Arthropod Struct Dev 2014 Jaoszyski P

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ASD593_proof 17 October 2014 1/22
Arthropod Structure & Development xxx (2014) 1e22
Contents lists available at ScienceDirect
Arthropod Structure & Development

journal homepage: www.elsevier.com/locate/asd
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

Pawe Jaoszyn
a
b
Museum of Natural History, Wrocaw University, Wrocaw, Poland

Institut fr 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 inating the
endophallus, and the long agellum does not penetrate the ductus spermathecae during copulation. The
structure of the agellum 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. Benets 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 specic intromittent structures
of the male reproductive system, e.g., the agellum in various
beetle families (e.g., Gack and Peschke, 2005; Matsumura et al.,
2013), the distiphallus in Tephritidae ies (Eberhard, 2005), the
processus gonopori in Lygaeidae bugs (Gschwentner and Tadler,
2000), the virga of earwigs (Kamimura, 2000, 2005), the agellum 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, Wrocaw University, Sienkiewicza 21, 50-335 Wrocaw, Poland.
ski).
E-mail address: scydmaenus@yahoo.com (P. Jaoszyn
mechanisms are broadly accepted to explain the genital diversication, 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 diversication 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: Jaoszyn
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. Jaoszyn
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,
Hnefeld 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 diversication processes
leading to extreme elongation of male genitalia are arguably lineages comprising closely related taxa differing signicantly 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 agellum 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; Jaoszyn
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) (Jaoszyn
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; Jaoszyn
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 rst author in South Africa). The
monophyly of Mastigini is strongly supported by an exceptional
ski, 2012d),
number of 12 unique character changes (Jaoszyn
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 agellum 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 agellum (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 Jaoszyn
genera remained unresolved. Only Stenomastigus and Palaeostigus
are well-dened 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 Jaoszyn
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 inatable 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; Jaoszyn
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 signicant 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
Wrocaw, the rst 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 rst 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
j.asd.2014.09.006
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P. Jaoszyn
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 Jaoszyn
ski (A), Mastigus spinicornis (Fabricius) (B), Palaeostigus micans Leleup (C); Papusus macer Casey (D); Leptomastax sp. (E),
for each genus: Stenomastigus berlinafricanus Jaoszyn
ski, Hlavac & Nomura (G); and Leptochromus agilis (Sharp) (H). Abbreviations: cp, copulatory piece; eph, permanently
Ablepton treforti Frivaldszky (F); Clidicus aliquantulus Jaoszyn
everted distal portion of endophallus; fg, agellum; pm, paramere.
and specically 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
j.asd.2014.09.006
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P. Jaoszyn
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 ination and movements function in an

aedeagus with extremely long and coiled agellum.
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);
Jaoszyn
Palaeostigus palpalis
(Latreille, 1804) (Genilla Riv. valley, Cordoba, Spain, leg. A. Castro &
ski); Palaeostigus micans Lelup, 1968 (Wilderness NaP. Jaoszyn
tional Park, West Cape, South Africa, leg. R. Ruta & M. Wanat) (all
preserved in the collection of Natural History Museum, University
of Wrocaw, 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
inated endophallus and the degree of agellum extension. The
maximum ination 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 eld 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 Scientic, 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,
Mnster, 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 les were then transferred to
MAYA7 (Alias Wavefront, Toronto/Ontario, Canada) in order to
rene 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 agellum and
ductus spermathecae were carried out using photographs of partly
fragmented agella 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 Hnefeld 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 agellum 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 prole 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 prole 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.
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
rst visible one) is broadly and rmly 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 conuent 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 inated and forms an elongated sac with two nger-like projections. The same degree of ination was obtained when isolated
aedeagi were placed under hypotonic conditions. The agellar
opening (or gonopore) (Fig. 4IeJ) is located subapically on the
inated endophallus, on the apex of an elongate conical projection.
The endophallic surface is irregularly covered with ne microtrichia locally forming dense patches (Fig. 4G). The median lobe
contains the agellar 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 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, agellum inside partly dissected median lobe; IeJ, aedeagus in right lateral view with
distal portion of permanently extruded endophallus fully inated (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, agellum; fgo,
agellar 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 200 mm; EeH 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 lled
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 articially 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, agellum; fgo, agellar 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 200 mm.
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Fig. 6. Stenomastigus longicornis, thoracic and abdominal organs, schematic view (A, B) and 3D-reconstructions (CeG). A, skeletal structures of male postabdomen, horizontal
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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
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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
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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*,
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M. tergoapodemo-distomembranalis (posterioris); 27, retractor of distal connecting membrane (not named); 28, M. tergoapodemo-phallobasicus major; acg, accessory glands; aed,
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aedeagus; apo9, tergal apodeme IX; cns, posterior central nervous system; cp, copulatory piece; dcm, distal connecting membrane; ed, ejaculatory duct; eph, permanently everted
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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 200 mm.
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1*: not named (and not reconstructed). O. ( origin): anterior
through the basal foramen. The ventral nerve cord forms a single
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region of tergal apodeme VIII; I. ( insertion): anterior region of
undivided ganglionic mass in the pterothorax and abdomen.
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tergal apodeme IX; F. ( function): retractor of segment IX.
The postabdomen of Stenomastigus is equipped with a complex
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2*: not named (and not reconstructed). O.: anterior region of
system of 18 muscles (Figs. 6CeH and 7AeD; summarized in Table 1),
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tergal apodeme VIII; I.: middle region of tergal apodeme IX; F.:
in and posterior to segment IX and outside of the aedeagus (the
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retractor of segment IX.
muscularis of internal organs is not treated as separate muscles here):
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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 200 mm.
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Table 1
Postabdominal muscles (on and behind segment IX) in Stenomastigus longicornis and
Palaeostigus palpalis compared to Tetraphalerus bruchi (after Hnefeld 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)
(submedialis)
(anterioris)
M. antecosta-antecostalis
uronotum medialis VIII
uronotum lateralis VIII
(medialis)
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)
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a
Unpaired, only on the left side.

b
Composed of multiple bers.
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 bers, 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
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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
lling large spaces between loosely arranged internal organs
around and anterad the short aedeagus with its enclosing connecting membrane. However, signicant 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 unconrmed.
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The latter muscle is possibly present but obscured by surrounding

muscle bers and not identiable 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 agellar
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 agellum in the median part of the capsular portion
of the median lobe (Figs. 8AeF). The agellum forms ve 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 agellum 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 agellum has solid and
smooth cuticular walls (Fig. 11AeB) and is surrounded by a twolayered agellar pouch (Figs. 8AeF, 9AeC, 10BeE, 11A). The
agellar 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 agellar opening
(in Figs. 8AeF and 9D the reconstruction of distal portion is
omitted). The agellar pouch membrane is continuous with the
cuticle of the endophallus and both are covered with microtrichia
(the endophallus externally, and the agellar pouch internally
along the entire length); the microtrichia inside the agellar pouch
are mostly bent and oriented along the longitudinal axis of the
agellum (Fig. 11AeB). The internal membrane of the agellar
pouch within the agellar loops is continuous with massive and
rigid internal sclerotizations rmly 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, agellum; fgo, agellar opening; fgp, agellar
pouch; ma, membranous area; pm, paramere; sat, subapical tooth of copulatory piece. Scale bars: AeD 200 mm; EeF 100 mm.
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Fig. 9. Stenomastigus longicornis, 3D-reconstruction of agellar 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, agellum; fgo, agellar opening; fgp, agellar pouch; pm, paramare; sat, subapical tooth of copulatory piece. Scale bars: 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 ne structure of the aedeagus, including the
musculature, does not differ from that of Stenomastigus, except for
the increased length of the agellum (Table 2), with six loops. The
agellum 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 agellar apex in the inated endophallus (Figs. 4IeJ). The
fully inated endophallus projects distally beyond the long paramere, but is only 2e3 times longer compared to the folded position.
The agellum does not extend beyond the apex of the agellar
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 laments 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 agellum (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 eld 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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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, agellum; fgp, agellar pouch; pcm, proximal connecting membrane; pm, paramere; rbf, rim of
basal foramen; scl, median sclerotization of agellar loops; spp, sperm pump. Scale bars: 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 exed 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 exed 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 inated 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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Fig. 11. Stenomastigus longicornis, transmission electron micrographs of cross-sectioned agellar loop. A, agellum in agellar pouch; B, internal wall of agellar pouch with
microtrichia. Abbreviations: fgl, agellar lumen; fgp, agellar pouch; fgw, agellar wall; mtr, microtrichia. Scale bars: 1 mm.
abdomen exed position, with the male grasping the female with
the fore legs between the prothorax and elytra. The aedeagus
(Fig. 13CeE) remains largely visible outside (conrmed 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
inated endophallus (with distal gonopore) was found close to (but
not inside) the insertion of the ductus spermathecae, and the agellum 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 diversication of genitalia are only fragmentarily
claried (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; Jaoszyn
tation to this function: the slightly emarginated or curved protibial
apex. Not only the protibiae are modied 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 modications show a considerable variability in species of Stenomastigus. In seven species
ski (2012a,b), the internal protibial margin is
illustrated by Jaoszyn
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 Jaoszyn
(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 Jaos ski) or is elongated and forms a tooth- or rod-like process (e.g.,
zyn
ski, Stenomastigus allaeri
Stenomastigus berlinafricanus Jaoszyn
ski, 2012a,b). All these modications may stabilize
Leleup) (Jaoszyn
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Table 2
Measurements of ductus spermathecae and agellum in S. longicornis and P. palpalis;
N 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 agellum
Coiled part
Total
Diameter of agellum
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; Jaoszyn
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; tl, terminal lament; vf, valvifer. Scale bars 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 1 mm; DeE 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, ts 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 xed 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 (Jaoszyn
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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18
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
Unmodied
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 at
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 ex 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 Jaoszyn
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 modications 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 rst place, gradually
changed its function to locking the aedeagus in a xed 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 sufcient 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 modication 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 (
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., Jaoszyn
The Mastigitae genera, except for Leptomastax Pirazzoli and
ski, 2012d and
Clidicus, do not have a metafurcal stalk (Jaoszyn
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; Jaoszyn
other lineages of Scydmaeninae the metafurca is much more
ski, 2013a),
frequently Y-shaped or nearly V-shaped (e.g. Jaoszyn
only rarely without a stalk and with broadly separated arms
ski,
(Eutheiini;
Jaoszyn
2014).
The
staphylinid
clade
Euaesthetinae Steninae was identied 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 (Jaoszyn
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 t between the paired meta- and mesofurcal projections.
4.3. Function of the agellum 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,
agellum (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
agellar origin were hypothesized, reecting 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 agellum 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 agellum 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 agellum is inserted into the ductus spermathecae and
reaches or nearly reaches the spermatheca; the safe withdrawal of
the long agellum 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 agellum 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 agellum from the coiled
region retained the same curved shape.
The agellum 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 agellar 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 laments are running spirally
around the central lumen and it is surrounded by a thick muscularis
composed of spirally and longitudinally arranged muscle bers
(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 agellum originates as a protrusion of the
epidermis and cuticle of the internal sac (i.e., the distal inatable
part of aedeagus corresponding to the endophallus in the present
study) at the distal end of the ejaculatory duct by an invagination.
Moreover, the agellum is contained in an invaginated apex of the
internal sac forming a spacious pouch in which the agellum is
stored in repose. In Mastigini, the agellum 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 agellum). It was
suggested that these spines might play a crucial role in the
insertion and withdrawal of the agellum by providing an adhesive
force interacting with the membranes (Matsumura and Yoshizawa,
2010). This idea was not further developed. The arrangement of the
agellar pouch and the microtrichia in Mastigini suggest that independent of the currently available range of movements of the
agellum, 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 agellum 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 verication.
The extremely long and coiled agellum 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 agellum was invariantly xed 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 agellum into the ductus spermathecae to any length
would be only possible (there are no length or diameter constraints) if the agellum would be able to uncoil. However, the
agellar 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
agellar 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
agellar movement during the intromission is likely caused by this
structural constraint.
The mechanism of extending (slightly) the distal portion of the
agellum 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 inating
the membranous endophallus. The forward movement of the
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endophallus with its conical projection causes a corresponding

extension of the agellum, 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., Jaoszyn
though the membranous area is located basally in some cases (e.g.,
ski, 2013b) or subapically (e.g., Jaoszyn
ski, 2012e), and the
Jaoszyn
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 inating, which also suggests this mechanism. Moreover, such a manipulation also caused the copulatory
piece to ick outward, as a consequence of inating the basal
membrane, in which this structure is eccentrically inserted
(Fig. 4E). Why the agellum 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 Jaoszyn
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 bers 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 exibility. In criocerine leaf beetles, for instance, it was found that the musculature
differs between taxa with and without agellum 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 bers 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 nally 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 rst part of this hypothesis is supported by our ndings; 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 tness of both taxa.
For the males of Stenomastigus, the stabilization of the enormously
elongated aedeagus may be crucial for the efciency 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 conicts 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 t of stabilizing structures in
males and females of Stenomastigus. The stabilizing adaptations in
females may be benecial in the same way as in corresponding
structures in males, i.e. by an increased control over the copulation
process, which is more difcult 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 specic 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 efciency 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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P. Jaoszyn
aedeagus whose position does not require strict mechanical control

mechanisms; some degree of movability of the inserted aedeagus
may be benecial 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, Rafa Ruta and Katarzyna
_
Zuk
(University of Wrocaw) and Ruth Mller (Ditsong National
Museum of National History, Pretoria) for their invaluable support
during the rst author's eld studies in South Africa. The eld 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. Rafa Ruta took photos 2B-C and Krzysztof
ski and Anna Siudzin
ska (Wrocaw 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 Wrocaw, Poland. This
is also gratefully acknowledged.
References
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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).
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Arthropod Structure & Development xxx (2014) 1e22

Contents lists available at ScienceDirect

Arthropod Structure & Development

Evolution of a giant intromittent organ in Scydmaeninae (Coleoptera:

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

Museum of Natural History, Wrocaw University, Wrocaw, Poland

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simple. The intromission and sperm transfer is a primary function

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

and specically the postabdomen. We address four questions in

and thorax of males (to clarify problems related to the available

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mechanism of endophallus ination and movements function in an

Female genitalia were dissected from 75% ethanol-preserved

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microscope (Zeiss Axioplan, Germany) equipped with a camera

3.2. Male abdomen and postabdomen e skeletal structures

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apical portion. The capsular basal part of the median lobe

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3.3. Male abdomen and postabdomen e internal organs

connecting membrane (Fig. 6CeH) is surrounded by tightly packed

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Stenomastigus Palaeostigus Tetraphalerus

M. sinister urotergodistomembranalis (posterioris)

Unpaired, only on the left side.

3*: M. sinister urotergo-distomembranalis (posterioris)

region (dorsally) of distal connecting membrane; F.: retractor of

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The latter muscle is possibly present but obscured by surrounding

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sclerotized structures, but also oviducts, each with only a pair of

3.5. Female genitalia of Palaeostigus and Stenomastigus

3.6. Copulation and intromission in Palaeostigus and

The abdominal terminalia of females in both genera are similar

Copulating pairs of S. longicornis were observed in the eld and

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apex of long paramere reaches the level of prothorax (Fig. 13A). In

between elytra and abdominal tergites, and only the permanently

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4.1. Copulation in Mastigini with short and long aedeagus e

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Length of ductus spermathecae

the mounting position during copulation by interlocking the male

the entire coupling) may play a less important role in Palaeostigus

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