The male postabdomen and reproductive system of Bibio marci Linnaeus, 1758 (Hexapoda: Diptera: Bibionidae)

Abstract

The male postabdomen and the internal parts of the male genital system of Bibio marci (Bibionomorpha) were examined and reconstructed 3-dimensionally. Several features differ from the presumptive dipteran groundplan. The bases of the gonopods are fused with each other and with tergite IX. The penis is not tube-shaped and only sclerotized on the ventral side. The vasa deferentia are S-shaped, and two pairs of accessory glands are present. In contrast to these characteristics, the arrangement of the internal parts is probably close to the ancestral condition. With its specific shape, the penis is well suited for the transfer of a spermatophore. The dorsal sclerite of the copulatory organ probably represents the medially fused parameres. A cladistic analysis of 27 characters of the postabdomen yielded two most parsimonious trees, with the strict consensus as follows: Nannochoristidae (outgroup)+(Culicidae [Culicomorpha]+((Nymphomyiidae+(Tipulidae+Trichoceridae))+(Tabanidae [Brachycera]+(Bibionidae, Anisopodidae, Axymyiidae [Bibionomorpha])))). Potential synapomorphies of Bibionomorpha (including Axymyiidae) and Brachycera are the fusion of sternum IX with the gonocoxites, the fusion of the parameres forming the dorsal sclerite and the presence of an entire series of postabdominal muscles (M4, M20, M23, M26, M27, M31, M35 and M37). The results of the analysis are preliminary as it is based on a single-character system with a limited taxon sampling. However, the main result - a clade Bibionomorpha+Brachycera - is fully compatible with current hypotheses on dipteran phylogeny.

Full text

Entomology group, Institut fu
¨r Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universita
¨t
Jena, Jena, Germany
The male postabdomen and reproductive system of Bibio marci Linnaeus, 1758
(Hexapoda: Diptera: Bibionidae)
Rico Spangenberg,Frank Hu
¨nefeld,Katharina Schneeberg and Rolf Georg Beutel
Abstract
The male postabdomen and the internal parts of the male genital system of Bibio marci (Bibionomorpha) were examined and reconstructed
3-dimensionally. Several features differ from the presumptive dipteran groundplan. The bases of the gonopods are fused with each other and with
tergite IX. The penis is not tube-shaped and only sclerotized on the ventral side. The vasa deferentia are S-shaped, and two pairs of accessory
glands are present. In contrast to these characteristics, the arrangement of the internal parts is probably close to the ancestral condition. With its
specific shape, the penis is well suited for the transfer of a spermatophore. The dorsal sclerite of the copulatory organ probably represents the
medially fused parameres. A cladistic analysis of 27 characters of the postabdomen yielded two most parsimonious trees, with the strict consensus
as follows: Nannochoristidae (outgroup) + (Culicidae [Culicomorpha] + ((Nymphomyiidae + (Tipulidae + Trichoceridae)) + (Tabanidae
[Brachycera] + (Bibionidae, Anisopodidae, Axymyiidae [Bibionomorpha])))). Potential synapomorphies of Bibionomorpha (including
Axymyiidae) and Brachycera are the fusion of sternum IX with the gonocoxites, the fusion of the parameres forming the dorsal sclerite and
the presence of an entire series of postabdominal muscles (M4, M20, M23, M26, M27, M31, M35 and M37). The results of the analysis are
preliminary as it is based on a single-character system with a limited taxon sampling. However, the main result – a clade Bibionomor-
pha + Brachycera – is fully compatible with current hypotheses on dipteran phylogeny.
Key words:Bibio – Bibionomorpha – diptera – morphology – male genitalia – spermatophore
Introduction
Bibionomorpha belongs to the basal dipteran lineages com-
monly referred to as the nematoceran-grade families (Bla-
schke-Berthold 1994; Ziegler 2003; Amorim and Yeates 2006).
According to current hypotheses, the group includes Aniso-
podidae, Canthyloscelidae, Scatopsidae, Bibionidae, Pachy-
neuridae, Ditomyiidae, Manotidae, Diadocidiidae, Sciaridae,
Cecidomyiidae, Lygistorrhinidae, Rangomaramidae, Myceto-
philidae, Keroplatidae and Bolitophilidae (e.g. Amorim and
Rindal 2007; Bertone et al. 2008; Wiegmann et al. 2011). The
position of Axymyiidae (inside or outside of Bibionomorpha)
and the status and position of Hesperinidae (separate family or
subgroup of Bibionidae) are still debated (Fitzgerald 2004;
Bertone et al. 2008; Papp and Krivosheina 2009; Wiegmann
et al. 2011). Bibionidae comprises ca. 700 species in seven
genera, the vast majority of them occurring in the Holarctic
(Sutou 2002; Fitzgerald 2010). The genus Bibio Geoffroy, 1762
contains ca. 240 species (Sutou 2002). Adults of Bibio marci
Linnaeus, 1758 are about 11–13 mm long and black. They bear
a dense vestiture of setae (Duda 1930) and display distinct
sexual dimorphism (Hennig 1973; Zeil 1983).
The phylogeny of Bibionomorpha is not sufficiently resolved
yet. Analyses of molecular data (Bertone et al. 2008) and
molecular data combined with morphological characters
(Wiegmann et al. 2011) tentatively confirm Bibionomorpha
as a clade. However, Axymyiidae is excluded in the former and
included in the latter. From the morphological point of view,
the monophyly is not sufficiently supported, and the placement
of the group is far from being settled. Hennig (1973) studied
characters of the adult thorax and wings. He suggested the
reduction in the costa as an autapomorphy of Bibionomorpha
and pointed out structural and possible phylogenetic affinities
with Brachycera. In both taxa, the second laterotergite is
enlarged and the postphragma of the thorax undivided
(Hennig 1973). A phylogenetic analysis of Amorim (1992),
mainly based on wing features, supported a clade Bibiono-
morpha + Brachycera. Michelsen (1996) documented four
skeleto-muscular modifications of the pronoto-cervical region
in adults to establish a new group named Neodiptera
(Brachycera + bibionomorph Nematocera). Blaschke-Bert-
hold (1994) added the presence of a dorsal sclerite as a part
of the male genital complex in Bibionomorpha and Brachy-
cera, but also in Blephariceridae. Another potentially relevant
feature is sternite IX located between the gonocoxites and
fused with them. This presumably derived condition is found
in Bibionomorpha and in the orthorrhaphous Brachycera
(Hennig 1973). However, Sinclair et al. (1994) assigned a
sternite IX (hypandrium) separated from the gonocoxites to
the groundplan of Brachycera.
Wood and Borkent (1989) compared 83 characters of adults,
pupae and larvae, but did not take Brachycera into consid-
eration. Like Hennig (1973), they suggested Tipulomorpha
as the sister group of the remaining Diptera. They placed
Bibionomorpha with Culicomorpha, Ptychopteromorpha, Psy-
chodomorpha, Axymyiomorpha and Blephariceromorpha.
Oosterbroek and Courtney (1995) increased the number of
characters of adults (36), pupae (6) and larvae (57). They
suggested a sister-group relationship between Bibionomopha
and a group ‘‘higher Nematocera + Brachycera’’ (HNB)
(containing also Tipulidae + Trichoceridae). However, some
character states were criticized by Sinclair et al. 2007. A recent
molecular study of Bertone et al. (2008) was based on sequence
data of 28SrDNA and the protein-coding genes CAD,
PGD and TPI. The results again suggested a clade Bibiono-
Corresponding author: Rico Spangenberg (rico.spangenberg@gmail.
com; rico.spangenberg@googlemail.com)
Contributing authors: Frank Hu
¨nefeld (frank.huenefeld@uni-jena.de),
Katharina Schneeberg (katharina.schneeberg@gmx.de), Rolf Georg
Beutel (rolf.beutel@uni-jena.de)
2012 Blackwell Verlag GmbH
Accepted on 20 June 2012
J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2012.00669.x
J Zool Syst Evol Res (2012) 50(4), 264–288

morpha + Brachycera as hypothesized by Hennig (1973).
Apparently, the phylogeny of Bibionomorpha and the
nematoceran-grade families in general is a topic of ongoing
debate (e.g. Oosterbroek and Courtney 1995; Yeates and
Wiegmann 2005; Yeates et al. 2007; Bertone et al. 2008;
Wiegmann et al. 2011).
A considerable number of studies provide information on the
male genital apparatus of bibionomorph taxa (Anisopodidae:
Peterson 1981; Haenni 1997, 2006; Axymyiidae: Wood 1981a;
Bolitophilidae: Polevoi 1996; Sevcik and Papp 2004; Polevoi
et al. 2006; Canthyloscelidae: Hutson 1977; Cecidomyiidae:
Gagne
´1981; Abe et al. 2011a,b; Diadocidiidae: Polevoi 1996;
Polevoi et al. 2006; Hesperinidae: Krivosheina 1997b; Sinclair
2000; Papp and Krivosheina 2009; Keroplatidae: Polevoi 1996;
Polevoi et al. 2006; Kjaerandsen et al. 2007; Lygistorrhinidae:
Papp 2002, 2005; Blagoderov et al. 2009, 2010; Mycetophil-
idae: Vockeroth 1981; Polevoi et al. 2006; Kjaerandsen et al.
2007; Pachyneuridae: Wood 1981b; Krivosheina 1997a; Sca-
topsidae: Cook 1981; Sciaridae: Steffan 1981). More compre-
hensive accounts were added by Blaschke-Berthold (1994) and
Fitzgerald (2004). A drawback of these studies is that they are
largely restricted to features of the exoskeleton and lack
comprehensive information on the internal softparts and the
mode of sperm transfer. Consequently, in the present contri-
bution, the male copulatory apparatus and the reproductive
organs of B. marci were examined in detail, with a complete
survey of the postabdominal musculature. The results are
compared with the conditions found in nematoceran taxa and
in one group of Brachycera, the Tabanidae. The main goals are
a detailed documentation of an additional character system,
functional interpretations, the homologization of structures
and an assessment of dipteran groundplan features. The results
also extend the spectrum of morphological data for resolving
the relationships of basal dipteran lineages. A formal analysis
of the characters with a limited taxon sampling is presented. A
more extensive analysis would be beyond the scope of this
contribution, which has a clear focus on morphology. The
presented data will be integrated in more extensive matrices in
subsequent studies in the framework of an ongoing morpho-
logical project on the basal branching events and early
evolutionary transformations in Diptera.
Material and Methods
Taxa examined
The taxa investigated are listed in Table 1.
Techniques
Specimens were fixed and stored in 70% ethanol. Macerated
and non-macerated individuals of B. marci were examined with a
Leica MZ 12.5 stereomicroscope. Line drawings were made using
an integrated drawing mirror (Leica Microsystems, Wetzlar,
Germany).
One specimen of each species was embedded in Araldite

(Huntsman Advanced Materials) for semi-thin cross-sectioning, cut
at 1.5 lm(Bibio), 1 lm(Nymphomyia,Sylvicola,Axymyia,Hybomi-
tra)or3lm(Limonia) with a glass knife on a microtom HM 360
(Microm, Walldorf, Germany) and stained with toluidine blue.
Photographs of sections for 3D reconstruction were taken with a
Zeiss Axioplan (Zeiss, Goettingen, Germany) and the AnalySIS

documentation system (Soft Imaging System GmbH, Mu
¨nster,
Germany). Figures were processed in adobe

photoshop

cs2
version 9.0 (Adobe Systems Incorporated, San Jose, CA, USA) and
adobe

illustrator

cs2 12.0.0 (Adobe Systems Incorporated).
Three-dimensional reconstructions of Bibio were prepared with
Mercury Amira

4.1.2 (Visage Imaging GmbH, Berlin, Germany),
and surfaces were smoothed with Autodesk Maya

7.0 (Autodesk
GmbH, Munich, Germany).
Confocal laser scanning micrographs (CLSM) of Bibio were taken
with a Zeiss LSM 510 (Zeiss), using an excitation wavelength of
488 nm and a 10·Apochromat lens (for autofluorescence of insect
cuticle see Klaus et al. 2003). Before scanning, the specimen was
macerated in 10% KOH for 36 h at 25C.
Scanning electron micrographs (SEM) were taken of three individ-
uals of B. marci with a Philips XL 30 ESEM (FEI Company,
Hillsboro, OR, USA) and scandium 5.0 Software (Soft Imaging
System GmbH). The specimens were dried at the critical point
(EmiTech K850; Emitech Ltd., Kent, UK), sputter-coated with gold
(EmiTech K500; Emitech Ltd.) and fixed on a specimen holder
designed by H. Pohl (Pohl 2010).
Cladistic analysis
For character coding and cladistic analyses, winclada version 1.00.08
(Nixon 2002) and nona software were used (Goloboff 1999)
(Ratchet ⁄Island Hopper, number of iterations ⁄rep: 1000). Additional
analyses were carried out with tree analyses using new technology
(TNT) (Goloboff et al. 2003) (implicit enumeration). All characters
were equally weighted and non-additive.
Terminology
The terminology used for the exoskeleton, cuticular structures and the
internal parts largely follows the studied of Snodgrass (1936), Matsuda
(1976) and McAlpine (1981). Muscles were numbered consecutively,
and an overview on muscle nomenclatures is given in Table 3. The
penis, the parameres and the phallobase combined are referred to as
the aedeagus.
Table 1. Taxa examined
Group Infraorder Family Genus ⁄species Locality ⁄collector
Lower Diptera Bibionomorpha Bibionidae Bibio marci Linnaeus, 1758 Warnemu
¨nde, Germany ⁄leg. H. Pohl
Lower Diptera Bibionomorpha Axymyiidae Axymyia furcata McAtee, 1921 USA, NC, Haywood Co, GSMNP,
seep across Rough Fk nr. trailhead,
3537¢N8307¢W 865m ⁄leg. et det.
GW Courtney
Lower Diptera Bibionomorpha Anisopodidae Sylvicola fenestralis
(Scopuli, 1763)
Jena, Germany ⁄leg. et det. E. Anton
Lower Diptera Tipulomorpha Tipulidae Limonia sp. Jena, Germany ⁄leg. F. Hu
¨nefeld
Lower Diptera – Nymphomyiidae Nymphomyia dolichopeza
Courtney, 1994
USA, NC, Macon Co.
Coweeta Hydrologic Lab.
Grady Branch (WS 18)
3503¢N8326¢W 730m
13.iii.2005 ⁄leg. GW Courtney
Brachycera Tabanomorpha Tabanidae Hybomitra sp. Gugny, Poland ⁄leg. H. Pohl
The male postabdomen and reproductive system of Bibio marci 265
J Zool Syst Evol Res (2012) 50(4), 264–288
2012 Blackwell Verlag GmbH

Results
Exoskeleton
The male abdomen of B. marci is elongate and round in cross
section. The abdominal segments are slightly tapering towards
the terminal region (Figs 1a–c). The sternites I–VIII cover the
ventrolateral margins of the corresponding tergites. Tergites
and sternites are separated by pleural membranes containing
the spiracles. Segment VIII appears largely unmodified exter-
nally, with the tergite and sternite clearly separated. The
spiracles VIII are shifted caudo-dorsad to the intersegmental
membrane between segments VIII and IX. They are func-
tional, even though they are reduced in size and modified in
shape compared with those of the previous segments.
The male genital segment IX distinctly differs from the
preceding ones. A pleural membrane is missing (Figs 2c and
5c). Tergite IX (epandrium) is crescent-shaped and approxi-
mately half as long and broad as segment VIII (Fig. 1a). Its
base is widened cranially. The caudal end bears two cusp-like
processes (Figs 2a, 5a and 7b) with membranous bases. The
rest of tergite IX is sclerotized (Figs 5a,c and 7a). A distinct
sternal sclerotization of segment IX is lacking.
Together, the sclerotized gonocoxites (gc) are about as wide
as segment VIII (Figs 1b, 2c, 8 and 9). The convex ventral
surface of the fused bases of the gonocoxites (bgp) is evenly
sclerotized (Figs 2c,d, 5e and 6g). A membranous gap (mbg) is
present mediocaudally. It is V-shaped, and its dorsal apex
forms a distinct angle (Figs 2a and 7a). The anterodorsal
regions of the gonocoxites are fused with the ventrolateral
margins of tergite IX; thus, the entire anterior part of segment
IX is encircled by a closed, ring-like sclerotization. Both
gonocoxites bear a gonocoxal apodeme (gcap) anteriorly
(Fig. 5c,d), which is almost three-fourth as long as the main
body of the gonocoxites and nearly reach the anterior margin
of the genital segment (Figs 7d and 11). The gonocoxal
apodemes are triangular, with a strongly sclerotized, bar-
shaped anterior apical region (ca. one-third of the length of the
whole apodeme) (Figs 5f, 6h, 7d and 12). The apodemes are
muscle attachment sites. The posterior parts of the gonocoxites
are tapering caudally. Their ventral and dorsal margins each
form a hump (Fig. 7b). The medial surfaces enclose a bowl-
shaped cavity. The completely sclerotized surfaces of the
caudal region merge seamlessly, with an increasing thickness of
the cuticle in cranial direction (compare Fig. 5a,c). The
gonostyli (gs) arise apically on the gonocoxites (Figs 2a,b, 7
and 10). They are completely sclerotized, and the surface is
smooth (Fig. 3b). They are sickle-shaped and tapered apically,
with the tips pointing antero-dorsad (Figs 2, 3B, and 10). At
the base of the gonostyli, the dorsolateral and ventrolateral
regions are more strongly sclerotized (hsr), each of these areas
forming a joint-like articulation with the dorsal and ventral
hump of the corresponding gonocoxite (dicondylic condition)
(Fig. 7a,b,d; distinct in macerated specimens).
The gonocoxal apodemes are medially connected by a
sclerotized plate (dorsal sclerite, ds; Blaschke-Berthold 1994;
see also Fitzgerald 2004) (Fig. 5d), which represents the (fused)
parameres. Its anterolateral regions are connected with the
gonocoxal apodemes by membranes (Fig. 5f). The dorsal
sclerite is crescent-shaped in cross section and arching above
the ductus ejaculatorius (de) and the endophallus (eph)
(Figs 5d, 8a, 10a and 12). The convex posterior margin of
the sclerite is slightly bent downwards and almost reaches the
tip of the penis (pe) (Figs 10 and 12c). The wing-shaped lateral
extensions of the dorsal sclerite comprise about two-third of its
total width and reach almost the base of the penis ventrally
(Fig. 12b–d). The anterolateral parts of the lateral extensions
merge gradually into membranes (mbds), which extend in
ventral direction and then turn dorsad, without getting in
touch with the sclerite again (Fig. 5f). Here, the membranes
and the lateral regions of the main part of the dorsal sclerite
form an oval chamber on each body side, serving as an
(c)
(a)
(b)
Fig. 1. Bibio marci, male, postabdomen, SEM. All views from indi-
vidual (=ind.) 1. (a) dorsal view; (b) lateral view; (c) ventral view. bgp,
fused bases of gonopods; gp, gonopod; pl, pleuron membrane; s,
sternite; t, tergite
266 Spangenberg, Hu
¨nefeld, Schneeberg and Beutel
J Zool Syst Evol Res (2012) 50(4), 264–288
2012 Blackwell Verlag GmbH

attachment site for muscle M9 (Figs 5f, 7d and 12a,c). The
membranes also fuse with the lateral regions of the ejaculatory
apodeme (ejap).
The ventral side of the penis (pe) is a longitudinal, arched
and plate-like structure (Figs 5d, 10, 11, 12a,d), which is
completely sclerotized. The caudal part is tapering, with a
rounded tip (Figs 7d and 12d). Anteriorly, the penis merges
with the ejaculatory apodeme (ejap) (Fig. 10). Both elements
form the Ôpenis–ejaculatory apodeme complexÕ. The major part
of the dorsal side of the penis is membranous (mbvd) (Figs 5d
and 10). The posterior part of this membrane does not reach
the tip of the penis. An oval opening is present where the short
endophallus (eph) invaginates (Fig. 10a). The penis is not
recognizable externally (Figs 2a, 3a, 7a and 10).
The ejaculatory apodeme (ejap) is tongue-shaped, with its
anterior part forming a ventrally directed hook (Figs 4, 7d, 10,
(a) (b)
(c) (d)
Fig. 2. Bibio marci, male, genital segment, SEM. Views B–D (ind. 1), view A (ind. 2). (a) caudal view; (b) dorsal view (detail of Fig. 1a); (c) lateral
view (detail of Fig. 1b); (d) ventral view (detail of Fig. 1c). ac, anal cone; bgp, fused bases of gonopods; gc, gonocoxite; gs, gonostylus; epr,
epiproct; mbg, membranous gap; s, sternite; sap, subapical plate; t, tergite
(a) (b)
Fig. 3. (a) Bibio marci, detail of male genital segment, caudal view, SEM (ind. 5). (b) gonostyli, lateral view, SEM (individual 1, detail of Fig. 2C).
ac, anal cone; gc, gonocoxite; gs, gonostylus; epr, epiproct; mbg, membranous gap; sap, subapical plate
The male postabdomen and reproductive system of Bibio marci 267
J Zool Syst Evol Res (2012) 50(4), 264–288
2012 Blackwell Verlag GmbH

11b, 12a,c and d). It almost reaches the anterior margin of
segment IX (Fig. 10). A shield-like structure is present ventrad
the transition region of penis and ejaculatory apodeme
(Fig. 12c,d). Titillators or other extensions are lacking.
Paired, sclerotized kidney-shaped structures laterad the
penis (dsdv) are completely surrounded by membranes (mbds;
Figs 4, 7d, 11b, 12a,c and d), which are also connected with
the ejaculatory apodeme and the dorsal sclerite (Figs 4, 12c,
and d).
Distinct sclerites of segment X are not present. The epiproct
(epr), the anal cone (ac) and the cerci (ce) are located between
the bases of the gonocoxites (Figs 2a, 5a,b and 7a,b).
Paraprocts are absent (Figs 5b and 7a). The epiproct covers
the anal cone dorsally. Its anterior margin is indented, and a
deep median cleft is present at the posterior margin, resulting
in the formation of a pair of rounded, plate-like terminal lobes
(Figs 2a,b, 5b, and 7a,b). The base of the epiproct is
conspicuously widened and disc-shaped. It forms a right angle
with the posterior parts (Figs 10 and 11a). The anal cone is
enclosed by the epiproct and the cerci (Figs 7a and 10). Its
distal end points dorsad (Fig. 10). The anal cone is membra-
nous except for the flattened crescent-shaped subapical plate
on the ventral side of its base (sap) (Figs 2a, 7a, 10 and 11).
The unsegmented cerci (ce) are tongue-shaped and medially
fused with each other (Figs 10 and 11). They insert on the
membrane between the anal cone and the bases of the gonopods
and are located ventrad the subapical plate (Fig. 10). They are
distinctly shorter than the anal cone (Fig. 10) and not visible
externally (Figs 2a and 3a). The phallotrema (pht) lies between
the cerci and subapical plate (Figs 5a and 10a).
The entire abdomen is covered with hair-like setae, and
stronger bristles are inserted on some areas. The longest bristles
are present on the mesal regions and on the marginal areas of the
sternites and tergites (Fig. 1). Long bristles are also inserted on
the posterior margin of tergite IX, while shorter bristles
originate between them on the mesal regions (Fig. 2a,b). Long
bristles are regularly distributed over most parts of the gono-
coxites, but are lacking on the ventral sides of the bases and are
replaced by shorter bristles caudally (Fig. 2). The gonostyli bear
only a loose vestiture of irregularly distributed short bristles
(Fig. 3). On the epiproct, the longest bristles insert on the caudal
margin, while the rest of this structure bears a uniform vestiture
of short setae (Fig. 2a,b). The anal cone and the cerci lack any
setation (Figs 2a,b and 3a).
Musculature
A total of 16 muscles were identified in the genital segment
(Figs 10b, 11 and 12a). Pleural muscles are absent. The
musculature of the ductus ejaculatorius, the vasa deferentia
and the vesiculae seminalis are treated in chapter ÔInternal
parts of the genital systemÕ.
The muscles are numbered consecutively, starting from
segment VI (Figs 5, 6, 9, 10b, 11 and 12a, Tables 2–4). They
are described with origin, insertion and function (Table 2). The
nomenclature follows the studies of Maki (1936) and von
Ke
´ler (1963), supplemented by a comparison with muscles
described by Blaschke-Berthold (1994) for B. marci and by
Ovtshinnikova (1989) for B. hortulans (Table 3).
Internal parts of the genital system
Testes (tst)
The paired testes are sac-shaped and unifollicular and located
in segments VI–VII (Figs 6k,l and 8). The right testis is
slightly larger and reaches further anteriorly (Fig. 8a,c). The
epithelial cells of the testes are flattened (sep) (Fig. 6k,l). The
lumen is densely filled with short, filiform spermatozoa (sp)
(Fig. 6K,L). A muscularis is lacking.
Vasa deferentia (vd)
The paired vasa deferentia are long and tubular (Figs 6J and
8). They extend into the intersegmental region between
segments VIII and IX where they are connected in a bridge-
like manner over a short distance (fvd). Beyond that, they form
a u-turn in anterior direction and become separated again. A
second turning point is present in about mid-length of segment
VII (Figs 8a,b and 10a). Between the two turning points, both
vasa deferentia are shifted from the midline to the left body
side in the examined specimens (Figs 6k,l and 8a,c). The vasa
deferentia are equipped with a single-layered squamose
epithelium (sep). Their extended regions in the intersegmental
area VII–VIII form a pair of seminal vesicles (vs) (Fig. 8).
Both regions are surrounded by a single-layered meshwork of
muscle fibres (mvd, mvs), which is slightly denser around the
vesicles (Fig. 6j,l).
Vesiculae seminales (vs)
The paired seminal vesicles have the shape of large tubes and
are in close contact with each other but not fused (Fig. 6j).
They are densely filled with sperm (sp) (Fig. 6j). The diameter
is five to seven times larger than that of the vasa deferentia
(Figs 6i,j and 8). The epithelium (epvs) is formed by large,
cubic cells with the nuclei close to the basal poles (Fig. 6j).
Ductus ejaculatorius (de)
The median, unpaired ductus ejaculatorius is tube-shaped and
runs between the dorsal sclerite, and the complex formed by
Fig. 4. Bibio marci, male genital segment, ventral view, CLSM (ind. 4).
dsdv, dorsal sclerite derivatives; ejap, ejaculatory apodeme; gc, gono-
coxite; gs, gonostylus; mbds, membranes associated with dorsal
sclerite; s, sternite
268 Spangenberg, Hu
¨nefeld, Schneeberg and Beutel
J Zool Syst Evol Res (2012) 50(4), 264–288
2012 Blackwell Verlag GmbH

(a) (b)
(c) (d)
(e) (f)
Fig. 5. Bibio marci, male postabdomen, cross sections from caudal (a) to cranial (e) (ind. 3). Overview on left side, enlarged details on right side.
Figs 8, 9 and 10 indicate the plane of sections. ac, anal cone; bgp, fused bases of gonopods; cdag, caudal accessory gland; ds, dorsal sclerite; ejap,
ejaculatory apodeme; eph, endophallus; epr, epiproct; fb, fat body; g, gut; gc, gonocoxite; gcap, gonocoxal apodeme; i, intima of ductus
ejaculatorius; M, muscle with appropriate number (number of muscle corresponds to number in text, Figs 6, 9, 10b, 11 and 12, Tables 1–3); mb,
membrane; mbg, membranous gap; mbds, membranes associated with dorsal sclerite; mbvd, membrane ventral of dorsal sclerite; mde, muscularis
of ductus ejaculatorius; ocdg, opening of caudal accessory gland; pe, penis; pht, phallotrema; t, tergite
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(g) (h)
(i) (j)
(k) (l)
Fig. 6. Bibio marci, male postabdomen, cross sections from caudal (g) to cranial (k) (ind. 3). Overview on left side, enlarged details on right side.
Figs 8, 9 and 10 indicate the plane of sections. bgp, fused bases of gonopods; crag, cranial accessory gland; dv, dorsal vessel; ejap, ejaculatory
apodeme; epvs, epithelium of vesiculae seminales; g, gut; gc, gonocoxite; gcap, gonocoxal apodeme; gep; glandular epithelium; gsc, glandular
secretion; i, intima of ductus ejaculatorius; M, muscle with appropriate number (number of muscle corresponds to number in text, Figs 5, 9, 10b,
11 and 12, Tables 1–3); mal, Malpighian tubule; mb, membrane; mde, muscularis of ductus ejaculatorius; mvd, muscularis of vasa deferentia;
mvs, muscularis of vesiculae seminales; nv, nervous system; ocrg, opening of cranial accessory gland; plmb, pleuron membrane; s, sternite; sep,
squamous epithelium; sp, spermatozoa; t, tergite; tr, trachea; tst, testis; vd, vas deferens; vs, vesicula seminalis
270 Spangenberg, Hu
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the penis and the ejaculatory apodeme (Fig. 10a). The dorsal
sclerite only covers the distal part of the duct, whereas the
penis–ejaculatory apodeme complex covers its entire length on
the ventral side (Fig. 10a). The ejaculatory duct is connected
with the dorsolateral distal end of the penis–ejaculatory
apodeme complex by membranes (Fig. 5f). Three successive
regions of the duct can be distinguished, each of approxi-
mately the same length. They differ morphologically and
histologically. From the area of fusion of the vesiculae
seminales, the proximal region abruptly bends downwards.
Its intima (i) is surrounded by a multiple-layered uniform
muscularis (mde, five to seven layers) (Figs 5f, 6g and 10a).
The orientation of the middle region is approximately parallel
to the postabdominal sternites and tergites. The muscularis
forms a hunch dorsally and is twice as thick as the layer on the
ventral side (Figs 5f and 10a). Beyond this thickened area, the
muscle layer ends on the dorsal side (Fig. 10a). A short
distance proximad the tip of the penis, the terminal tongue-
shaped region of the duct is continuous with the endophallus.
On the ventral side, the muscularis reaches the ventral base of
the endophallus (Fig. 10a).
Endophallus (eph)
The endophallus (eph) is an unpaired, membranous, dorso-
ventrally flattened tube. It is suspended by membranes on the
dorsal edges of the penis (Fig. 5d). Its surface is slightly
sinuate. A virga is not present (Fig. 5d).
Accessory glands
Two pairs of accessory glands are present. The anterior glands
(crag) are lobular and extend to segment VIII anteriorly
(Fig. 8). Both glands almost completely fill out the ventral
postabdominal region (Fig. 6i). The left gland overlaps the
right one ventrally (Figs 6i and 8c). The epithelium (gep) is
formed of cubic cells with the oval nuclei close to the basal poles
(Fig. 6j). The lumen is sparsely filled with granular secretions
(gsc) (Fig. 6j). The size of the lobes decreases slightly anteriorly,
whereas the posterior apical regions in the border region of
segments VIII and IX appear truncate (Fig. 8a,c). The short
ducts of the accessory glands (ocrg) originate at the postero-
median edges of the gland lobes (Fig. 8b,c). The ducts run
ventrad to the ejaculatory apodeme and form a partially
membranous unit with the distal end of these structures
(Figs 6h and 10a). The posterior glands (cdag) are distinctly
different and oval, and their size is only ca. 5% of that of the
anterior glands (Figs 8a,b and 10a). The lumen is compara-
tively narrow (Fig. 5f). The epithelium is similar to that of the
anterior glands (Figs 5f and 6j). The gland openings (ocdg) are
located in the dorsal region of the mid-part of the ductus
ejaculatorius (Figs 5f and 10a). Distinct gland ducts are
lacking.
Other organs such as the nervous system (nv), tracheae
(tr + dv), gut (g) or the Malpighian tubules (mal) are not
described here but shown in Figs 5, 6 and 10a. They are
indicated in the figures and facilitate the orientation.
(a) (b)
(c) (d)
Fig. 7. Bibio marci, male genital segment, schematic drawing, setae omitted. (a) caudal view (ind. 2); (b) dorsal view; (c) lateral view; (d) ventral
view into slightly evaginated genital segment, line drawing combined from CLSM and stereomicroscopic images (b–d: ind. 4). ac, anal cone; bgp,
fused bases of gonopods; dsdv, dorsal sclerite derivatives; ejap, ejaculatory apodeme; epr, epiproct; gc, gonocoxite; gcap, gonocoxal apodeme; gs,
gonostylus; hsr, heavily sclerotized region; mb, membrane; mbg, membranous gap; pe, penis; s, sternite; sap, subapical plate; t, tergite
The male postabdomen and reproductive system of Bibio marci 271
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(a)
(b)
(c)
Fig. 8. Bibio marci, male postabdomen with reproductive system, 3D reconstruction (ind. 3). Setae, muscularis of vasa deferentia and vesiculae
seminales omitted. (blue: sclerites, grey: membranes, orange: musculature). Dotted lines indicating planes of sections see Figs 5 and 6. (a) Dorsal
view, dorsal part of postabdomen including gonocoxal apodemes partly omitted; (b) mediosagittal view, left part of postabdomen omitted; (c)
ventral view, ventral part of postabdomen including sternal parts omitted. bgp, fused bases of gonopods; cdag, caudal accessory gland; ce, cerci;
crag, cranial accessory gland; de, ductus ejaculatorius; dorsal sclerite (=fusion of parameres); eph, endophallus; fvd, bridge-like fusion of vasa
deferentia; gc, gonocoxite; gcap, gonocoxal apodeme; gs, gonostylus; mde, muscularis of ductus ejaculatorius; ocrg, opening of cranial accessory
gland; pe, penis; plmb, pleuron membrane; s, sternite; t, tergite; tst, testis; vd, vas deferens; vs, vesicula seminalis
272 Spangenberg, Hu
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(a)
(b)
(c)
Fig. 9. Bibio marci, male postabdomen with musculature, 3D reconstruction (ind. 3). Setae omitted, only muscles of right side shown, pleural
muscles not reconstructed. Musculature of genital segment illustrated with greater magnification in Figs 10b, 11 and 12 (blue: sclerites, grey:
membranes, orange: musculature). Dotted lines indicate planes of sections in Figs 5 and 6. (a) Dorsal view, dorsal part of postabdomen including
parts of gonocoxal apodemes omitted; (b) parasagittal view, left part of postabdomen omitted; (c) ventral view, ventral part of postabdomen
including sternal parts sternites omitted. bgp, fused bases of gonopods; ce, cerci; epr, epiproct; gc, gonocoxite; gcap, gonocoxal apodeme; gs,
gonostylus; M, muscle with number (numbers corresponds to those in the text, Figs 5, 6, 10b, 11 and 12, Tables 2–4); plmb, pleuron membrane; s,
sternite; t, tergite
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Table 2. Bibio marci, male, musculature of postabdomen and segment IX
Origin Insertion Morphological description Function
M1 Membrane adjacent
to antero lateral margin
of segment IX, lateral
base of gonocoxite
(Figs 10b and 11)
Medially on anterior margin
of gonostylus (Figs 11 and 12a)
Largest muscle of segment IX,
flattened, almost filling entire
lateral half of gonocoxite
(Figs 5e, 6g, 10b, 11 and 12a);
longitudinal muscle
Adductor of gonostylus
M2 Membrane adjacent
to mediolateral side
of gonocoxite, anterior
1⁄3 of segment IX
(Figs 10b and 11)
Membranes adjacent with base
of gonostylus (Figs 11 and 12a)
Second largest muscle of segment
IX, flattened, filling half of lateral
part of gonocoxite (Figs 5a,c, 11
and 12a); longitudinal muscle
Abductor of gonostylus
M3 Membrane at dorsolateral
side of gonocoxite, anterior
lateral region of tergite IX
(Figs 5e and 10b)
Membrane adjacent with cusp-like
processes of tergite IX (Figs 5a,
10b and 12a)
Flattened, attached to gonocoxite
(Figs 5a,c,e and 12a ); dorsolateral
longitudinal muscle
(levator of gonocoxites)
M4 Membranes associated
with gonocoxal apodeme ⁄base
of gonocoxite, right body half
(Figs 5b, 11b and 12a)
Anterior margin of fused ventral
bases of gonopods left body half
(Figs 5b, 11b and 12a)
Disc-shaped, unpaired, located
between bases of gonocoxites
(Figs 5b and 12a); transversal muscle
(contraction of bases of
gonocoxites)
M5 Membranes ventrad to
base of epiproct, above
gut (Figs 5b and 10b)
Mediodorsal region of anal cone
(Fig. 12a)
Short transversal muscle (contraction of anal cone)
M6 Posterior margin of membranes
connected with gonocoxal
apodeme (Fig. 12a)
Apophysis of anal cone (Fig. 5b) Flattened, both halves arranged
asymmetrically (Fig. 12a); transverse
muscle (Fig. 10b)
(contraction of anal cone)
M7 Membranes ventrolaterad
with regard to penis
(Figs 5e, 10b and 11b)
Dorsal margin of fused posterior
parts of gonopods (Figs 5a,
11b and 12a)
Flattened (Figs 10b and 11b);
ventrolateral longitudinal muscle
(supporting of M8)
M8 Ventral region of proximal
part of ejaculatory apodeme
(Figs 6h, 10b and 11)
Membranes ventrolaterad to
penis, near origin of M7 (Figs
10b and 11b)
V-shaped, unpaired, base of V
enclosing ventral region of proximal
part of ejaculatory apodeme, arms are
flattened (Figs 10b and 11); ventral
longitudinal muscle
Penis–ejaculatory apodeme
complex is pushed caudad
during contraction
M9 Membranes associated
with dorsal sclerite (mbds),
in boundary region of
ejaculatory apodeme and
penis (Figs 5e, 10b and 11b)
Ventrolateral region of dorsal
sclerite, approximately half length
of penis, partly enclosing ‘‘wings’’
of dorsal sclerite (dsdv) (Fig. 11b)
Oval (Fig. 11b), ventral longitudinal
muscle
(stabilization ⁄fixation
of dorsal sclerite while
copulation)
M10 Ventral region of proximal
part of gonocoxal apodeme
(Figs 6h, 10b and 11)
Mediolateral boundary region of
ejaculatory apodeme and penis
(Figs 10b and 11a)
Bar-shaped (Fig. 10b); transverse
muscle
Movement of ejaculatory
apodeme (retreating and
raising during contraction;
lead it back in rest position
after copulation)
M11 Dorsal region of proximal
part of gonocoxal apodeme
(Figs 6h, 10b and 11)
Membrane at anterior, dorsolateral
region of genital segment (Figs
10b and 11b)
Bar-shaped (Fig. 10b); transverse
muscle
(depressor of gonocoxites)
274 Spangenberg, Hu
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Table 2. (Continued)
Origin Insertion Morphological description Function
M12 Inner proximal region
of gonocoxal apodeme
(Figs 10b and 11a)
Inner proximal region of gonocoxal
apodeme (Figs 10b and 11a)
Rod-shaped, curved over dorsal
region of muscularis of ductus
ejaculatorius (Fig. 12a), transverse
muscle
Stabilization of gonocoxal
apodemes
M13 Inner proximal region
of gonocoxal apodeme
(Figs 10b and 11a)
Anterior, lateral region of epiproct
(Fig. 10b)
Cone-shaped (Fig. 10b), longitudinal
muscle
(retraction ⁄stabilization of
epiproct during copulation)
M14 Membrane adjacent
to anterior margin of
tergite IX (Fig. 10b)
Two insertions: mid-part of dorsal
margin of gonocoxal apodeme and
anterolateral corner of epiproct
(Fig. 12a)
Propeller-shaped, asymmetrical muscle
pair: an additional fibre bundle of right
muscle reaches near to mid-part of
posterior margin of gonocoxal apodeme,
transverse muscle (Fig. 12a)
(retraction of epiproct ⁄anal
region, supporting of M13)
M15 Mid-part of inner
dorsal margin of
gonocoxal apodeme
(Figs 11a and 12a)
Membranous region between lateral
margin of epiproct and lateral margin
of gonocoxite (Fig. 12a)
Hammer-shaped (‘‘head’’ inserts at
ejaculatory apodeme), crosses M14;
transversal muscle (unclear if paired
or unpaired) (Figs 11a and 12a)
Unclear
M16 Membrane at anterior,
dorsolateral region of
genital segment (Fig. 11b)
Membranous region between lateral,
outer margin of epiproct and lateral,
inner margin of gonocoxite (Fig. 12a)
Tubular, crosses M14 (Figs 11a and 12a),
transversal muscle (unclear if paired
or unpaired)
Unclear
M17 Membrane at
ventrolateral side of
segment VIII (Fig. 9b,c)
Lateral at antecosta of genital
segment (Figs 10b and 11)
Flattened, tapering in caudal direction
(Fig. 9), longitudinal muscle
Contraction of sternal region
for breathing and haemolymph
flow
M18 Membrane at medioventral,
anterior region of segment
VIII (Fig. 9a,b)
Membrane at medioventral, posterior
region of segment VIII (Fig. 9)
Flattened, single muscle fibres of
right muscle end arch-shaped
medially (Figs 6j and 9a,b), ventral
longitudinal muscle
Contraction of sternal region
for breathing and haemolymph
flow
M19 Membrane at dorsolateral,
middle region of segment
VIII (Fig. 9c)
Dorsomedian margin of genital segment,
membrane at border region of tergite
VIII to IX (Figs 10b and 12a)
Bar-shaped (Figs 6i and 9b,c),
dorso-dorsolateral longitudinal
muscle
Contraction of tergal region for
breathing and haemolymph flow
M20 Membrane at posterior,
mediodorsal region of
segment VIII (Fig. 9c)
Lateral at antecosta of genital segment
(Figs 9b, 10b, 11 and 12a)
Bar-shaped, (two-armed) (Fig. 10b),
transversal muscle
Contraction of tergal region for
breathing and haemolymph flow
M21 Membrane at posterior,
mediodorsal region of
segment VIII
(Figs 10b and 12a)
Apophysis in mediodorsal border region
of segment IX to VIII (Figs 10b and 12a)
Relatively short muscle,
(Figs 10b and 12a), dorsal
longitudinal muscle
Extension of abdomen, pushing
segments apart (in interaction
with M24)
M22 Mediolateral, dorsal
region of segment
VII (Fig. 9b,c)
Mediodorsal border region of segment
VIII to VII (Fig. 9b,c)
Bar-shaped (Fig. 6k), dorso-dorsolateral
longitudinal muscle
Contraction of tergal region
for breathing and haemolymph
flow
M23 Membrane at
mediodorsal,
posterior region of
segment VII
(Fig. 9b,c)
Membrane at dorsolateral, anterior,
region of segment VIII (Fig. 9b,c)
Bar-shaped (Fig. 9c), transversal muscle Contraction of tergal region for
breathing and haemolymph flow
M24 Mediodorsal, posterior
region of segment
VII (Fig.9b,c)
Membrane at mediodorsal border
region of
segment VII to VIII (Fig. 9b,c)
Bar-shaped (Fig. 9c), relatively short
muscle, dorsal longitudinal muscle
Extension of abdomen, pushing
segments apart (in interaction
with M21)
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Table 2. (Continued)
Origin Insertion Morphological description Function
M25 Membrane at posterior,
dorsolateral
region of segment
VII (Fig. 9b,c)
Dorsolateral border region of segment
VII to VIII (Fig. 9b)
Bar-shaped (Fig. 9b), dorsolateral
longitudinal muscle
Extension of abdomen,
pushing segments apart
M26 Membrane at
dorsolateral, median region
of segment VII (Fig. 9b,c)
Lateral, posterior region of segment
VII (Fig. 9b,c)
Rod-shaped (Fig. 6k), insertion not
clearly, lateral longitudinal muscle
(probably supporting of
breathing and haemolymph
flow)
M27 Ventrolateral, anterior region
of segment VII (Fig. 9b,c)
Lateral pleuron membrane at posterior
region of segment VII (Fig. 9b,c)
Rod-shaped (Fig. 9b), insertion not
clearly, dorsoventral muscle
Unclear
M28 Membrane at anterior,
ventral region of
segment VII (Fig. 9)
Apophysis at anterior, ventral margin
of segment VIII (Fig. 9a,b)
Flattened (Fig. 9a), ventral
longitudinal muscle
Contraction of sternal region
for breathing and haemolymph
flow
M29 Antecosta at
ventrolateral region of
segment VIII (Fig. 9a,b)
Membrane at ventral, posterior region
of segment VII (Fig. 9a,b)
Flattened (Fig. 9a,b), transversal
muscle
Contraction of sternal region
for breathing and haemolymph
flow
M30 Apophysis at mediodorsal
region of segment
VI (Fig. 9b,c)
Trachea at dorsolateral, posterior region
of segment VI (Fig. 9b,c)
Bar-shaped (Fig. 9b,c), transversal
muscle
Contraction of tergal region
for breathing and haemolymph
flow
M31 Membrane at dorsolateral,
anterior region
of segment VI (Fig. 9b,c)
Mediodorsal, posterior region of
segment VI (Fig. 9b,c)
Bar-shaped (Fig. 9b), insertion
not clearly, dorso-dorsolateral
longitudinal muscle
Contraction of tergal region for
breathing and haemolymph flow
M32 Dorsolateral,
median region of
segment VI (Fig. 9b,c)
Dorsolateral, posterior region of
segment VI (Fig. 9b,c)
Bar-shaped (Fig. 9b), insertion
and origin not clearly, lateral
longitudinal muscle
Extension of abdomen, pushing
segments apart
M33 Dorsolateral, anterior
region of
segment VI (Fig. 9b,c)
Lateral, posterior region of segment
VI (Fig. 9b,c)
Bar-shaped (Fig. 9b), insertion
and origin not clearly, lateral
longitudinal muscle
Probably supporting of
breathing and haemolymph
flow
M34 Apophysis at
mediodorsal region of
segment VI (Fig. 9b,c)
Mediodorsal, posterior region
of segment
VI (Fig. 9b,c)
Bar-shaped (Fig. 9c), insertion
not clearly, dorsal longitudinal
muscle
Extension of abdomen,
pushing segments apart
M35 Membrane at
ventrolateral, anterior
region of segment
VI (Fig. 9b,c)
Membrane at lateral, median region
of segment VI (Fig. 9b,c)
Rod-shaped (Fig. 9b),
dorsoventral muscle
Unclear
M36 Membrane at
medioventral,
anterior region
of segment
VI (Fig. 9a,b)
Membrane at ventral border region of
segment VI to VII (Fig. 9a,b)
Flattened, posterior tongue-shaped
extension overlaps M37 (Fig. 9a),
dorsal longitudinal muscle
Contraction of sternal region for
breathing and haemolymph flow
M37 Membrane at
medioventral,
posterior region
of segment VI
(Fig. 9a,b)
Membrane at ventro- to ventrolateral
border region of segment VI to
VII (Fig. 9a,b)
Flattened (Fig. 9a); transversal
muscle
Contraction of sternal region for
breathing and haemolymph flow
The functional interpretations are hypothetical, and uncertain interpretations are in parentheses. Numbers correspond with those in the text, in Figs 5, 6, 9, 10b, 11 and 12a, and in Tables 3, 4 and 5.
276 Spangenberg, Hu
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Results of the phylogenetic analyses
The analyses with nona (ratchet, 1000 replicates) yielded two
minimum-length trees with 44 steps (CI: 0.68). A clade
Bibionidae (Bibionomorpha) + Tabanidae (Brachycera) was
unambiguously confirmed and is supported by six unambig-
uous synapomorphies, four of which are non-homoplasious.
Two identical minimum-length trees with the same number of
steps (44) were obtained with TNT (Fig. 13; list of phyloge-
netically relevant characters; see Appendix S1).
Potential synapomorphies of Bibionmorpha (represented by
Bibio,Axymyia and Sylvicola) and Brachycera retrieved with
the limited character set and taxon sampling are the fusion of
sternum IX with the gonocoxites (1), the fusion of the
parameres forming the dorsal sclerite (7) and the presence of
Table 3. Bibio marci, musculature of male postabdomen, nomenclatures following Blaschke-Berthold (1994), Ovtshinnikova (1989), Maki (1936)
and von Ke
´ler (1963)
Present
study
Blaschke-Berthold
(1994)
Ovtshinnikova
(1989) Maki (1936)
von Ke
´ler
(1963)
M1 M1 M27 ? ?
M2 M2 M28 ? ?
M3 – M5
1
??
M4 M8 – ? ?
M5 – M29 ? M. tergorectalis dorsalis
M6 – – ? M. tergorectalis lateralis
M7 – M2 ? ?
M8 M5 M31 ? ?
M9 M9 M39 ? ?
M10 M3 M30 ? ?
M11 M7 M5
2
??
M12 – – ? ?
M13 M4 – ? ?
M14 – M21 ? ?
M15 – – ? ?
M16 – – ? ?
M17 ? M18
2
258 M. antecostaantecostalis
urosterni medialis VIII
M18 ? M18
1
259 M. antecostaantecostalis
urosterni lateralis VIII
M19 ? M19 250 M. antecostaantecostalis
uronotum lateralis VIII
M20 ? M20 249 M. antecostaantecostalis
uronotum medialis VIII
M21 ? – 251 M. uronotoantecostalis
obliquomedialis VIII
M22 ? ? 250 M. antecostaantecostalis
uronotum lateralis VII
M23 ? ? 249 M. antecostaantecostalis
uronotum medialis VII
M24 ? ? 251 M. uronotoantecostalis
obliquomedialis VII
M25 ? ? 252 M. uronotoantecostalis
obliquolateralis VII
M26 ? ? 255 –
M27 ? ? 268 M. apertor spiraculi VII
M28 ? ? 259 M. antecostaantecostalis
urosterni lateralis VII
M29 ? ? 258 M. antecostaantecostalis
urosterni medialis VII
M30 ? – 249 M. antecostaantecostalis
uronotum medialis VI
M31 ? M41 250 M. antecostaantecostalis
uronotum lateralis VI
M32 ? – 252 M. uronotoantecostalis
obliquolateralis VI
M33 ? M42 255 –
M34 ? – 251 M. uronotoantecostalis
obliquomedialis VI
M35 ? – 268 M. apertor spiraculi VI
M36 ? M44 259 M. antecostaantecostalis
urosterni lateralis VI
M37 ? – 258 M. antecostaantecostalis
urosterni medialis VII
(–) muscle absent; (?) not mentioned by author or unambiguous figure missing in the cited works. It is assumed that the musculature within Bibio
is constant. Some minute muscles cannot be visualized by dissections and a stereomicroscope (e.g. Ovtshinnikova 1989).
The male postabdomen and reproductive system of Bibio marci 277
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Table 4. Bibio marci, male, musculature of postabdomen and genital segment and the homologous muscles of selected species
Bibio marci
Sylvicola
fenestralis
Axymyia
furcata
Tabanus
sulcifrons
1
Trichocera
annulata
2
Limonia
tripunctata
3
Culiseta inornata
4
Nymphomyia
Nannochorista
holostigma
5
M1 M1 M1 187 d M27 M. gonocoxito-gonostylaris
basalis
M1 M. add. gonst.
M2 M2 M2 188 e M28 M. gonocoxito-gonostylaris
basimedialis
M2 M. abd. gonst.
M3 M3 M3 184 ? (ps. a) M16 – M3 –
M4 M4 M4 186 – – – – –
M5 M5 M5 196 b – – – –
M6 M6 M6 195 c – – – –
M7 M7 M7 ? (ps. 190) f – ? (ps. M. sterno-basalis lobalis) M7 ? (ps. M. retr. phal.)
M8 M8 M8 192 n M31 ⁄m
6
– ? (ps. M8) ? (ps. M. depr. cam.)
M9 M9 M9 – k – – – –
M10 M10 M10 193 g M2 ? (ps. M. gonocoxitalis
apodemo-parameralis)
M10 ? (ps. M. lev. cam)
M11 M11 M11 185 j – – ? (ps. M11) –
M12 M12 – – – – – – –
M13 M13 – ? (ps. 191) ? (ps. i) – – – –
M14 M14 M14 ? (ps. 183) ? (ps. h) ? (ps. M5) – ? (ps. M14) –
M15 – – – – – – – –
M16 – – – – – – – –
M17 M17 M17 – o ? (ps. M18) – ? (ps. M17) ?
M18 M18 M18 180 p ? (ps. M18) ? (ps. M. sternalis
antecosto-sternalis)
M18 ?
M19 M19 M19 174 ? (ps. t) ? ? (ps. M. tergalis
antecosto-tergalis)
M19 ?
M20 M20 M20 176 – ? – – ?
M21 M21 M21 – ? (ps. u) ? – – ?
M22 M22 M22 160 q ? M. tergalis
antecosta-antecostalis
M22 ?
M23 M23 M23 162 ? ? – ? ?
M24 – M24 – ? ? ? (ps. M. Tergo-
acrotergitalis)
M24 ?
M25 M25 M25 – ? ? – ? ?
M26 M26 M26 161 ? (ps. s) ? – ? ?
M27 M27 M27 165 ? ? – ? ?
M28 M28 M28 166 ? ? M. sternalis antecosto-
antecostalis
M28 ?
M29 M29 M29 – ? ? – M29 ?
M30 ? M30 149 ? ? – ? ?
M31 M31 M31 150 ? ? – ? ?
M32 – ? (ps. M32) – ? ? – ? ?
M33 – M33 – ? ? – ? ?
M34 – M34 – ? ? ? (ps. M. Tergo-
acrotergitalis)
??
M35 M35 M35 155 ? ? – ? ?
278 Spangenberg, Hu
¨nefeld, Schneeberg and Beutel
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Table 4. (Continued)
Bibio marci
Sylvicola
fenestralis
Axymyia
furcata
Tabanus
sulcifrons
1
Trichocera
annulata
2
Limonia
tripunctata
3
Culiseta inornata
4
Nymphomyia
Nannochorista
holostigma
5
M36 M36 M36 156 ? ? M. sternalis antecosto-
antecostalis
??
M37 M37 M37 157 ? ? – ? ?
– – ? (ps.189) 189 – – – – –
– – – 176 – – – – ?
– – – 170 – – – – ?
– – – 175 – – – – –
– – – 163 – – – – –
– – – – – M30 – – –
–––––M1– – –
– – – – – M8 (1) – – –
– – – – m M8 (2) – – –
––––r? – – ?
– – – – – – M. sterno-aedeagalis – –
– – – – – – M. sterno-tergalis
annularis
––
– – – – – – M. gonocoxitalis
apodemo-sternalis
––
– – – – – – M. gonocoxitalis
apodemo-tergalis
apicalis
––
– – – – – – M. gonocoxitalis apodemo-tergalis basalis – –
– – – – – – M. tergo-dorsalis armalis – –
– – – – – – – – M. teg. mpo.
– – – – – – – – M. teg. lpo.
– – – – – – – – M. dil. dej.
– – – – – – – – M. protr. phal.
(–) muscle absent; (?) not mentioned by the author or figures and descriptions for a clear conclusion are absent in the cited works. ps, possibly.
1
Bonhag (1951).
2
Neumann (1958).
3
Paramonov (2004).
4
Owen (1980).
5
Mickoleit (2008).
6
Hu
¨nefeld and Beutel (2005).
The male postabdomen and reproductive system of Bibio marci 279
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the muscles M4 (11) and M20 (20) (Fig. 13). The presence of
M21 (char. 21) and M25 (char. 23) (and probably M14) are
potential autapomorphies of Bibionomorpha. However, this
requires verification in the other families of this large lineage
(so far, only confirmed for Bibionidae, Axymyiidae and
Anisopodidae).
Discussion
Function
The shape of the penis of Bibio markedly differs from the
condition suggested for the dipteran groundplan (Wood
1991) (elongate sclerotized tube with terminal opening): it is
short and stout, dorsoventrally flattened, anteriorly produced
into a stout ejaculatory apodeme and only ventrally covered
by a huge, pan-shaped sclerite, whereas the dorsal surface is
membranous and includes the phallotrema. This clearly
indicates the mode of sperm transfer involving a spermato-
phore (see Wood 1991; Blaschke-Berthold 1994; Sinclair et al.
2007). At least three sclerotized elements and two muscles are
directly involved in operating the copulatory apparatus and
in the spermatophore transfer: the gonocoxal apodemes, the
dorsal sclerite, the penis–ejaculatory apodeme complex, and
M8 and M9. The following tentative interpretations of the
movements during copulation are based on the morpholog-
ical conditions and not on direct observations. The penis–
ejaculatory apodeme complex is pulled posteriorly by M8,
resulting in the extrusion of the penis (see Fig. 11). At the
same time, the posterior part of the dorsal sclerite is lifted,
which results in the release of the spermatophore. In the
resting position, the entire dorsal surface of the penis
including the phallotrema is concealed by the dorsal sclerite.
The dorsal sclerite forms paired rotational joints with the
gonocoxal apodemes (Fig. 12b,c; indicated by arrows).
Therefore, its movability is restricted to a rotation in a single
plane. M9 extends between membranes adjacent to the basal
region of the penis and the apices of the wing-like lateral
extensions of the dorsal sclerite. Resulting from its contrac-
tion, the anterior part of the dorsal sclerite is pulled ventrad.
Rotation around the joints then results in the elevation of the
posterior part of the sclerite. The spermatophore is pressed
out by the muscularis of the ejaculatory duct. During
copulation, the femaleÕs postabdomen is probably only
clasped by the male gonostyli. The fusion of the gonocoxites
with each other and with tergite IX results in a strong
limitation of their movability (see Wood 1991).
Musculature
Compared to the preceding segments, three muscles are
missing in segment VIII of Bibio. These are the equivalents
of M35 ⁄M27, M33 ⁄M26 and M32 ⁄M25 (see Fig. 9b). The
position in the preceding segments suggests that the homo-
logue of M32 ⁄M25 may fused with M20, and this would be
consistent with its bifurcate condition (Fig. 9b). Considering
the different placement of the homologues of M35 ⁄M27 and
M33 ⁄M26, a fusion appears less likely in their case. Appar-
ently, the clarification of these issues requires ontogenetic
investigations. M17 (equivalent to M37 ⁄M29) in segment VIII
is also modified. It is larger and inserted on the mediolateral
margin of the following segment (Fig. 9b).
In some cases, the homologization of the postabdominal
muscles of Bibio with those occurring in other potentially related
taxa is problematic (see also Table 4, uncertain homologues in
parentheses). For a detailed discussion, see Appendix S2.
Dipteran groundplan, phylogeny and character evolution
The putative groundplan configuration of the external male
genitalia of Diptera was outlined as follows by Wood (1991):
(a)
(b)
Fig. 10. Bibio marci, male genital segment, sagittal section, left part of
segment omitted, 3D-reconstruction (ind. 3). Setae omitted (blue:
sclerites, grey: membranes, orange: musculature). Vertical dotted lines
indicating planes of sections see Figs 5 and 6. (a) Reproductive organs
and sclerotized parts; (b) Musculature and sclerotized parts. ac, anal
cone; bgp, fusion bases of gonopods; cdag, caudal accessory gland; ce,
cerci; crag, cranial accessory gland; de, ductus ejaculatorius; ds, dorsal
sclerite (=fusion of parameres); ejap, ejaculatory apodeme; eph, en-
dophallus; epr, epiproct; fvd, bridge-like fusion of vasa deferentia; g,
gut; gc, gonocoxite; gonocoxal apodeme; gs, gonostylus; M, muscle
with appropriate number (number of muscle corresponds to number in
text, Figs 5, 6, 9, 11 and 12, Tables 2–4); mbvd, membrane ventral of
dorsal sclerite; mde, muscularis of ductus ejaculatorius; ocrg, opening
of cranial accessory gland; pe, penis; pht, phallotrema; sap, subapical
plate; t, tergite with appropriate number; vs, vesicula seminalis
280 Spangenberg, Hu
¨nefeld, Schneeberg and Beutel
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(a)
(b)
Fig. 11. Bibio marci, male, genital segment, musculature and sclerotized parts of the genital apparatus, 3D reconstruction (individual 3). Setae
omitted (blue: sclerites, grey: membranes, orange: musculature). a: dorsal view, obliquely sectioned surface: dorsad the subapical plate to the
dorsal margin of the genital segment, muscle on left side of M13, M17 and M20 omitted; b: ventral view, external membranes and sclerotized
exoskeleton semi-transparent, muscle on left side of M1, M2, M13, M17, M19 and M20 omitted. ce, cerci; ds, dorsal sclerite (=fusion of
parameres); dsdv, dorsal sclerite derivatives; ejap, ejaculatory apodeme; gc, gonocoxite; gcap, gonocoxal apodeme; gs, gonostylus; M, muscle with
appropriate number (number of muscle corresponds to number in text, Figs 5, 6, 9, 10b and 12, Tables 2–4); mb, membrane; pe, penis; sap,
subapical plate; se, segment with appropriate number; t, tergite
The male postabdomen and reproductive system of Bibio marci 281
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(a)
(b) (c) (d)
Fig. 12. (a) Bibio marci, male, genital segment, caudal view, 3D reconstruction (individual 3). Setae omitted (blue: sclerites, grey: membranes,
orange: musculature), external membranes and sclerotized exoskeleton semi-transparent, muscles on left side of M13, M17, M19, M20 and M21
omitted. Arrows indicate the hinge joints; (b–d) aedeagus, gonocoxal apodemes and associated membranes, membranes semi-transparent (b: dorsal
view; c: lateral view; d: ventral view). ds, dorsal sclerite (=fusion of parameres); dsdv, dorsal sclerite derivatives; ejap, ejaculatory apodeme; gc,
gonocoxite; gcap, gonocoxal apodeme; gs, gonostylus; M, muscle with appropriate number (number of muscle corresponds to number in text, Figs 5,
6, 9, 10b and 11, Tables 2–4); mb, membrane; mbds, membranes associated with dorsal sclerite; pe, penis; s, sternite; t, tergite
282 Spangenberg, Hu
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tergite and sternite IX (=epandrium and hypandrium)
strongly converging or fused, gonopods paired and 2-seg-
mented, median tubular penis, paired plate-like parameres,
anal cone distinct, with epiproct and hypoproct. This was
further specified by McAlpine (1981): Ô[…] a reduced tergite 10,
which is closely associated with tergite 9 […]Õ,Ô[…] a simple
sternite 10 (ventral epandrial plate); the vestigial tergite and
sternite of segment 11, more or less consolidated to form a
proctiger bearing the cerci and the anus […]Õ.Bibio markedly
differs from this condition. A distinct sternite IX is not present.
It is either fused with the ventral sides of the gonocoxites or
reduced. The gonocoxites are fused with each other and with
tergum IX (Wood 1991), the parameres are medially fused
(Ôdorsal scleriteÕ; Wood 1991), the shape of the penis is
modified, the epiproct is divided along the median line
(McAlpine 1981), and the cerci are shifted ventrad (Wood
1991). Moreover, a distinct tergite X is absent in B. marci. The
dorsal sclerite is part of the groundplan of Bibionomorpha
(Blaschke-Berthold 1994).
Blaschke-Berthold (1994) investigated the closely related
B. leucopterus, which is characterized by a tergite X placed
between the epandrium and the cerci on the evaginated anal
complex (Blaschke-Berthold 1994: Fig. 54). As the specimens
available to us were not fixed with the anal complex evaginat-
ed, the comparison with the results of Blaschke-Berthold
(1994) turned out as difficult. However, semi-thin sections
revealed that tergite X is indeed absent in B. marci. Fig. 54 of
the study of Blaschke-Berthold (1994) suggests that tergite X is
either fused with the cerci or with tergite IX. Considering its
relative position, it appears likely that the Ôsubapical plateÕ(see
chapter ÔExoskeletonÕ) represents the hypoproct. Epiproct and
hypoproct are modified elements of segment XI or derivatives
of a product of fusion of segments X and XI (see Snodgrass
1936; Seifert 1999).
Even though a considerable degree of variation can be
observed, the general configuration of the postabdominal
sclerites in Bibio is similar to what is found in other
bibionomorph families. The posterior margin of the plate-like
tergite IX is often produced into a pair of cusp-like processes,
as it is the case in Anisopodidae (Haenni 1997), Cecidomyiidae
(Abe et al. 2011a,b), Diadocidiidae (Polevoi 1996) and My-
cetophilidae (processes minute in Exechia; Kjaerandsen et al.
2007). Tergite IX and the dorsolateral margins of the
gonocoxites converge strongly or are fused in Anisopodidae
(Haenni 1997), Cecidomyiidae (Feltiella, Abe et al. 2011a;
Endaphis, Abe et al. 2011b) and Lygistorrhinidae (Blagoderov
et al. 2010). In Pachyneuridae, sternite and tergite IX are fused
and form a ring (Wood 1981b; Krivosheina 1997a). In
Cramptonomyia spenceri, sternite IX is separated from the
anteroventral margin of the gonocoxite (Wood 1981b),
whereas it is fused in Pachyneura fasciata (Krivosheina
1997a). There is also a high degree of variation within
Brachycera: in some Rhagionidae, Bombyliidae, Therevidae
and Asilidae, sternite IX is a separate structure (Sinclair et al.
1994). The gonocoxites are ventromedially fused to varying
degrees in most of bibionomorph representatives studied so
far. At least the bases are fused in representatives of Aniso-
podidae (Sylvicola; Peterson 1981; Haenni 1997), Bolitophil-
idae (Bolitophila; Polevoi 1996), Cecidomyiidae (Abe et al.
2011a,b), Diadocidiidae (Diadocidia; Polevoi 1996) and Lyg-
istorrhinidae (Asiorrhina; Papp 2002). Extensive fusion (more
than two-third of the total length) is documented for the
members of Mycetophylidae (Rymosia,Neuratelia; Polevoi
et al. 2006). Gonostyli are generally present and unsegmented
in all families studied yet. However, they display a high degree
of structural variation, as for instance conspicuous apical
bifurcations in Lygistorrhinidae (Asiorrhina; Blagoderov et al.
2009). Conspicuous modifications occur in some mycetophi-
lids, such as, for instance, a gonostylus with a largely separated
dorsal and ventral ÔarmÕin Neuratelia (Polevoi et al. 2006), or a
trifurcate gonostylus in Exechia (dorsal, medial and ventral
branch; Kjaerandsen et al. 2007).
The penis is short, and its sclerotized element appears plate-
or pan-like in Bolitophilidae (Polevoi et al. 2006), Diadocidii-
dae (Polevoi et al. 2006), Lygistorrhinidae (Blagoderov et al.
2009) and Mycetophilidae (Kjaerandsen et al. 2007). This
Nannochoristidae
Bibionidae
Culicidae
Tipulidae
Trichoceridae
Tabanidae
Nymphomyiidae
Anisopodidae
Axymyiidae
8
1
6
0
3
1
14
1
13
1
12
1
22
0
8
1
6
1
2
0
22
0
9
1
27
1
5
0
3
1
9
1
23
1
21
1
14
1
2
0
20
1
13
1
12
1
11
1
7
1
1
1
10
1
Bibionomorpha
Fig. 13. Phylogenetic diagram[eight
dipteran taxa and Nannochorista
(Mecoptera)] generated with nona
and tree analyses using new tech-
nology (strict consensus of two
trees). Full circles show unambigu-
ous apomorphic character states,
and white circles show homoplas-
ious changes. The numbers corre-
spond to those in chapter 4 and
Table 5
The male postabdomen and reproductive system of Bibio marci 283
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Nannochoristidae
Siphonaptera
Deuterophlebiidae
Ptychopteridae
Perissomatidae
Trichoceridae
Tipulidae
Blephariceridae
Tanyderidae
Psychodidae
Axymyiidae
Nymphomyiidae
Thaumaleidae
Simulidae
Chironomidae
Ceratopogonidae
Dixidae
Corethrellidae
Chaoboridae
Culicidae
Brachycera
Anisopodidae
Canthyloscelidae
Scatopsidae
Mycetophilidae
Pachyneuridae
Bibionidae
Cecidomyiiade
Mycetophilidae
Sciaridae
Nannochoristidae
Deuterophlebiidae
Nymphomyiidae
Trichoceridae
Tipulidae
Ptychopteridae
Psychodomorpha
Ceratopogonidae
Chironomidae
Thaumaleidae
Simuliidae
Dixidae
Culicidae
Corethrellidae
Chaoboridae
Perissommatidae
Anisopodidae
Canthyloscelidae
Scatopsidae
Axymyiidae
Bibionidae
Pachyneuridae
Ditomyiidae
Manotidae
Diadocidiidae
Sciaridae
Cecidomyiidae
Lygistorrhinidae
Mycetophilidae
Keroplatidae
Bolitophilidae
Nemestrinidae
Xylophagidae
Vermileonidae
Rhagionidae
Rhagionidae
Pelecorhynchidae
Oreoleptidae
Athericidae
Tabanidae
Acroceridae
Hilarimorphidae
Stratiomyomorpha
Asiloidea
Eremoneura
BIB
CUL
PTY
PSY
TIP
TIP
CUL
2
1
1
3
2
4
3
5
6?
6?
6
6
6
6
4
6
6
6
6
5
BIB
BRA
(a) (b)
Fig. 14. (a) Phylogeny after Bertone et al. 2008 (modified); (b) Phylogeny after Wiegmann et al. 2011 (modified). BIB, Bibionomorpha; BRA,
Brachycera; CUL, Culicomorpha; PSY, Psychodomorpha; TIP, Tipulomorpha. Potential apomorphies of male postabdomen mapped on trees.
[1] sternite IX adjacent to tergite IX or fused with it to form a ring-shaped structure; tergite IX not fused with gonocoxites; hypoproct absent;
cerci absent; penis tubular; paired parameres, not fused with each other; muscularis of ductus ejaculatorius single-layered to slightly developed;
homologous sperm pump; M4 absent; muscle m present. [2] gonocoxites not fused with each other; presence of M5, M6, M9, M11, M17, M18 and
r; M20 absent. [3] absence of M5, M6 and M11. [4] sternite IX adjacent to tergite IX or fused with it forming a ring-shaped structure; tergite IX
fused with gonocoxites; gonocoxites fused with each other; cerci present; paired parameres, not fused with each other; penis tubular; muscularis of
ductus ejaculatorius single-layered to slightly developed; probably liquid sperm; presence of M3, M7, M10, M18, M19, M22, M24, M28, M29. [5]
sternite IX adjacent to tergite IX or fused with it to form a ring-shaped structure; tergite IX not fused with gonocoxites; gonocoxites not fused
with each other; penis formed by two single sclerites; paired parameres, not fused with each other; sperm transfer via spermatophore; absence of
M3, M4, M5, M6, M9, M11, M17, M19, M20, M21, M23, M25, M26, M27, M29, M30, M31, M32, M33, M35, M37, muscle m and r. [6] sternite
IX fused with gonocoxites; hypoproct present; parameres fused to a single sclerite [but parameres varying from two separated structures to a
fused shield-like complex in Anisopodidae (Peterson 1981)]; presence of muscles M4, M20, M23, M26, M27, M31, M35 and M37
284 Spangenberg, Hu
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suggests sperm transfer via spermatophores as it is also the
case in Bibionidae. According to Blaschke-Berthold (1994),
there are three different modes of sperm transfer in Bibiono-
morpha: (1) spermatophore (e.g. Bibionidae), (2) ‘‘simple
device for pressing out free sperm’’ in Sciaridae and (3) more
complex sperm pumps in Ditomyiinae (Ditomyia) and Bolito-
philiniae. The presently available information suggests that a
sperm pump is also present in Canthyloscelidae (Hyperoscelis;
Hutson 1977) and Scatopsidae (Coboldia,Apiloscatopse; Cook
1981; Sinclair et al. 2007). However, this is only documented
with line drawings without lettering (Hutson 1977: figs. 15–17;
Cook 1981: figs. 18, 19; no figure in Sinclair et al. 2007). A
short functional interpretation is given by Sinclair et al. (2007).
Therefore, further investigations of males of these families are
required. The tube-like penis and the muscularis of the
ejaculatory duct in Axymyia (Axymyiidae) also suggest the
transfer of liquid sperm by a pumping mechanism. As an
additional exception within Bibionomorpha, Wood (1991)
mentioned the genus Hesperinus, which he considered as the
sister group of the remaining Bibionomorpha, and for which
he assumed sperm transfer with a sperm pump. However,
WoodÕs placement of this taxon was not supported by specific
arguments, and a position as sister group of Bibionidae
(Blaschke-Berthold 1994) or inclusion in Bibionidae (Fitzger-
ald 2004) is more likely (see also Bertone et al. 2008;
Wiegmann et al. 2011). In recent studies on Hesperinus, no
direct evidence for the presence of a sperm pump was
presented (Sinclair 2000; Sinclair et al. 2007; Papp and
Krivosheina 2009). The mode of sperm transfer in Sylvicola
(Anisopodidae) is unclear. The weakly developed muscularis
of the ejaculatory duct and its coiled condition (which would
impede the transport of a spermatophore) suggest the transfer
of liquid sperm. However, this would also require a long tube-
like penis, which is absent in the studied specimen. Peterson
(1981) documented a tubular ÔaedeagusÕin connection with an
Ôaedeagal guideÕ. This, in comparison with the studies of Dahl
(1980) and Sinclair et al. (2007), indicates indeed liquid sperm
transferred by a sperm pump.
Considering the presently available information, we agree
with Blaschke-Berthold (1994), who suggested a spermato-
phore in combination with a powerful muscularis of the ductus
ejaculatorius as a groundplan feature of Bibionomorpha.
Transfer of liquid sperm and a specific type of sperm pump
(differing from that of Mecoptera [excl. Boreidae] and Sipho-
naptera, respectively; Hu
¨nefeld and Beutel 2005) are arguably
apomorphic groundplan features of Diptera (Hennig 1973;
Hu
¨nefeld and Beutel 2005). Reduction in this structural
complex and a secondary shift to sperm transfer via sperma-
tophores took apparently place several times independently
(Brachycera: e.g. Drosophila [Drosophilidae], Glossina [Glos-
sinidae], Pollock 1972; Sepsis cynipsea [Sepsidae], Martin and
Hosken 2002; Culicomorpha, Wood 1991; see also Sinclair
et al. 2007 and Kotrba 1996). As the absence of the pumping
mechanism is likely a bibionomorph groundplan autapomor-
phy (see above), liquid sperm transfer and the presence of a
sperm pump have to be addressed as secondary character
reversals, which probably occured several times independently
within the group.
The arrangement of the internal parts in Bibio largely
corresponds with the situation suggested for the dipteran
groundplan by Zissler (1999). Differences and derived condi-
tions are the S-shaped vasa deferentia and the presence of a
second pair of accessory glands. In contrast to the study of
Blaschke-Berthold (1994) and the results of this study, Sinclair
et al. (2007) interpreted the paired seminal vesicles of B. flavi-
halter as a fused 2-chambered Ôaccessory gland complexÕ.
Caudal and cranial accessory glands were not illustrated (Figs
3C, 4 in Sinclair et al. 2007). However, it is likely that in all
species of the genus Bibio, two pairs of such glands are present.
These differences are probably due to different techniques of
examination. Thus, the glands could not be identified by
dissection alone, a technique applied by Sinclair et al. (2007).
Section series allow an unambiguous classification of the
accessory glands. We agree with Sinclair et al. (2007) that at
least different densities in the lumen of the seminal vesicles
indicate a kind of compartimentation.
For a more reliable interpretation of character evolution, we
carried out a formal analysis with a limited data set of 27
postabdominal characters of eight dipteran terminals and
Nannochorista as an outgroup (see chapter 4, Table 5; Fig. 13:
strict consensus of two trees). Bibio,Sylvicola and Axymyia
form a clade (presence of M14, M21, M25) placed as sister
group of Tabanus (Tabanidae) representing the Brachycera.
This result is fully compatible with the hypothesized sister
group relationship between the two large dipteran lineages
postulated by Hennig (1973) and Wiegmann et al. (2011). The
close affinity between Bibionomorpha and Brachycera is also
conform with Bertone et al. (2008). However, Axymyiidae,
which form a clade with Bibio and Sylvicola in our analyses
(see above), were placed outside of Bibionomorpha in that
study.
Whether the fusion of sternum IX with the gonocoxites (1)
and the fusion of the parameres with the dorsal sclerite (7) are
indeed groundplan features of Bibionomorpha (and then a
Table 5. Character states of selected taxa of lower diptera with the outgroup Nannochorista
111111111122222222
123456789012345678901234567
Nannochoristidae 0 1 0 ? 1 0 ? ? 2 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? 0
Bibionidae 1 0 0 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0
Axymyiidae 1 0 0 1 1 3 1 0 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 0
Anisopodidae 1 0 0 1 1 3 – 0 ? 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 0 0
Tabanidae 1 1 0 1 1 1 1 1 0 1 1 1 1 0 1 0 ? ? 0 1 0 0 0 0 0 0 0
Culicidae 0 1 1 ? 1 2 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 ? 0
Tipulidae 0 1 1 0 0 3 0 0 1 1 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? 1
Trichoceridae 0 1 1 0 0 3 0 0 1 ? 0 1 1 1 1 0 ? ? 1 0 ? ? ? ? ? ? 1
Nymphomyiidae 0 0 0 0 1 3 0 0 1 1 0 0 0 0 ? 0 0 ? ? 0 0 1 ? 1 ? ? 0
The numbers of characters correspond with those in Appendix S1.
The male postabdomen and reproductive system of Bibio marci 285
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synapomorphy with Brachycera) is still ambiguous, as there is
no reliable information on the condition in most bib-
ionomorph families.
In addition to our own analysis, character states were
mapped on two recently published phylogenies (Bertone et al.
2008; Wiegmann et al. 2011; Fig. 14). As already suggested by
Hennig (1973), in both studies, Bibionomorpha (with or
without Axymyiidae) turned out as the sister group of
Brachycera. Thus, the following characters can be considered
as new potential synapomorphies of Bibionomorpha and
Brachycera: presence of the hypoproct and of M4, M20,
M23, M26, M27, M31, M35 and M37. Sperm transfer with a
spermatophore occurs in Bibionomorpha, Brachycera and
Culicomorpha. The presence of a sperm pump and transfer of
liquid sperm can be assumed for the groundplan of Diptera
(see Hennig 1973; Wood 1991; Hu
¨nefeld and Beutel 2005).
Thus, the acquisition of a spermatophore as a character
reversal probably took place several or multiple times inde-
pendently within Diptera, that is, in several groups of
Bibionomorpha (sperm pump preserved in Sciaridae, Dito-
myia,Axymyia and Bolitophilinae, and probably also in
Canthyloscelidae, Scatopsidae, Sylvicola and Hesperinus), in
Brachycera, and also in culicomorph lineages [e.g. Chironom-
idae (Nielsen 1959); Simuliidae (Davies 1965)].
It is evident that characters of the male genital apparatus are
not sufficient to reconstruct the phylogenetic relationships of
basal dipteran lineages. Evaluations of limited character sets
and taxa can easily lead to incorrect and misleading conclu-
sions (see, for example, Beutel and Baum 2008). In the
framework of a project on the phylogeny of Diptera, the data
presented here will be incorporated in a much more extensive
morphological data set with a much broader taxon sampling.
This, in combination with molecular data [Bertone et al. 2008;
Wiegmann et al. 2011; 1KITE: (http://1kite.wikispaces.com/)],
will be a sound basis for a reconstruction of evolutionary
transformations of morphological features in the basal dip-
teran lineages.
Acknowledgements
The authors are grateful to Hans Pohl (Institut fu
¨r Spezielle Zoologie
und Evolutionsbiologie, FSU Jena) for providing specimens. We also
thank Benjamin Wipfler (Institut fu
¨r Spezielle Zoologie und Evolu-
tionsbiologie, FSU Jena) for his technical support and the reviewers of
this study for many helpful criticisms and suggestions. Financial
support from the German Science Foundation (BE1789 ⁄6-1) and the
VolkswagenStiftung is also gratefully acknowledged.
Zusammenfassung
Das ma
¨nnliche Postabdomen und das Reproduktionssystem von Bibio
marci Linnaeus, 1758 (Hexapoda: Diptera: Bibionidae)
Das ma
¨nnliche Postabdomen und die inneren Organe des ma
¨nnlichen
Genitalsystems von Bibio marci (Bibionomorpha) wurden ras-
terelektronenmikroskopisch, mit einem konfokalen Lasermikroskop
und mit Hilfe von Schnittserien untersucht. Abweichend vom postu-
lierten Grundmuster der Diptera sind die Basen der Gonopoden
miteinander und mit dem Tergit IX verschmolzen. Der Penis ist nicht
ro
¨hren-, sondern schalenfo
¨rmig und somit fu
¨r die U
¨bertragung von
Spermatophoren geeignet. Das Dorsalsklerit des Genitalapparats
geht vermutlich auf median verschmolzene Parameren zuru
¨ck.
Die inneren Organe entsprechen weitgehend dem angenommen
Zustand im Grundmuster, abgesehen von den S-fo
¨rmigen Vasa
deferentia und dem Vorhandensein von zwei Paar akzessorischen
Dru
¨sen. Parsomonieanalysen von 27 Merkmalen des ma
¨nnlichen
Postabdomens von acht Vertretern der Diptera und Nannochorista
(Außengruppe) ergab zwei sparsamste Ba
¨ume. Der strict consensus
treeÕzeigt folgendes Verzweigungsmuster: Nannochoristidae + (Cu-
licidae [Culicomorpha] + ((Nymphomyiidae + (Tipulidae + Tricho-
ceridae)) + (Tabanidae [Brachycera] + (Bibionidae, Anisopodidae,
Axymyiidae [Bibionomorpha])))). Potentielle Synapomorphien der
Bibionomorpha (einschließlich Axymyiidae) und Brachycera sind die
Verschmelzung des Sternum IX mit den Gonocoxiten, die Vers-
chmelzung der Parameren zum Dorsalsklerit und das Vorhandensein
von M4, M20, M23, M26, M27, M31, M35 und M37. Die Aussagek-
raft der Analyse ist begrenzt, da nur ein Merkmalssystem und eine
geringe Anzahl von Taxa beru
¨cksichtigt wurde. Das Hauptergebnis,
ein Monophylum Bibionomorpha + Brachycera, stimmt jedoch mit
aktuellen Hypothesen u
¨berein.
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Supporting Information
Additional supporting information may be found in the online
version of this article:
Appendix S1. 4. List of phylogenetically relevant characters.
Appendix S2. The homologisation of muscles of Bibio with
those occurring in other potentially related taxa
Please note: Wiley Blackwell are not responsible for the
content or functionality of any supporting information
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material) should be directed to the corresponding author for
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