeF) is less sclerotized than in Palaeostigus and the short paramere is entirely reduced, while the long paramere is extremely elongated and darkly sclerotized at its permanently projected


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Fig. 3. Palaeostigus palpalis (A, B) and Stenomastigus longicornis (CeF). Male in dorsal view (A, C); female in dorsal (B, D) and lateral (F) view; and elytra of female in posterior view (E). Arrows showing anterior elytral impressions, posterior gap, raised elytral suture and roof-like shape of elytra. Scale bars ¼ 1 mm.

apical portion. The capsular basal part of the median lobe (Fig. 5EeF) is long and slender, and the rim surrounding the basal foramen is as narrow as the median notch of sternite IX. The copulatory piece is equipped with a large subapical tooth (Fig. 5EeF) directed toward the paramere, and the endophallus is

located near the submedian region of the paramere. Some variability in the shape and length of the entire aedeagus and especially in the shape of the copulatory piece and paramere were found within and between several isolated populations of S. longicornis.


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Fig. 4. Palaeostigus palpalis (AeH), and P. micans (IeJ). A, abdominal sternites and posterior portion of metaventrite of male in ventral view; B, metafurca in dorsal view; C, abdominal segments IXeX with aedeagus in ventral view; D, aedeagus in abparameral view; E, base of copulatory piece; F, permanently erected and folded distal portion of endophallus in abparameral view; G, details of erected endophallus, abparameral view; H, flagellum inside partly dissected median lobe; IeJ, aedeagus in right lateral view with distal portion of permanently extruded endophallus fully inflated (obtained by dissection of a couple preserved in copula). Abbreviations: aed, aedeagus; apo9, tergal apodeme IX; bm, basal membrane; cop, conical projection of endophallus; cp, copulatory piece; ed, ejaculatory duct; eph, permanently everted distal portion of endophallus; fg, flagellum; fgo, flagellar opening; ht9, abdominal hemitergite IX; lmfa, lateral metafurcal arm; ma, membranous area; ml, median lobe of aedeagus; pm, paramere; st3e9, abdominal sternite IIIeIX; v3; metaventrite. Scale bars: AeD, IeJ ¼ 200 mm; EeH ¼ 20 mm.

3.3. Male abdomen and postabdomen e internal organs (Figs. 6AeH and 7AeD) Internal organs of Stenomastigus were reconstructed 3dimensionally, while those of Palaeostigus were studied using digitalized and aligned stacks of serial cross-sections. The aedeagus of Stenomastigus (Fig. 6AeB) is contained in a membranous pouch divided in its middle region by a telescopic fold into a proximal and distal portion (here referred to as a connecting membrane; possibly homologous with ‘pretegminal membrane’ of Wanat, 2007). The proximal part is connected to the rim surrounding the basal aedeagal foramen, the distal part ventrally to sternite IX and dorsally to tergite X. The aedeagus with its

connecting membrane (Fig. 6CeH) is surrounded by tightly packed internal organs; small remaining spaces between organs are filled with spongy parenchyma and diffused streaks of the fat body. The large hindgut (Fig. 6C, EeF) widens posteriorly were it opens into a spacious dorsal rectum. Close to its pre-rectal loop large clusters of fat bodies (Fig. 6C, E) are present. The large paired and asymmetrical testes (Fig. 6EeG) are ventrally placed, together with a pair of large accessory glands (Fig. 7EeG; closely adjacent and therefore reconstructed as a single solid structure), and short vasa deferentia (Fig. 6H). The latter are connected to a long ejaculatory duct (Fig. 6CeD, H) which extends as a loop from the ventral to the dorsal region of the abdomen and forms a distinctly thickened and elongate sperm pump (Fig. 6CeD, H) before entering the aedeagus


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Fig. 5. Stenomastigus longicornis. A, abdominal sternites and posterior portion of metaventrite of male in ventral view; B, pterothorax and abdomen with aedeagus in dorsal view (aedeagus artificially shifted posteriorly during preparation, in intact specimens its base is rested between mesofurcal arms near anterior margin of mesothorax); C, abdominal segments IXeX with aedeagus in dorsal view; D, distal portion of paramere in ventral view; E, aedeagus in abparameral view; F, aedeagus in left lateral view. Abbreviations: aed, aedeagus; apo9, tergal apodeme IX; bc, basal capsule of median lobe; bf, basal foramen; cp, copulatory piece; ed, ejaculatory duct; eph, permanently everted distal portion of endophallus; fg, flagellum; fgo, flagellar opening; ht9, abdominal hemitergite IX; ms, membranous area; msf, mesofurca; mtf, metafurca; pm, paramere; sat, subapical tooth of copulatory piece; st3e9, abdominal sternite IIIeIX; t8, 10, abdominal tergite VIII, X; v3, metaventrite. Scale bars ¼ 200 mm.


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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 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 61 62 63 64 65



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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 Fig. 6. Stenomastigus longicornis, thoracic and abdominal organs, schematic view (A, B) and 3D-reconstructions (CeG). A, skeletal structures of male postabdomen, horizontal 114 section in dorsal view; B, same, sagittal section in lateral view; C, internal organs and terminal abdominal segments in dorsal view; D, same but with intestine and fat bodies 115 removed; E, internal organs and terminal abdominal segments in lateral view; F, same, sagittal section with fat bodies removed; G, internal organs and terminal abdominal 116 segments in ventral view; H, same but with testis and accessory glands removed. Abbreviations: 3*, M. sinister urotergo-distomembranalis (posterioris); 5*, M. sinister urotergo117 distomembranalis (anterioris); 7*, M. urotergo-distomembranalis (posterioris); 8*, M. urotergo-distomembranalis (submedialis); 9*b, branch b of M. urotergo-distomembranalis (anterioris); 12*, M. urotergo-distomembranalis (medialis); 14*, M. tergoapodemo-proximomembranalis (posterioris); 15*, M. tergoapodemo-distomembranalis (medialis); 16*, 118 M. tergoapodemo-distomembranalis (posterioris); 27, retractor of distal connecting membrane (not named); 28, M. tergoapodemo-phallobasicus major; acg, accessory glands; aed, 119 aedeagus; apo9, tergal apodeme IX; cns, posterior central nervous system; cp, copulatory piece; dcm, distal connecting membrane; ed, ejaculatory duct; eph, permanently everted 120 distal portion of endophallus; fb, fat body; hg, hindgut; ht9; hemitergite IX; pcm, proximal connecting membrane; pm, paramere; spp, sperm pump; st9; sternite IX; t10, tergite X; Q2 121 tst, testis; vd, vasa defferens. Scale bar ¼ 200 mm. 122 123 124 1*: not named (and not reconstructed). O. (¼ origin): anterior through the basal foramen. The ventral nerve cord forms a single 125 region of tergal apodeme VIII; I. (¼ insertion): anterior region of undivided ganglionic mass in the pterothorax and abdomen. 126 tergal apodeme IX; F. (¼ function): retractor of segment IX. The postabdomen of Stenomastigus is equipped with a complex 127 2*: not named (and not reconstructed). O.: anterior region of system of 18 muscles (Figs. 6CeH and 7AeD; summarized in Table 1), 128 tergal apodeme VIII; I.: middle region of tergal apodeme IX; F.: in and posterior to segment IX and outside of the aedeagus (the 129 retractor of segment IX. muscularis of internal organs is not treated as separate muscles here): 130 ski, P., et al., Evolution of a giant intromittent organ in Scydmaeninae (Coleoptera: Staphylinidae): Please cite this article in press as: Jałoszyn Functional morphology of the male postabdomen in Mastigini, Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.09.006

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Fig. 7. Stenomastigus longicornis, 3D-reconstructions of postabdominal skeleton and muscles. A, right lateral view; B, right dorsolateral view; C, dorsal view; D, same but symmetrical dorsal muscles removed. Abbreviations: 3*, M. sinister urotergo-distomembranalis (posterioris); 4*, M. sinister urotergo-distomembranalis (medialis); 5*, M. sinister urotergo-distomembranalis (anterioris); 7*, M. urotergo-distomembranalis (posterioris); 8*, M. urotergo-distomembranalis (submedialis); 9*a, branch a of M. urotergodistomembranalis (anterioris); 9*b, branch b of M. urotergo-distomembranalis (anterioris); 12*, M. urotergo-distomembranalis (medialis); 14*, M. tergoapodemoproximomembranalis (posterioris); 15*, M. tergoapodemo-distomembranalis (medialis); 16*, M. tergoapodemo-distomembranalis (posterioris); 27, retractor of distal connecting membrane (not named); 28, M. tergoapodemo-phallobasicus major; apo9, tergal apodeme IX; bf, basal foramen of aedeagus; cp, copulatory piece; dcm, distal connecting membrane; eph, permanently everted distal portion of endophallus; pcm, proximal connecting membrane; pm, paramere; st9, sternite IX; t10, tergite X. Scale bar ¼ 200 mm.


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ski et al. / Arthropod Structure & Development xxx (2014) 1e22 P. Jałoszyn Table 1 Postabdominal muscles (on and behind segment IX) in Stenomastigus longicornis and Palaeostigus palpalis compared to Tetraphalerus bruchi (after Hünefeld et al., 2011). No Name

Stenomastigus Palaeostigus Tetraphalerus

1* 2* 3*

Present Present Presenta

Present Present Absent

Absent Absent Absent

Presenta

Absent

Absent

Presenta

Absent

Absent

Present Present

Present Absent

Absent Absent

Present

Present

Absent

Present

Unknownf

Absent

Present

Present

Present

Present

Present

Present

Present

Present

Absent

Present

Present

Present

Present

Present

Present

Present

Present

Absent

Present

Present

Absent

Presentb Presentc

Presentb Presentc

absent Absent

Absent Absent

Absent Absent

Present Present

Presentd Presente

Presentd Presente

Present Present

Absent

Absent

Present

Absent

Absent

Present

Absent

Absent

Present

4* 5* 6* 7* 8* 9* 10 11 12* 13 14*

15* 16*

M. sinister urotergodistomembranalis (posterioris) M. sinister urotergodistomembranalis (medialis) M. sinister urotergodistomembranalis (anterioris) M. urotergo-rectalis M. urotergo-distomembranalis (posterioris) M. urotergo-distomembranalis (submedialis) M. urotergo-distomembranalis (anterioris) M. antecosta-antecostalis uronotum medialis VIII M. antecosta-antecostalis uronotum lateralis VIII M. urotergo-distomembranalis (medialis) M. antecosta-antecostalis urosterni VIII M. tergoapodemoproximomembranalis (posterioris) M. tergoapodemodistomembranalis (medialis) M. tergoapodemodistomembranalis (posterioris)

17* 18* M. phallobaso-endophallicus (basalis) 21 M. urotergo-sternalis IX 24: M. urotergo-phallicus (medialis) 27 28 M. tergapodemo-phallobasicus major 46: M. phallobasophalloapodemalis 51 M. phalloapodemoendophallicus (dorsalis) 62 a

Unpaired, only on the left side. b Composed of multiple fibers. c Distally bifurcate. d Homology with Tetraphalerus uncertain; shifted from sternite IX to connecting membrane? e Homology with Tetraphalerus uncertain. f Possibly hidden among other muscle fibers, impossible to separate on sections.

3*: M. sinister urotergo-distomembranalis (posterioris) (Fig. 7CeD). O.: anterior region of tergite IX, laterally; I.: middle region (dorsolaterally) of distal connecting membrane; F.: rotator of aedeagus. 4*: M. sinister urotergo-distomembranalis (medialis) (Fig. 7CeD). O.: anterior region of tergite IX, laterally; I.: middle region (dorsolaterally) of distal connecting membrane; F.: rotator of aedeagus. 5*: M. sinister urotergo-distomembranalis (anterioris) (Fig. 7CeD). O.: anterior region of tergite IX, laterally; I.: posterior region (dorsolaterally) of distal connecting membrane; F.: rotator of aedeagus. 6*: M. urotergo-rectalis (not reconstructed). O.: middle region of tergite IX, submedially; I.: posterior end (dorsally) of rectum; F.: unclear, possibly dilator of rectum. 7*: M. urotergo-distomembranalis (posterioris) (Fig. 6CeD, C). O.: anterior margin of tergite IX, dorsolaterally; I.: posterior

11

region (dorsally) of distal connecting membrane; F.: retractor of distal connecting membrane. 8*: M. urotergo-distomembranalis (submedialis) (Figs. 6D and 7C). O.: anterior margin of tergite IX, subdorsolaterally; I.: submedian region (dorsolaterally) of distal connecting membrane; F.: retractor of distal connecting membrane. 9*: M. urotergo-distomembranalis (anterioris) (Figs. 6D and 7C). O.: anterior margin of tergite IX, subdorsolaterally; I.: branch 9*a: middle region (dorsolaterally) of distal connecting membrane, branch 9*b: anterior region (dorsally) of distal connecting membrane; F.: possibly rectractor of distal connecting membrane. 10: M. antecosta-antecostalis uronotum medialis VIII (not reconstructed). O.: anterior region (submedially) of tergite VIII; I.: anterior margin (submedially) of hemitergite IX; F.: retractor of segment IX. 11: M. antecosta-antecostalis uronotum lateralis VIII (not reconstructed). O.: subanterior region of tergite VIII; I.: anterior margin (sublaterally) of hemitergite IX; F.: retractor of segment IX. 12*: M. urotergo-distomembranalis (medialis) (Fig. 7A, C). O.: anterior margin of tergite IX, subdorsolaterally; I.: submedian region (dorsolateral) of distal connecting membrane; F.: retractor of distal connecting membrane. 13: M. antecosta-antecostalis urosterni VIII (not reconstructed). O.: subanterior region (submedially) of sternite VIII; I.: intersegmental membrane VIIIeIX (ventrally); F.: retractor of segment IX. 14*: M. tergoapodemo-proximomembranalis (posterioris) (Figs. 6CeE, G and H, 7AeB). O.: base of tergal apodeme IX (mesally); I.: posterior region (right: dorsolaterally; left: ventrolaterally) of proximal connecting membrane; F.: retractor of aedeagus (via proximal connecting membrane). 15*: M. tergoapodemo-distomembranalis (medialis) (Figs. 6GeH and 7AeB). O.: subapical region of tergal apodeme IX (mesoventrally); I.: median region (lateroventrally) of distal connecting membrane; F.: retractor of distal connecting membrane. 16*: M. tergoapodemo-distomembranalis (posterioris) (Figs. 6CeD and 7AeD). O.: subapical region of tergal apodeme IX (mesoventrally); I.: posterior region (dorsolaterally) of distal connecting membrane; F.: retractor of distal connecting membrane. 27: not named (Fig. 7AeD). O.: base of tergal apodeme IX (mesally); I.: posterior region (laterally) of distal connecting membrane; F.: retractor of distal connecting membrane. 28: M. tergoapodemo-phallobasicus major (Figs. 6CeD, H and 7AeB). O.: apex of tergal apodeme IX (mesally); I.: submedian region (dorsally) of proximate connecting membrane; F.: retractor of aedeagus (via proximal connecting membrane). The internal architecture of the thorax and abdomen of Palaeostigus is essentially similar to that of Stenomastigus, except for voluminous spongy parenchyma tissue and diffused fat body lobes filling large spaces between loosely arranged internal organs around and anterad the short aedeagus with its enclosing connecting membrane. However, significant differences were found in the musculature of postabdomen. Three asymmetrical muscles: 3* (M. sinister urotergo-distomembranalis (posterioris)), 4* (M. sinister urotergo-distomembranalis (medialis)) and 5* (M. sinister urotergo-distomembranalis (anterioris)), all rotators of the aedeagus, are missing in Palaeostigus. Additionally, the retractor of the distal part of the connecting membrane, muscle 7* (M. urotergodistomembranalis (posterioris)) is also missing, and the presence of 9* (M. urotergo-distomembranalis (anterioris)) is unconfirmed.


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The latter muscle is possibly present but obscured by surrounding muscle fibers and not identifiable unambiguously on sections. 3.4. Aedeagus e internal structures (Figs. 8AeF, 9AeD, 10AeE, 11AeB) Internal components of the aedeagus of both genera were studied in transparent preparations and on digitalized and aligned stacks of serial sections; the aedeagus of Stenomastigus was additionally reconstructed 3-dimensionally. Details of the flagellar pouch were examined using TEM. In Stenomastigus (Figs. 8AeF, 9AeD, 10AeE, 11AeB) the ejaculatory duct enters the aedeagus through the basal foramen and connects to the flagellum in the median part of the capsular portion of the median lobe (Figs. 8AeF). The flagellum forms five broad oval loops (Figs. 8EeF, 9AeC, 10B) and extends along the copulatory piece to enter the permanently everted membranous endophallus (Figs. 8AeC, 9D, 10E), where it opens on top of a conical projection

(Fig. 9D). The flagellum in its distal portion (in middle of copulatory piece) is distinctly thicker than in the loops, while the diameter is smallest near the gonopore (Table 2). The flagellum has solid and smooth cuticular walls (Fig. 11AeB) and is surrounded by a twolayered flagellar pouch (Figs. 8AeF, 9AeC, 10BeE, 11A). The flagellar pouch surrounds all loops as one unit (Figs. 8EeF and 9AeC) with a complicated arrangement of its membranes (Fig. 10B) and, as a narrow tube, extends up to the flagellar opening (in Figs. 8AeF and 9D the reconstruction of distal portion is omitted). The flagellar pouch membrane is continuous with the cuticle of the endophallus and both are covered with microtrichia (the endophallus externally, and the flagellar pouch internally along the entire length); the microtrichia inside the flagellar pouch are mostly bent and oriented along the longitudinal axis of the flagellum (Fig. 11AeB). The internal membrane of the flagellar pouch within the flagellar loops is continuous with massive and rigid internal sclerotizations firmly connecting the loops in their center (Fig. 10B). Two intrinsic aedeagal muscles were found:

Fig. 8. Stenomastigus longicornis, 3D-reconstruction of aedeagal skeleton and musculature. A, parameral view; B, left lateral view; C, parameral view; D, right lateral view; E, basal capsule in parameral view; F, basal capsule in left lateral view. Abbreviations: 17*, aedeagal membranous area retractor (not named); 18*, M. phallobaso-endophallicus (basalis); bf, basal foramen of aedeagus; cp, copulatory piece; ed, ejaculatory duct; eph, permanently everted distal portion of endophallus; fg, flagellum; fgo, flagellar opening; fgp, flagellar pouch; ma, membranous area; pm, paramere; sat, subapical tooth of copulatory piece. Scale bars: AeD ¼ 200 mm; EeF ¼ 100 mm.


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13

Fig. 9. Stenomastigus longicornis, 3D-reconstruction of flagellar loop region (AeC) and distal portion of endophallus (D). A, parameral and slightly lateral view; B, left lateral view; C, right lateral view; D, left lateral view. Abbreviations: 18*, M. phallobaso-endophallicus (basalis); cp, copulatory piece; ed, ejaculatory duct; eph, permanently everted distal portion of endophallus; fg, flagellum; fgo, flagellar opening; fgp, flagellar pouch; pm, paramare; sat, subapical tooth of copulatory piece. Scale bars: ¼ 100 mm.

17*: not named (Figs. 8EeF and 10BeC). O.: parameral wall (internally) of aedeagus; I.: abparameral wall (internally) of aedeagus: F.: retractor of membranous area. 18*: M. phallobaso-endophallicus (basalis) (Figs. 8EeF, 9AeB, 10A). O.: base (internally) of aedeagus; I.: base of endophallus. F.: retractor of endophallus. In Palaeostigus the fine structure of the aedeagus, including the musculature, does not differ from that of Stenomastigus, except for the increased length of the flagellum (Table 2), with six loops. The flagellum is also distinctly thicker, especially in its distal portion, i.e. inside the copulatory piece. In specimens of P. micans preserved in copula it was also possible to examine the extent of the relocation of the flagellar apex in the inflated endophallus (Figs. 4IeJ). The fully inflated endophallus projects distally beyond the long paramere, but is only 2e3 times longer compared to the folded position. The flagellum does not extend beyond the apex of the flagellar pouch and uncoiling or a relocation of the loops inside the median lobe are not recognizable.

sclerotized structures, but also oviducts, each with only a pair of ovarioles with extremely long and thin terminal filaments and a single large maturing egg (Fig. 12A). In both genera tergite IX is divided into hemitergites (Fig. 12B) and in repose contained inside the abdomen; the paraprocts (Fig. 12C) are fused and equipped with elongate valvifers (Fig. 12C); the bursa copulatrix (Fig. 12BeC) is membranous, short and broad, with the insertion of the ductus spermathecae located on its anterior margin (Fig. 12BeC). The ductus spermathecae is elongate and thin in both cases (Fig. 12BeC), but it is distinctly longer in P. palpalis (measurements given in Table 2). In Palaeostigus the ductus spermathecae has the largest diameter at the insertion to the bursa copulatrix; near the median line it is much thinner and again broadens directly before it connects to the spermatheca; in Stenomastigus the diameter at the proximal and distal end is comparable, but the large median portion of the duct is also thinner (and distinctly thinner than in P. palpalis) (Table 2). In both studied species the ductus spermathecae is much shorter than the male flagellum (even than its coiled portion; see Table 2).

3.5. Female genitalia of Palaeostigus and Stenomastigus (Fig. 12AeC)

3.6. Copulation and intromission in Palaeostigus and Stenomastigus (Fig. 2B and 13AeF)

The abdominal terminalia of females in both genera are similar and do not differ in any fundamental features. Since live females of P. palpalis were available, it was possible to observe not only

Copulating pairs of S. longicornis were observed in the field and shortly after catching them from grass and bushes using a beating net. When projected, the aedeagus is anteriorly directed and the


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14


Fig. 10. Stenomastigus longicornis, histological cross-sections of aedeagus and associated structures. AeE, aedeagus; F, submedian region of sperm pump; GeH, chart of sections. Abbreviations: 17*, aedeagal membranous area retractor (not named); 18*, M. phallobaso-endophallicus (basalis); bf, basal foramen of aedeagus; dcm, distal connecting membrane; ed, ejaculatory duct; eph, permanently everted distal portion of endophallus; fg, flagellum; fgp, flagellar pouch; pcm, proximal connecting membrane; pm, paramere; rbf, rim of basal foramen; scl, median sclerotization of flagellar loops; spp, sperm pump. Scale bars: ¼ 50 mm.

apex of long paramere reaches the level of prothorax (Fig. 13A). In copula (Figs. 2B and 13F), the male adopts a position on top of female (the “male-above, male abdomen flexed” position of Huber et al., 2007), with the fore legs tightly grasping the female laterally in the constriction between the prothorax and elytra while the protrochanters are resting in the elytral impressions on either side of the suture. The middle legs of the male are lifted upward, the tip of the abdomen is flexed toward the female genital opening, and contact with the ground is only maintained by the hind legs. The aedeagus appears entirely inserted into the female abdomen. However, a closer examination revealed that the major part of it including the entire long paramere, is inserted into the empty space

between elytra and abdominal tergites, and only the permanently everted distal portion of the inflated endophallus is inserted into the female genital opening (Fig. 13F). The subelytral space in females is formed by the roof-like raised median (adsutural) part and is accessible through the posterior gap between each elytron. Moreover, the median position of the inserted aedeagus is ensured by the elongate anterolateral impressions of the female elytra, which prevent the apical portion of the paramere from moving in the horizontal plane. In P. palpalis the projected aedeagus is anteroventrally directed and not reaching midlength of the abdomen (Fig. 13B). Copulating pairs of Palaeostigus (Fig. 13C) also adopt the “male-above, male


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15

Fig. 11. Stenomastigus longicornis, transmission electron micrographs of cross-sectioned flagellar loop. A, flagellum in flagellar pouch; B, internal wall of flagellar pouch with microtrichia. Abbreviations: fgl, flagellar lumen; fgp, flagellar pouch; fgw, flagellar wall; mtr, microtrichia. Scale bars: ¼ 1 mm.

abdomen flexed” position, with the male grasping the female with the fore legs between the prothorax and elytra. The aedeagus (Fig. 13CeE) remains largely visible outside (confirmed by direct observations of copulating pairs of P. palpalis and in preserved couples of P. micans, not showing any substantial differences) and only the permanently everted endophallus is inserted into the female genital opening. The apices of both parameres rest against tergite VIII of the female and the aedeagus is slightly twisted around its longitudinal axis. The distal portion of the copulatory piece with the endophallus is inserted between tergite X and hemitergite IX. The angle between the copulatory piece and the parameres is distinctly larger than in repose. In dissected couples of P. micans the conical projection of the inflated endophallus (with distal gonopore) was found close to (but not inside) the insertion of the ductus spermathecae, and the flagellum was not extended beyond the conical projection, as shown in Fig. 4IeJ. 4. Discussion One of many outstanding features of the extremely species rich Hexapoda is the unparalleled diversity of the genitalia, especially of males. Investigating evolutionary mechanisms inducing and modulating the megadiverse genital morphology in insects is still a challenging task. Various authors emphasized that these mechanisms still remain obscure and exceptionally poorly known, and problems related to the diversification of genitalia are only fragmentarily clarified (e.g., Polak and Rashed, 2010). In the current study we focused on elucidating the functional morphology of the male terminalia (postabdomen and aedeagus) in Scydmaeninae, a successful subgroup of the megadiverse rove-beetles, the largest beetle family (Grebennikov and Newton, 2009). By studying two closely related genera of Mastigini with striking differences in the length of the aedeagus, we postulate that a remarkable elongation of the genitalia can develop by relatively minor morphological transformations and might have been possible due to preadaptations that removed structural constraints present in other tribes of Scydmaeninae.

4.1. Copulation in Mastigini with short and long aedeagus e additional stabilizing structures evolved in Stenomastigus Stenomastigus with its extremely long aedeagus shows a number of differences in structural adaptations to copulation, compared to the less specialized Palaeostigus. These adaptations (summarized in Table 3) can be divided into two subsets: i) structures stabilizing the pre-intromission mounting; and ii) structures directly involved in inserting and stabilizing the intromittent organ. In males of Palaeostigus and Stenomastigus mounting on the female is stabilized by a tight grip of the fore legs around her ‘waist’, i.e., the distinct constriction between the prothorax and elytra (Figs. 2B and 13C, F). The fore legs of P. palpalis (and all species of ski, unpublished data) show only one adapPalaeostigus; Jałoszyn tation to this function: the slightly emarginated or curved protibial apex. Not only the protibiae are modified in S. longicornis. Their protrochanters are characterized by a slightly projecting and angulate distoventral margin (Fig. 13A). During mounting they are placed against the anterior longitudinal elytral impressions of the female, and the projecting part of trochanter is then secured in this recess. Both tibial and trochanteral modifications show a considerable variability in species of Stenomastigus. In seven species ski (2012a,b), the internal protibial margin is illustrated by Jałoszyn slightly (e.g., in Stenomastigus franzi Leleup) to deeply (e.g., in Stenomastigus kochi Leleup) emarginated or indented, the tibial ski) to strongly apex is weakly (e.g., Stenomastigus kosianus Jałoszyn (e.g., S. kochi) curved, and additionally in some species the proximal margin of indentation bears an angulate expansion (e.g., Stenomastigus basilewskyi Leleup) or tooth (e.g., S. kochi) that may ensure a tighter grip on the female. The male protrochanter is often subquadrate or subrectangular and an angulate distoventral margin projecting like that of S. longicornis can be present (e.g., in Stenomastigus jeanneli Leleup). In some species it forms a short rounded distoventral projection (e.g., in Stenomastigus pseudofranzi Jałos ski) or is elongated and forms a tooth- or rod-like process (e.g., zyn ski, Stenomastigus allaeri Stenomastigus berlinafricanus Jałoszyn ski, 2012a,b). All these modifications may stabilize Leleup) (Jałoszyn


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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 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 61 62 63 64 65



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Table 2 Measurements of ductus spermathecae and flagellum in S. longicornis and P. palpalis; N ¼ 3 [mm]; in parenthesis % of body length.

Length of ductus spermathecae Diameter of ductus spermathecae At insertion to bursa copulatrix In middle At insertion to spermatheca Length of flagellum Coiled part Total Diameter of flagellum In median part of loops In middle of copulatory piece At base of endophallal conical projection

S. longicornis

P. palpalis

750.36e762.97 (15.53e16.58%)

1885.32e1895.12 (36.19e38.21%)

8.08e9.34 5.41e5.78 8.09e8.43

15.10e16.34 9.12e9.97 6.11e6.34

1087.22e1375.23 (24.11e33.14%) 2083.09e2315.44 (46.19e55.79%)

2653.01e2797.12 (55.27e64.15%) 3308.36e3.587.76 (68.92e82.29%)

5.52e6.89 7.23e7.87 4.87e5.11

4.44e6.22 9.65e10.11 6.75e7.02

the mounting position during copulation by interlocking the male trochanters with the female elytral impressions. Interestingly, shallow and short anterior impressions can also be found on the elytra of females of Palaeostigus. They are usually inconspicuous in Mediterranean species (Bordoni and Castellini, 1973) and more distinct in South African ones (Leleup, 1968), but never as long and deep as those in Stenomastigus. They also mark the position of the male's protrochanters during the mounting (Fig. 13C) and even when shallow may increase the stability of the male's copulating position. However, this pre-intromission stabilization (lasting for

the entire coupling) may play a less important role in Palaeostigus compared to Stenomastigus, where the extremely elongated aedeagus requires additional mechanisms to ensure successful mating. The enormously elongated aedeagus of Stenomastigus (Fig. 13A) with its rigid and immovable paramere and the submedian endophallus, cannot be positioned during copulation as is the case of Palaeostigus with its short genitalia (Fig. 13BeE). Instead of resting the parameral apices against the exposed terminal abdominal tergites, the male Stenomastigus inserts a major part of the aedeagus into the subelytral space of the female, i.e., between the abdominal tergites and the inner surface of the elytra, a strategy not known in any other beetles. This is possible due to: i) lack of wings that normally would have been folded in this space; and ii) the presence of a subapical gap between the female elytra (Fig. 3DeE). The gap is absent in females of Palaeostigus, all other Mastigitae and also in the remaining Scydmaeninae, while the lack of wings is shared by most Mastigitae (Leleup, 1968; Bordoni and Castellini, 1973; Castellini, ski et al., 2003; O'Keefe, 2003; Jałoszyn ski, 2009, 1996; Jaoszyn 2012a,b,c and unpublished data), with the only exceptions of Leptochromus Motschulsky (O'Keefe, 2002) and possibly one species of Clidicus Laporte (O'Keefe and Monteith, 2000). During copulation, the position of the aedeagus itself is stabilized by unique structures of the male and female of Stenomastigus, which are indistinctly developed or missing in Palaeostigus. In the female of S. longicornis these are: i) the raised elytral suture (Fig. 3EeF), below which a subtriangular ‘ceiling’ forms an elongate space medially positioning the aedeagus; and ii) the elongate anterior impressions along the elytral suture (Fig. 3DeE). The apical portion of the long paramere is stabilized during copulation below the female's raised elytral suture and between these impressions,

Fig. 12. Female terminal abdominal segments and associated structures of Palaeostigus palpalis (AeB) and Stenomastigus longicornis (C). A, segments VIIeIX with associated soft tissues; B, segments VIIIeIX with sclerotized genital structures; C, paraprocts with bursa copulatrix and spermatheca. Abbreviations: ag, accessory gland; bc, bursa copulatrix; ds, ductus spermathecae; ht9, hemitergite IX; ids, insertion of ductus spermathecae; ovd, oviduct; ovr, ovariole; ppr, paraproct; rec, rectum; sp, spermatheca; st8, sternite VIII; t10, tergite X; tfil, terminal filament; vf, valvifer. Scale bars ¼ 200 mm.


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Fig. 13. Stenomastigus longicornis (A, F), Palaeostigus palpalis (B), and P. micans (CeE). Male with projected aedeagus in lateral view (A, B); mating couple (C, F) and aedeagus during copulation (D, E). Arrows showing emarginated protibial apex and angulate protrochanter. Abbreviations: cp, copulatory piece; ht9, hemitergite IX; pm, paramere; st8, sternite VIII; t10, tergite X; vf, valvifer. Scale bars: AeC, F ¼ 1 mm; DeE ¼ 0.25 mm.

and therefore its lateral movements are limited. In the male of S. longicornis, three unique structural adaptations are present: i) the general shape of the aedeagus; ii) the narrow notch of sternite VIII; and iii) the subapical tooth of the copulatory piece. The long aedeagus is narrow and when projected (Fig. 13A) its subbasal portion, and more precisely the rim around basal foramen, fits into the long and narrow emargination of sternite VIII (Fig. 5A); additionally this has raised margins forming an elongated pocket that

receives and locks the aedeagus in a fixed position, thus preventing lateral movements. In Palaeostigus this emargination is much shorter and broader (Fig. 4A). It is likely that instead of stabilizing the aedeagus it rather ensures a greater freedom of movements. It is noteworthy that within Scydmaeninae the apical emargination of ski, 2012d), sternite VIII is a unique character of Mastigitae (Jałoszyn but is reversed in taxa with the shortest aedeagus in relation to the abdomen, i.e. in Leptomastacini (Fig. 1EeF) and some species of


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Table 3 Comparison of the pre-intromission and intromission stabilizing structures of Palaeostigus and Stenomastigus. Stenomastigus

Function

Structures stabilizing pre-intromission mounting Males Emarginated or curved protibial apex Always weakly developed Protrochanters Unmodified

Structure

Palaeostigus

Usually strongly developed, in some cases with additional tooth Often subrectangular or projecting

Grasping female around her ‘waist’ Interlocking with the female elytral impressions

Females Anterior elytral impressions

Long and deep

Interlocking with the male protrochanters

Narrow Extremely elongated

Stenomastigus: interlocking with notch of sternite VIII Stenomastigus: interlocking with female's subapical elytral gap, raised elytral suture and anterior elytral impressions; clasping abdominal dorsum Stenomastigus: interlocking with the rim of basal foramen

Short and shallow

Structures stabilizing intromittent organ during copulation Males Basal foramen of aedeagus Broad Longer paramere Relatively short

Subapical tooth of copulatory piece

Broadly and shallowly emarginated, margins of emargination flat Small

Narrowly and deeply emarginated, margins of emargination raised Large

Parameral tooth

Absent

Present in some species

Palaeostiogus and Stenomastigus: anchoring copulatory piece in bursa copulatrix Stenomastigus: clasping female's abdominal dorsum Stenomastigus: probably clasping female's abdominal dorsum

Females Elytral impressions Subapical gap between elytra Elytra in cross-section

Short and shallow Absent Rounded

Long and deep Present, usually deep Subtriangular, with suture raised

Stenomastigus: locking distal portion of long paramere Stenomastigus: locking basal portion of long paramere Stenomastigus: locking distal portion of long paramere

Sternite VIII

Clidicus, also showing the least elongate aedeagi within the genus ski et al., 2003). Moreover, the emargination is only narrow (Jaoszyn and elongated in Stenomastigus, compared to the distinctly shorter and broader condition in the remaining Mastigitae. It might have evolved as a mechanism increasing the freedom of movements of the genitalia, especially to protrude and flex the abdominal segments IXeX with the projected aedeagus and reach into the female genital opening in strongly convex and elongate beetles. A further elongation of the male genitalia in the ancestor of Stenomastigus was consequently facilitated by this preadaptation, i.e., by the already existing emargination of sternite VIII. The copulatory piece in Stenomastigus is equipped with a large subapical tooth (Fig. 6EeF), a structure which is short and inconspicuous in Palaeostigus. It is directed toward the paramere and during copulation (Fig. 13F) may function as a clasping device that additionally stabilizes the aedeagus. The female's abdominal dorsum is clasped between this tooth and the paramere. An additional tooth is present on the paramere in several species of Stenomastigus, usually slightly or distinctly distad the one on the ski, copulatory piece (illustrated for seven species in Jałoszyn 2012a,b). This second tooth can provide an additional stabilization. During copulation it is directed downward, presumably pressing against the female's abdominal tergites. In Palaeostigus, the asymmetrical parameres are apparently the only stabilizing structures. They are in close contact with the abdominal tergites of the female (Fig. 13DeE), but it is also possible that their main function is sensory and not stabilization. The elongation of the aedeagus in Stenomastigus required a development of additional stabilizing devices. When the stability of mounting and copulating is considered, it seems that none of them is a true novelty, but they rather evolved as slight modifications of pre-existing structures. The emargination of the male sternite VIII, presumably ensuring a greater degree of movements and a larger range of reach of segments IXeX in the first place, gradually changed its function to locking the aedeagus in a fixed position along the body axis. The female's impressions on the elytra, initially increasing the stability of the mounting position, possibly co-

evolved with the enlarging male's protrochanters, which further stabilized the mounting. When the elongating aedeagal paramere reached a sufficient size to be inserted under the elytra and also between the elytral impressions, the function of the latter extended to stabilizing the position of the aedeagus. The peculiar insertion of the aedeagus in Stenomastigus was possible due to a pre-existing lack of wings and a slight modification of female's elytra, i.e., the development of the median gap and the roof-like shape. 4.2. Thoracic morphology facilitated evolution of elongated aedeagus In repose the extremely long aedeagus in Stenomastigus is rested along the median part of abdomen and pterothorax, between the metafurcal and mesofurcal arms. Typically, the metendosternite (¼ metafurca) is composed of a variously developed stalk and two lateral furcal arms diverging from its apex, thus forming a Y-shaped (long stalk) or nearly V-shaped (short stalk) sclerotized structure between the metacoxae (Lawrence et al., 2011). The Y- or V-shaped metendosternite constitutes a constraint for elongation of the aedeagus beyond the abdomen and into the thorax. In many groups of Scydmaeninae with a tendency to elongate the aedeagus (e.g., in Cephenniini) the male genitalia do not project beyond the anterior ski, 2011a, 2012e, 2014). abdominal margin (e.g., Jałoszyn The Mastigitae genera, except for Leptomastax Pirazzoli and ski, 2012d and Clidicus, do not have a metafurcal stalk (Jałoszyn unpublished data). The metafurcal arms are broadly separated and inserted near the mesal margins of the metacoxae (Fig. 5B). A very short and broad metafurcal stalk with broadly separated furcal arms (and metacoxae) also occurs (with few exceptions) in Scydmaenini, a tribe recognized as a sister group of Mastigitae ski, 2012d,e, 2014). In (Grebennikov and Newton, 2009; Jałoszyn other lineages of Scydmaeninae the metafurca is much more ski, 2013a), frequently Y-shaped or nearly V-shaped (e.g. Jałoszyn only rarely without a stalk and with broadly separated arms ski, (Eutheiini; Jałoszyn 2014). The staphylinid clade Euaesthetinae þ Steninae was identified as the sister group of


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Scydmaeninae (Grebennikov and Newton, 2009). In Euaesthetinae the metafurcal stalk can be absent or present (Clarke and Grebennikov, 2009) but the insertions of the furcal arms are approximate and in the majority of genera the metacoxae are contiguous or nearly contiguous (Clarke and Grebennikov, 2009). In contrast, in Steninae the metacoxae and metafurcal arms (without a stalk) are broadly separated. It is conceivable that separated metacoxae and broadly separated metafurcal arms without stalk in ski, Mastigini, a presumably basal lineage of Mastigitae (Jałoszyn 2012d), are a preserved plesiomorphic condition inherited from the common ancestor of the supertribe (with reversal in two genera). This would imply that the process of reducing the metafurcal stalk and separating the arms (possibly correlated with the broad separation of the metacoxae) took place in the stemgroup of Mastigitae, long before the elongation of the aedeagus in Stenomastigus occurred. This suggests that the structure of the metendosternite was a preadaptation that made it possible to elongate the aedeagus until its basal portion exceeded the abdominalethoracic border. Finally its apical part could rest between the mesofurcal arms and reach the anterior margin of the narrow mesothorax (Fig. 6B), providing that its shape was narrow enough to fit between the paired meta- and mesofurcal projections. 4.3. Function of the flagellum in Mastigini and mechanism of intromission and sperm transfer In unrelated groups of insects a coiled internal structure can be found in the male genital organ. In beetles it is usually called a n, flagellum (e.g., Lawrence and Britton, 1991; Lindroth and Palme 1970). The same name was applied to a structure known in Zoraptera (Mashimo et al., 2013), but clear differences in the origin, structure and function (not sperm transfer but probably removing rival sperm from the female's genitalia) were demonstrated and thus this ambiguous term was replaced by ‘elongated tube’ (Matsumura et al., 2014). Even in beetles, several scenarios of the flagellar origin were hypothesized, reflecting distinct differences in the structure and position during copulation (summarized by Wanat, 2007). It was demonstrated in criocerine Chrysomelidae and in aleocharine Staphylinidae that the primary function of the flagellum is transporting the sperm into the spermatheca (Gack and Peschke, 2005; Matsumura and Yoshizawa, 2010). In Chrysomelidae the eversion of the aedeagus from the abdomen and the eversion of the endophallus with the flagellum is primarily caused by contractions of the abdomen resulting in an increased hemolymph pressure maintained by a bundle of muscles at the base of the aedeagal median lobe (Verma and Kumar, 1972; Matsumura and Yoshizawa, 2010). The flagellum is inserted into the ductus spermathecae and reaches or nearly reaches the spermatheca; the safe withdrawal of the long flagellum is usually achieved within a few seconds by criocerine males (Matsumura and Yoshizawa, 2010), whereas in Aleochara tristis it requires an elaborate behavior and is a gradual process to prevent entangling (Gack and Peschke, 2005). The exact mechanism of the withdrawal in beetles is not fully understood. Gack and Peschke (2005) observed that the coiled condition is the relaxed state and the flagellum spontaneously retains this shape after it has been freed from the endophallic pouch. Likewise, in dissected aedeagi of Palaeostigus and Stenomastigus we observed that the coiled state is stable and independent of the central interconnecting sclerotizations (Fig. 10B) e when dissected, even fragmented and separated parts of the flagellum from the coiled region retained the same curved shape. The flagellum in Mastigini is proximally connected with the ejaculatory duct, has a lumen, and distally enters the endophallus, where it opens on top of a conical projection. The flagellar structure

19

suggests that its primary function is the sperm transfer, as in other Staphylinidae (Gack and Peschke, 2005). The ejaculatory duct is equipped with an elongated sperm pump, a structure presumably functionally associated with the sperm transfer. In beetles sperm pumps were only found in Chrysomelidae (Suzuki, 1988), Mastigi ski et al., 2003), in euaesthetine Staphylinidae (e.g., tae (e.g., Jaoszyn Puthz, 1973) which are the sistergroup of Scydmaeninae, and in the unrelated Lepiceride (Navarrete-Heredia et al., 2005). In the sperm pump occurring in Mastigini chitin filaments are running spirally around the central lumen and it is surrounded by a thick muscularis composed of spirally and longitudinally arranged muscle fibers (Fig. 10F). Although the actual role of this structure in the sperm transfer remains unstudied, it seems that changes of length and therefore the volume of the lumen may be responsible for (or at least facilitate) this process. Recently, Matsumura et al. (2013) demonstrated that in criocerine leaf beetles the flagellum originates as a protrusion of the epidermis and cuticle of the internal sac (i.e., the distal inflatable part of aedeagus corresponding to the endophallus in the present study) at the distal end of the ejaculatory duct by an invagination. Moreover, the flagellum is contained in an invaginated apex of the internal sac forming a spacious pouch in which the flagellum is stored in repose. In Mastigini, the flagellum is also contained in a membranous pouch (Figs. 8AeF, 9AeC, 10B), the wall of which is continuous with the endophallic membrane and covered with microtrichia (Fig. 11AeB). Microtrichia were also found in Chrysomelidae on the internal surface of the membrane that tightly surrounds a ‘median ejaculatory guide’ (i.e., the flagellum). It was suggested that these ‘spines’ might play a crucial role in the insertion and withdrawal of the flagellum by providing an adhesive force interacting with the membranes (Matsumura and Yoshizawa, 2010). This idea was not further developed. The arrangement of the flagellar pouch and the microtrichia in Mastigini suggest that independent of the currently available range of movements of the flagellum, the endophallic microtrichia might have been conserved during the process of gradual invagination of the endophallic epidermis and cuticle because of their presumable role in centering the flagellum in the pouch. Moreover, they prevent a direct contact with the delicate pouch membrane and thus reduce the risk of injury during intromission. Presently this hypothesis is only suggested by structural evidence and requires a functional verification. The extremely long and coiled flagellum in Mastigini is an intriguing structure, as our results show that the loops are not uncoiled during the intromission. In dissected couples of Palaeostigus micans the tip of flagellum was invariantly fixed at the end of the subapical endophallal projection (Fig. 4IeJ), only slightly more distally than in the folded endophallus in repose. A penetration of the flagellum into the ductus spermathecae to any length would be only possible (there are no length or diameter constraints) if the flagellum would be able to uncoil. However, the flagellar loops inside the median lobe in Mastigini, unlike in Aleaochara or Criocerinae, are interconnected by large median sclerotizations originating from the internal membrane of the flagellar pouch (Fig. 10B). These sclerotizations prevent the loops from uncoiling, but not the entire coiled region from a limited rotation around a perpendicular axis. The very limited extent of the flagellar movement during the intromission is likely caused by this structural constraint. The mechanism of extending (slightly) the distal portion of the flagellum in Mastigini can be inferred from the architecture of the aedeagus. The intrinsic transverse muscle 17* (Fig. 8EeF) retracts the large abparameral membranous area. The remaining walls of the median lobe are rigid and therefore the retraction reduces the volume and increases the internal pressure, unfolding and inflating the membranous endophallus. The forward movement of the


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endophallus with its conical projection causes a corresponding extension of the flagellum, which is possibly due to a limited rotation of the coiled region around the central sclerotization. The retraction requires a contraction of the longitudinal intrinsic muscle 18* (Figs. 8EeF and 9AeC). This relatively simple mechanism is apparently common in Scydmaeninae. Similar muscles ski, 2011b), even were illustrated for various genera (e.g., Jałoszyn though the membranous area is located basally in some cases (e.g., ski, 2013b) or subapically (e.g., Jałoszyn ski, 2012e), and the Jałoszyn transverse muscle may have adopted an oblique or nearly longitudinal orientation. The changes of internal pressure require that the aedeagus is tightly sealed, and indeed it is likely that the basal foramen ensures such a closure around the ejaculatory duct. When dissected aedeagi were gently squeezed with tweezers between the parameral and abparameral walls of the median lobe, the endophallus was observed inflating, which also suggests this mechanism. Moreover, such a manipulation also caused the copulatory piece to flick outward, as a consequence of inflating the basal membrane, in which this structure is eccentrically inserted (Fig. 4E). Why the flagellum became extremely elongated and coiled in Mastigini remains an open question. Presently our results do not allow a reasonably well supported interpretation. 4.4. Origins of postabdominal muscle asymmetry in Stenomastigus Our study revealed characteristic differences in the postabdominal muscle apparatus of Stenomastigus and Palaeostigus. The elongated genitalia are equipped with three asymmetrical and thin muscle bundles (3*, 4*, 5*), all presumably functioning as rotators of the aedeagus (Fig. 7D). During the retraction of the aedeagus these muscles may participate in its proper positioning within the postabdomen. These muscles were not found in Palaeostigus, a genus with short aedeagus. Biogeography suggests that Palaeostigus was branching off the ancestral line of Mastigini earlier than Stenomastigus and in the northern hemisphere, while the latter genus most likely originated during or after the dispersal of Mas ski, 2012d). tigini from Europe to South Africa (discussed in Jałoszyn Therefore, it appears likely that the asymmetrical muscles in Stenomastigus are morphological novelties, presumably ensuring a proper positioning of the extremely long aedeagus during withdrawal. Muscles 3*, 4* and 5* may have originated as groups of fibers initially forming a part of another muscle, namely 9* (Fig. 7C). This issue remains an open question and requires further study. However, it should be noted that at least within the aedeagus, the musculature may show some developmental flexibility. In criocerine leaf beetles, for instance, it was found that the musculature differs between taxa with and without flagellum and is constructed in the late morphogenetic stage, and the authors concluded that “the muscles are adjustable during the entire morphogenesis in this group” (Matsumura et al., 2013). Compared to this observation, a separation of small groups of muscle fibers on one side driven by a distinctly asymmetrical aedeagus withdrawal into abdominal segments seems a simpler process that requires relatively small transformations. Another muscle was also found in Stenomastigus, but not in Palaeostigus, the symmetrical 7* (Fig. 7C). This is one of the retractors of the distal connecting membrane that possibly assists in the retraction and positioning of the withdrawing aedeagus (it should be kept in mind that the muscles reconstructed in Figs. 6 and 7 are in repose, while when the aedeagus is fully protruded their arrangement is different and all of them are stretched and lie anterior to the aedeagus). Based on our results, it is not possible to explain the lack of this muscle in Palaeostigus. However, it can be hypothesized (to facilitate further research of this phenomenon), that the ancestral state is the presence of 7*, and that it was

conserved in Stenomastigus. In Palaeostigus a possible evolutionary trend to reduce the size of the genitalia and a related increase in the freedom of movements (see below) might have resulted in the reduction and finally the loss of 7*. 4.5. Disruptive selection of sexual characters in Mastigini? It appears likely that the stabilization of the coupling and position of the inserted aedeagus (enabled by existing preadaptations) was (and is) crucial for the copulation in Stenomastigus, while an increased movability of the aedeagus might have determined the structures found in extant Palaeostigus. The first part of this hypothesis is supported by our findings; the evolution towards increased freedom of movement in the ancestral line of Palaeostigus remains merely a speculation. The taxonomically unclear and small genus Mastigus includes species with aedeagi showing a shape and relative size intermediate between Palaeostigus and Stenomastigus, and the assumption that such a condition may be plesiomorphic bears intriguing consequences for explaining the evolution of both long and short aedeagi. Unfortunately, species of the South African genus Mastigus are very rare and so far we did not succeed in obtaining properly preserved specimens for detailed study or to make observations of the mating behavior and the insertion of the aedeagus. The structural and functional differences between Stenomastigus and Palaeostigus seem to increase the fitness of both taxa. For the males of Stenomastigus, the stabilization of the enormously elongated aedeagus may be crucial for the efficiency of fertilization and therefore structures ensuring a stable coupling were favored. Restrictions of lateral movements of the aedeagus and a stable mating position may impede disturbing and interrupting of the copulation by rivals or female's movements and ensure a greater reproductive success in dense populations, where encounters between individuals are relatively frequent. The co-evolution of males and females resulting from conflicts of interest over control of copulation and fertilization, postulated as an important mechanism of sexual selection (e.g., Rice, 1996; Holland and Rice, 1998, 1999; Chapman et al., 2003; Gavrilets et al., 2001; Pitnick et al., 2001; Moore et al., 2001; but see also critical discussion in Eberhard, 2004), may explain the mutual fit of stabilizing structures in males and females of Stenomastigus. The stabilizing adaptations in females may be beneficial in the same way as in corresponding structures in males, i.e. by an increased control over the copulation process, which is more difficult for the male with large aedeagus to initiate (thus decreasing male harassment during feeding or oviposition) and less prone to energetically costly disturbance (e.g., caused by other males during mating) (Alexander et al., 1997). In the case of Palaeostigus, the observed lack of stabilizing structures can be explained on the same evolutionary ground. The short aedeagus does not require specific stabilization mechanisms, but rather an increased freedom of movements, especially as the female is larger than the male and has strongly convex elytra (Fig. 13C). Smaller genitalia may ensure a greater reproductive success by a facilitated initiation of the copulation (the insertion of short aedeagus may be simpler and faster). Therefore the males 'win' the sex competition with females at the stage of controlling the initiation of copulation. This suggests two diametrically opposed evolutionary processes in Mastigini: i) the structural stabilization of the copulation by elongating the aedeagus and developing (co-evolving) associated and corresponding structures in males and females; this may ensure a higher efficiency of sperm transfer and decreases the chances for interruption of the copulation, thus increasing the probability of successful reproduction for both partners; ii) facilitating the copulation initialization by males, favoring a small


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aedeagus whose position does not require strict mechanical control mechanisms; some degree of movability of the inserted aedeagus may be beneficial to counteract female movements; moreover, the formation of this type in the ontogenesis is less costly. Consequently, if the ancestral condition was a medium-sized aedeagus, these diverging processes might have resulted in disruptive selection (Bolnick and Fitzpatrick, 2007) that favors either extreme short or strongly elongated genitalia, causing the ancestral line to split into the Palaeostigus- and Stenomastigus- lines. We do not develop this hypothesis further, as our morphological results do not justify such far-reaching conclusions. However, this direction of further studies (covering the behavioral and genetic aspects of sexual selection remaining beyond scope of the present work) seems promising to clarify the intriguing evolution of the extremely elongate genitalia in Mastigini. Acknowledgments Great thanks are due to Marek Wanat, Rafał Ruta and Katarzyna _ Zuk (University of Wrocław) and Ruth Müller (Ditsong National Museum of National History, Pretoria) for their invaluable support during the first author's field studies in South Africa. The field work in South Africa was possible thanks to the research permits issued by the Ezemvelo KZN Wildlife and the South African National Parks. We also thank Augustin Castro (Cabra, Spain) for his help in collecting Iberian Mastigini. Rafał Ruta took photos 2B-C and Krzysztof ski and Anna Siudzin ska (Wrocław University of EnvironKalin mental and Life Sciences, Poland) took SEM images, Rommy Peterson (FSU Jena) prepared histological sections, and TEM images were taken by Marta Mazurkiewicz-Kania in the Department of Animal Developmental Biology, University of Wrocław, Poland. This is also gratefully acknowledged. References Alexander, R.D., Marshall, D.C., Cooley, J.R., 1997. Evolutionary perspectives on insect mating. In: Choe, J., Crespi, B. (Eds.), The Evolution of Mating Systems in Insects and Arachnids. Cambridge Univ. Press, Cambridge, pp. 4e31. Bolnick, D.I., Fitzpatrick, B.M., 2007. Sympatric speciation: models and empirical evidence. Ann. Rev. Ecol. Evol. Syst. 38, 459e487. Bordoni, A., Castellini, G., 1973. Sulle specie Paleartiche del genere Mastigus Latreille con osservazioni su due specie dell'Africa Australe (Coleoptera Scydmaenidae). Redia 54, 295e323. Castellini, G., 1996. Revisione del genre Leptomastax Pirazzoli, 1855 (Coleoptera, Scydmaenidae). Atti Mus. Civ. Storia Nat. Grosseto 15, 1e137. Chapman, T., Arnqvist, G., Bangham, J., Rowe, L., 2003. Sexual conflict. Trends Ecol. Evol. 18, 41e47. Clarke, D.J., Grebennikov, V.V., 2009. Monophyly of Euaesthetinae (Coleoptera: Staphylinidae): phylogenetic evidence from adults and larvae, review of austral genera, and new larval descriptions. Syst. Entomol. 34, 346e397. Dallai, R., Del Bene, G., Lupetti, P., 1997. Fine structure of the male reproductive organs of the western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Int. J. Ins. Morphol. Embryol. 26, 97e112. Eberhard, W.G., 1985. Sexual Selection and Animal Genitalia. Harvard University Press, Cambridge, MA. Eberhard, W.G., 2004. Maleefemale conflict and genitalia: failure to confirm predictions in insects and spiders. Biol. Rev. 79, 121e186. Eberhard, W.G., 2005. Threading a needle with reinforced thread: intromission in Ceratitis capitata (Diptera,Tephritidae). Can. Entomol. 137, 174e181. Gack, C., Peschke, K., 2005. ‘Shouldering’ exaggerated genitalia: a unique behavioural adaptation for the retraction of the elongate intromittant organ by the male rove beetle (Aleochara tristis Gravenhorst). Biol. J. Linn. Soc. 84, 307e312. Gavrilets, S., Arnqvist, G., Friberg, U., 2001. The evolution of female choice by sexual conflict. Proc. R. Soc. Lond. B 268, 531e539. Grebennikov, V.V., Newton, A.F., 2009. Good-bye Scydmaenidae, or why the ant-like stone beetles should become megadiverse Staphylinidae sensu latissimo (Coleoptera). Eur. J. Entomol. 106, 275e301. Gschwentner, R., Tadler, A., 2000. Functional anatomy of the spermatheca and its duct in the seed bug Lygaeus simulans (Heteroptera: Lygaeidae). Eur. J. Entomol. 97, 305e312. Hadley, A., 2010. Combine ZP software, new version, [WWW document]. URL: http://www.hadleyweb.pwp.blueyonder.co.uk/CZP/News.htm (accessed December 2010).

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Hieke, F., 1966. Vergleichende funktionelle Anatomie der Abdominalmuskulatur einiger m€ annlicher Coleopteren unter besonderer Berücksichtigung des Genitoanalkomplexes. Deut. Entomol. Z. N.F. 13, 1e168. Holland, B., Rice, W.R., 1998. Chase-away selection: antagonistic seduction vs. resistance. Evolution 52, 1e7. Holland, B., Rice, W.R., 1999. Experimental removal of sexual selection reverses intersexual antagonistic coevolution and removes a reproductive load. Proc. Natl. Acad. Sci. U.S.A. 96, 5083e5088. Hosken, D.J., Stockley, P., 2004. Sexual selection and genital evolution. Trends Ecol. Evol. 19, 87e93. Hotzy, C., Arnqvist, G., 2009. Sperm competition favors harmful males in seed beetles. Curr. Biol. 19, 404e407. €nn, J.L., Arnqvist, G., 2012. Phenotypic engineering unveils Hotzy, C., Polak, M., Ro the function of genital morphology. Curr. Biol. 22, 2258e2261. House, C.M., Simmons, L.W., 2003. Genital morphology and fertilization success in the dung beetle Onthophagus taurus: an example of sexually selected male genitalia. Proc. R. Soc. Lond. B 270, 447e455. Huber, B.A., Sinclair, B.J., Schmitt, M., 2007. The evolution of asymmetric genitalia in spiders and insects. Biol. Rev. 82, 647e698. Hünefeld, F., Marvaldi, A.E., Müller, B., Lawrence, J.F., Beutel, R.G., 2011. The male postabdomen of the “ancestral” archostematan beetle Tetraphalerus bruchi Heller, 1913 (Ommatidae) and its phylogenetic significance. Arthr. Struct. Dev. 40, 146e158. ski, P., 2009. Clidicus crocodylus sp. nov. from Mindanao, the first repreJałoszyn sentatives of the Mastiginae from the Philippines (Coleoptera, Scydmaenidae). Genus 20, 203e207. ski, P., 2011a. Cephenniini with prothoracic glands and internal ‘cavities’: Jałoszyn new taxa, enigmatic characters and phylogeny of the Cephennomicrus group of genera (Coleoptera, Staphylinidae, Scydmaeninae). Syst. Entomol. 36, 470e496. ski, P., 2011b. Discovery of Eutheiini (Coleoptera: Staphylinidae, ScydmaeJałoszyn ninae) in Australia, with implications for phylogeny and biogeography of Paraneseuthia Franz. Eur. J. Entomol. 108, 687e696. ski, P., 2012a. Stenomastigus Leleup (Staphylinidae, Scydmaeninae): status Jałoszyn of subgenus Acanthostigus Leleup and revision of species with elongated male protrochanters. Zootaxa 3153, 39e56. ski, P., 2012b. Stenomastigus pseudofranzi sp. n., from South Africa (StaphJałoszyn ylinidae, Scydmaeninae). Zootaxa 3268, 55e62. ski, P., 2012c. New synonymy and redescription of Mastigus deustus Jałoszyn (Thunberg) (Coleoptera, Staphylinidae, Scydmaeninae). Zootaxa 3482, 68e76. ski, P., 2012d. Description of Euroleptochromus gen.n. (Coleoptera, StaphJałoszyn ylinidae, Scydmaeninae) from Baltic amber, with discussion of biogeography and mouthpart evolution within Clidicini. Syst. Entomol. 37, 346e359. ski, P., 2012e. Beetles with 'trochantelli': phylogeny of Cephenniini (ColeJałoszyn optera: Staphylinidae: Scydmaeninae) with focus on Neotropical genera. Syst. Entomol. 37, 448e477. ski, P., 2013a. Revision of subgenera of Stenichnus Thomson, with review of Jałoszyn Australo-Pacific species (Coleoptera, Staphylinidae, Scydmaeninae). Zootaxa 3630, 39e79. ski, P., 2013b. Revision of the Neotropical genus Alloraphes Franz (ColeopJałoszyn tera: Staphylinidae: Scydmaeninae). Zootaxa 3750, 549e568. ski, P., 2014. Phylogeny of a new supertribe Cephenniitae with generic reJałoszyn view of Eutheiini and description of a new tribe Marcepaniini (Coleoptera: Staphylinidae: Scydmaeninae). Syst. Entomol. 39, 159e189. ski, P., Hlav Jaoszyn a c, P., Nomura, S., 2003. Contribution to the knowledge of the genus Clidicus (Coleoptera, Scydmaenidae), with descriptions of four new species from Vietnam and Laos. Bull. Nat. Sci. Mus. Tokyo, Ser. A 29, 21e38. Kamimura, Y., 2000. Possible removal of rival sperm by the elongated genitalia of the earwig, Euborellia plebeja. Zool. Sci. 17, 667e672. Kamimura, Y., 2005. Last-male paternity of Euborellia plebeja, an earwig with elongated genitalia and sperm removal behavior. J. Ethol. 23, 35e41. Krell, F.-T., 1996. Die Kopulationsorgane des Maik€ afers Melolontha melolontha (Insecta: Coleoptera: Scarabaeidae) e Ein Beitrag zur vergleichenden und funktionellen Anatomie der ektodermalen Genitalien der Coleoptera. Stuttgart. Beitr Nat. Ser. A 537, 1e101. Lawrence, J.F., Britton, E.B., 1991. Coleoptera (Beetles). In: CSIRO (Ed.), The Insects of Australia, second ed., vol. 2. Melbourne University Press, Melbourne, Australia, pp. 543e683. ski, A., Seago, A.E., Thayer, M.K., Newton, A.F., Marvaldi, A.E., Lawrence, J.F., Slipi n 2011. Phylogeny of the Coleoptera based on morphological characters of adults and larvae. Ann. Zool. 61, 1e217. vision des Mastigini de l'Afrique du Sud. Ann. Mus. R. Afriq. Leleup, N., 1968. Re Centre Tervuren 166, 1e107 (Series 8). n, E., 1970. Coleoptera. In: Tuxen, S.L. (Ed.), Taxonomist's Glossary Lindroth, C.H., Palme of Genitalia in Insects, second ed., pp. 80e99 Munksgaard, Copenhagen, Denmark. Mashimo, Y., Yoshizawa, K., Engel, M.S., Ghani, A.B., Dallai, R., Beutel, R.G., Machida, R., 2013. Zorotypus in Peninsular Malaysia (Zoraptera: Zorotypidae), with the description of three new species. Zootaxa 3717, 498e514. Matsumura, Y., Yoshizawa, K., 2010. Insertion and withdrawal of extremely elongated genitalia: a simple mechanism with a highly modified morphology in the leaf beetle, Lema coronata. Biol. J. Linn. Soc. 99, 512e520. Matsumura, Y., Yoshizawa, K., 2012. Homology of the internal sac components in the leaf beetle subfamily Criocerinae and evolutionary novelties related to the extremely elongated flagellum. J. Morphol. 273, 507e518. Matsumura, Y., Machida, R., Wipfler, B., Beutel, R.G., Yoshizawa, K., 2013. Parallel evolution of novelties: extremely long intromittent organs in the leaf beetle subfamily Criocerinae. Evol. Dev. 15, 305e315.


66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22


22


Matsumura, Y., Yoshizawa, K., Machida, R., Mashimo, Y., Dallai, R., Gottardo, M., Kleinteich, T., Michels, J., Gorb, S., Beutel, R., 2014. Two intromittent organs in Zorotypus caudelli (Insecta, Zoraptera): a paradox coexistence of an extremely long tube and a large spermatophore. Biol. J. Linn. Soc. 112, 40e54. Moore, A.J., Gowaty, P.A., Wallin, W.G., Moore, P.J., 2001. Sexual conflict and the evolution of female mate choice and male social dominance. Proc. R. Soc. Lond. B 268, 517e523. s-Aguilar, J., Beutel, R.G., 2005. New findings on the Navarrete-Heredia, J.L., Corte enigmatic beetle family Lepiceridae (Coleoptera: Myxophaga). Entomol. Abhandl. 62, 193e201. O'Keefe, S.T., 2002. Revision of the Neotropical genus Leptochromus Motschulsky (Coleoptera: Scydmaenidae). Syst. Entomol. 27, 211e234. O'Keefe, S.T., 2003. Revision of the Nearctic genus Papusus Casey (Coleoptera, Scydmaenidae). In: Cuccodoro, G., Leschen, R.A.B. (Eds.), Systematics of Cole€bl. Mem. Entomol. Int. 17, optera: Papers Celebrating the Retirement of Ivan Lo 257e310. O'Keefe, S.T., Monteith, G.B., 2000. Clidicus abbotensis O'Keefe, a new species of Scydmaenidae (Coleoptera: Staphylinoidea) from Australia with description of the larva. Mem. Qld. Mus. 46, 211e223. Pitnick, S., Reagan, J., Holland, B., 2001. Males' evolutionary responses to experimental removal of sexual selection. Proc. R. Soc. Lond. B 268, 1e10. Polak, M., Rashed, A., 2010. Microscale laser surgery reveals adaptive function of male intromittent genitalia. Proc. R. Soc. Lond. B 277, 1371e1376. Puthz, V., 1973. On some Neotropical Euaesthetinae (Coleoptera, Staphylinidae). Stud. Neotrop. Fauna 8, 51e73.

Reynolds, E., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208e212. Rice, W.R., 1996. Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. Nature 361, 232e234. Richmond, M.P., Johnson, S., Markow, T.A., 2012. Evolution of reproductive morphology among recently diverged taxa in the Drosophila mojavensis species cluster. Ecol. Evol. 2, 397e408. Rodriguez, V., 1995. Copulatory courtship in Chelymorpha alternans Boheman (Coleoptera: Chrysomelidae: Cassidinae). Coleopt. Bull. 49, 327e331. Sharp, D., Muir, F., 1912. The comparative anatomy of the male genitalia tube in Coleoptera. Trans. Entomol. Soc. Lond. 1912, 477e642. Soulier-Perkins, A., 2002. The phylogeny of the Lophopidae and the impact of sexual selection and coevolutionary sexual conflict. Cladistics 17, 56e78. Suzuki, K., 1988. Comparative Morphology of the Internal Reproductive System of Chrysomelidae (Coleoptera). In: Jolivet, P., Petitpierre, E., Hsiao, T.H. (Eds.), Biology of Chrysomelidae. Kluwer Academic Publishers, Dordrecht, Boston and London, pp. 317e355. Verma, K.K., Kumar, D., 1972. The aedeagus, its musculature, and ‘retournement’ in Aspidomorpha miliaris F. (Coleoptera, Phytophaga, Chrysomelidae). J. Nat. Hist. 6, 699e719. Verma, K.K., Kumar, D., 1980. Aedeagal musculature in Phytophaga (Coleoptera). J. Nat. Hist. 14, 237e270. Wanat, M., 2007. Alignment and homology of male terminalia in Curculionoidea and other Coleoptera. Invert. Syst. 21, 147e171. Yoshizawa, K., Ferreira, R., Kamimura, Y., Lienhard, C., 2014. Female penis, male vagina, and their correlated evolution in a cave insect. Curr. Biol. 24, 1006e1010.


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