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Molecular phylogeny of flat-footed flies (Diptera:
Platypezidae): main clades supported by new
morphological evidence
MICHAL TKO
C,ANDREA T
OTHOV
A,GUNILLA ST
AHLS,PETER J. CHANDLER &JAROM
IR VA
NHARA
Submitted: 4 April 2016
Accepted: 17 September 2016
doi:10.1111/zsc.12222
Tko
c, M., T
othov
a, A., St
ahls, G., Chandler, P. J. & Va
nhara, J. (2016). Molecular phy-
logeny of flat-footed flies (Diptera: Platypezidae): main clades supported by new morpho-
logical evidence. —Zoologica Scripta,00, 000–000.
The molecular phylogeny of flat-footed flies is inferred from analysis of DNA sequence data
from the five mitochondrial genes 12S, 16S, COI, COII and CytB, and the nuclear gene 28S
and discussed with the recent systematics based on morphological features. The Bayesian
inference, maximum likelihood and maximum parsimony analyses included 42 species of 18
genera, representing all four extant subfamilies (Microsaniinae, Melanderomyiinae, Callomy-
iinae and Platypezinae) and all known genera except one (Metaclythia). Representatives of the
brachycerous taxa Lonchopteridae, Phoridae, Sciadocerinae (Phoridae) and Opetiidae are
used as outgroups, and Lonchoptera was used to root the trees. Our results show Platypezidae
consisting of two well-supported clades, the first with the subfamilies Melanderomyi-
inae +Callomyiinae and the second formed by subfamily Platypezinae. Genus Microsania
was resolved as a separate lineage distant from Platypezidae which clustered with Opetiidae
as its sister group, both together forming a sister group to Platypezidae. At the generic level,
the genus Agathomyia proved not to be monophyletic in any of the analyses. The species
Chydaeopeza tibialis is sister to Agathomyia sexmaculata, and consequently, the genus Chy-
daeopeza Shatalkin, 1992 is a new junior synonym of Agathomyia Verrall, 1901. Bifurcated
setae on legs of adult Platypezidae are documented as a new synapomorphy of the family,
exclusive of Microsania. Outstretched wings and only a small overlap of their surfaces at rest-
ing position are considered a new synapomorphy for the subfamily Platypezinae. Other phy-
logenetically important characters defining main clades are documented, and their relevance/
validity in phylogenetic studies is discussed. The current systematic concept of Platypezidae
is discussed, and new phylogenetic hypotheses are proposed.
Corresponding author: Michal Tko
c, Department of Entomology, National Museum, Cirkusov
a
1740, CZ-193 00 Praha 9, Czech Republic and Department of Zoology, Faculty of Science, Charles
University, Vini
cn
a 7, CZ-128 00 Praha 2, Czech Republic. E-mail: michaltkoc@gmail.com
Michal Tko
c, Department of Entomology, National Museum, Cirkusov
a 1740, CZ-193 00 Praha
9, Czech Republic and Department of Zoology, Faculty of Science, Charles University, Vinicn
a7,
CZ-128 00 Praha 2, Czech Republic. E-mail: michaltkoc@gmail.com
Andrea T
othov
a, Department of Botany and Zoology, Faculty of Science, Masaryk University in
Brno, Kamenice 753/5, CZ-625 00 Brno, Czech Republic. E-mail: tothova@sci.muni.cz
Gunilla St
ahls, Zoology Unit, Finnish Museum of Natural History, University of Helsinki, PO Box
17, 00014 Helsinki, Finland. E-mail: gunilla.stahls@helsinki.fi
Peter J. Chandler, Melksham, UK. E-mail: chandgnats@aol.com
Jarom
ırVa
nhara, Department of Botany and Zoology, Faculty of Science, Masaryk University in
Brno, Kamenice 753/5, CZ-625 00 Brno, Czech Republic. E-mail: vanhara@sci.muni.cz
Introduction
General introduction
The Platypezidae, flat-footed flies, are a small family of
brachycerous flies comprising more than 250 species in 19
extant genera of worldwide distribution. The highest num-
ber of known genera occurs in the Nearctic and Palaearctic
regions, 15 and 13, respectively (Table 1), and the platype-
zid fauna is best known in these two regions. The adults
ª2016 Royal Swedish Academy of Sciences 1
Zoologica Scripta
live mostly in forested habitats and can be usually observed
sitting or running rapidly on broad leaves or hovering
above them. The adults of Microsania Zetterstedt, 1837
exhibit a special type of behaviour: both sexes are attracted
to smoke of bonfires, sometimes forming swarms in the
smoke, where they have been reported to mate (Snoddy &
Tippins 1968; Chandler 1978; Milberg et al. 2015). The
known larvae are mycophagous, feeding on fruiting bodies
or fungal mycelia of a diverse array of fungal hosts (Chan-
dler 2001; St
ahls et al. 2014, 2015). Although several cases
of polyphagy are known, the majority of the species are
mono- or oligophagous. Larval morphology and biology of
species from the following genera are unknown: Microsania;
Grossoseta Kessel and Kirby, 1968; Metaclythia Kessel, 1952;
Platypezina Wahlgren, 1910; Chydaeopeza Shatalkin, 1992
(Chandler & Shatalkin 1998). The European flat-footed
flies were treated by Chandler (2001), and some other
recent studies of the world fauna (Rotheray et al. 2004;
Krivosheina 2008a,b; Tko
c&Va
nhara 2008; Chandler
2010; Cumming & Cumming 2011; Tko
c 2012; St
ahls
et al. 2014, 2015) have resulted in relatively detailed assess-
ment of the morphology of adults and immature stages,
including data on their biology, behaviour, phenology and
distribution. Rotheray et al. (2004) provided generic
descriptions of immature stages of 11 Holarctic genera.
Krivosheina (2008a,b) reviewed the biology and provided
detailed descriptions of the immature stages of Palaearctic
species of Callomyia Meigen, 1804, Polyporivora Kessel and
Maggioncalda, 1968, Seri Kessel and Kessel, 1966 and Bolo-
pus Enderlein, 1932.
Platypezidae belong to the group of cyclorrhaphous fam-
ilies without ptilinal fissure (together with Lonchopteridae,
Opetiidae, Ironomyiidae, Phoridae, Syrphidae and Pipun-
culidae), formerly named Aschiza, currently known as
lower Cyclorrhapha (Yeates & Wiegmann 1999). McAlpine
(1989) divided lower Cyclorrhapha into Platypezoidea
(Lonchopteridae, Opetiidae, Platypezidae, Ironomyiidae,
Phoridae, Sciadoceridae) and Syrphoidea (Syrphidae,
Pipunculidae). Other authors classified lower cyclorrhaphan
families differently, and thus, the concept of this group is
not uniform (e. g. Hennig 1948; Griffiths 1972; Disney
1988). Brown (1992) summarized previous concepts of
taxon names Hypocera, Phoroidea and Phoridea and
considered Phoroidea to include families Sciadoceridae
and Phoridae. After detailed study of morphology (mainly
on pleuron and wings), Brown et al. (2015) changed the
former family Sciadoceridae to Sciadocerinae, a subfamily
of Phoridae. Relationships of lower cyclorrhaphan families
and concepts of Platypezoidea/Platypezidae, Hypocera/
Phoroidea/Phoridea and Syrphoidea/Syrphidae were reviewed,
analysed and debated by many authors (Griffiths 1972;
McAlpine 1989; Brown 1992, 1995; Disney 1994;
Cumming et al. 1995; Zatwarnicki 1996; Yeates & Wieg-
mann 1999; Collins & Wiegmann 2002; Rotheray & Gil-
bert 2008) who suggested often different hypotheses of
their relationships, resulting in nonconsistent phylogenies
among lower cyclorrhaphan families. In this study, we fol-
low the classification of lower Cyclorrhapha and Platype-
zoidea as presented by Collins & Wiegmann (2002) and
Wiegmann et al. (2011). According to their analyses of
molecular data matrices, the Platypezoidea are considered
to represent a sister group to all other Cyclorrhapha, whilst
Syrphidae are not sister to Pipunculidae. Platypezoidea are
probably an old cyclorrhaphan lineage, formed and radiated
in the Early Cretaceous (145–100 Mya), which took place
much before the early Tertiary radiation of Schizophora
(65–40 Mya) (Wiegmann et al. 2011).
Sister group of Platypezidae and their phylogenetic position
within Platypezoidea
Griffiths (1972) placed Platypezidae (including Opetia Mei-
gen, 1830) as sister group to Syrphoidea +Schizophora,
and these all together forming sister group to Ironomyi-
idae +Phoridae +Sciadoceridae. McAlpine (1989) consid-
ered Platypezidae (incl. Opetia) sister to all other families of
Platypezoidea. A molecular study of Collins & Wiegmann
(2002) based on maximum-likelihood analysis of 28S rDNA
sequences placed Opetia as sister group to Lonchopteridae
which together represent the sister group to Platypezi-
dae +Phoridae; their maximum parsimony analyses showed
different composition or unresolved polytomy, but in all
cases, Opetia was found to be more related to Lonchopteri-
dae than to the Platypezidae clade. In a multigene molecu-
lar phylogenetic hypothesis, Platypezidae were shown as a
sister group to Phoridae +Ironomyiidae, all together form-
ing a sister clade to Opetiidae, all together showing as sis-
ter to Lonchopteridae (Wiegmann et al. 2011). From the
above information, it is clear that there is no unified view
on the phylogenetic position of Platypezidae and also its
sister group has not been definitively established. Addi-
tional studies are needed focusing on the gaps in the
knowledge of the higher-level taxonomy of lower Cyclor-
rhapha to affirm the sister group of the family Platypezi-
dae.
Monophyly of Platypezidae
Griffiths (1972) defined the monophyly of Platypezidae
based on a single apomorphic character, the compressed
hind tarsi. McAlpine (1989) did not exclude Opetia and sug-
gested that Platypezidae in this concept are supported by
apomorphies as follows: reduced prothoracic pupal spiracu-
lar horns; the absence of a filter apparatus in the pharynx
of the larva; and the presence of pad-like empodia on all
tarsi. However, inasmuch as the immature stages of Opetia
2ª2016 Royal Swedish Academy of Sciences
Phylogeny of Platypezidae M. Tko
cet al.
spp. and Microsania spp. are unknown, two of these three
apomorphic characters cannot be verified. Chandler (2001)
debated several plesiomorphic characters common to all
Platypezidae genera, but he stated that there is no real
synapomorphy for the family with respect to Cyclorrhapha.
As probable synapomorphies he considered: uniserial acros-
tichal setae (lost in Platypezinae), compressed hind tarsi
(however, the ʻsolesʼ, which are the depressed less sclero-
tized areas without setulae, are present only in Callomyi-
inae and Platypezinae) and M
1+2
forking beyond cross-vein
dm-cu (fork, cross-vein or both may be lost in some
genera). The presence of uniserial acrostichal setae was also
used as synapomorphy in studies of Hennig (1976), Brown
(1992) and Cumming et al. (1995). Additionally, the com-
pressed hind tarsi were also considered to be a synapomor-
phy by Hennig (1976) and Cumming et al. (1995).
Modern systematic history of Platypezidae and position of
Opetia
Kessel & Maggioncalda (1968) in their first modern generic
revision of Platypezidae erected three new subfamilies based
on morphological evidence: Opetiinae (containing Opetia,
Atelestus Walker, 1837, Microsania and Melanderomyia Kessel,
1960), Platypezininae (Platypezina,Grossoseta,Callomyia and
Agathomyia Verrall, 1901) and Platypezinae (Protoclythia Kes-
sel, 1950, Platypeza Meigen, 1803, Paraplatypeza Kessel and
Maggioncalda, 1968, Plesioclythia Kessel and Maggioncalda,
1968, Symmetricella Kessel, 1965, Grossovena Kessel and
Maggioncalda, 1968, Penesymmetria Kessel and Maggion-
calda, 1968, Polyporivora,Calotarsa Townsend, 1894, Seri,
Metaclythia and Lindneromyia Kessel, 1965). Griffiths (1972)
was the first to note the numerous differences of Opetia from
other platypezids, and Hennig (1976) also commented on its
distinctness from other Muscomorpha on the basis of the
male genitalia and antennal structure. Chv
ala (1981)
accepted these findings and proposed the monotypic family
Opetiidae of which a detailed diagnosis was published later
(Chv
ala 1983). However later, Disney (1987) considered
Opetia not to belong to Cyclorrhapha based on the presence
of two-jointed arista and very short ejaculatory duct, which
does not allow the circumversion of the genitalia. Cumming
et al. (1995), proved this observation (Disney 1987) incorrect
by finding in Opetia a short vasa deferentia but a long ejacula-
tory duct, thus demonstrating that the circumversion of the
genitalia is also present in Opetia.
The genus Atelestus was affiliated to the subfamily
Hybotinae within Empididae by Collin (1961) in contrast
to Kessel (1960, as Platycnema Zetterstedt) and Kessel &
Maggioncalda (1968) who placed it in Platypezidae. This
view was not accepted by Griffiths (1972) and consequently
Chv
ala (1981, 1983) erected a separate family Atelestidae
within Empidoidea. Currently, the Atelestidae is considered
either a sister group to remaining Empidoidea (Collins &
Wiegmann 2002; Moulton & Wiegmann 2004, 2007) or a
sister group to the Hybotidae (Sinclair & Cumming 2006).
Shatalkin (1985) and Chandler & Shatalkin (1998)
reviewed the classification of Opetiidae and Platypezidae
and subsequently Chandler (1998, 2001) developed the cur-
rently accepted classification of Platypezidae on the basis of
analysis of adult and larval morphological characters. A
review of all currently valid genera with their distribution
in the main biogeographical regions is provided in Table 1.
Relationships among the subfamilies and genera of
Platypezidae
The family is presently subdivided into four subfamilies:
Melanderomyiinae Chandler, Microsaniinae Enderlein, Cal-
lomyiinae Rondani and Platypezinae Fall
en. Chandler (2001)
divided the four subfamilies of platypezids into two basic
clades on the basis of 19 morphological characters, but iden-
tified no true synapomorphy for the family itself. The first
clade consists of Microsaniinae (Microsania) and its sister
group Melanderomyiinae (Melanderomyia kahli Kessel,
1960); second clade is composed of Callomyiinae (Platypez-
ina,Grossoseta,Callomyia,Agathomyia,Chydaeopeza,Bertamyia
Kessel, 1970) and its sister group Platypezinae (Protoclythia,
Calotarsa,Seri,Bolopus,Polyporivora,Metaclythia,Kesselimyia
Va
nhara, 1981, Platypeza,Paraplatypeza,Lindneromyia).
According to the phylogenetic scheme of Chandler (2001),
Microsaniinae share the following six adult apomorphies
with Melanderomyiinae: (i) cross-vein dm-cu absent, (ii) vein
R
1
short, (iii) hairs on anal lobe and alula thickened, (iv) sides
of prothorax bare, (v) mouth margin medially produced and
(vi) scape bare. The sister pair Callomyiinae +Platypezinae
is supported by a single synapomorphy: tarsi of females with
depressed less sclerotized areas devoid of setulae (=ʻsolesʼ).
The Callomyiinae clade is defined by four apomorphies: (i)
tibia and hind tarsus having uniformly dark setulae; (ii) the
presence of strong bent posteroventral seta (=ʻoxhornʼseta)
on the base of front femur of males and corresponding
anteroventral notch on middle femur (missing in Platypez-
ina); (iii) the presence of strong posteroventral seta near the
base of hind femur (missing in Bertamyia) and (iv) eight pro-
cesses (=lappets) per segment in the larva. The Platypezinae
clade is delimited by three apomorphies: (i) first tarsomere
on hind leg shortened; (ii) acrostichal setae absent and scu-
tum bare between dorsocentral rows of setae; (iii) frons of
female with only sparsely distributed short frontal setae.
Rotheray et al. (2004) analysed the immature characters of
15 species belonging to 10 genera of Palaearctic Callomyi-
inae and Platypezinae and the Nearctic species Melandero-
myia kahli, but their parsimony analysis results supported
only the monophyly of Platypezinae. A summary of these
two phylogenetic hypotheses is shown in Fig. 1.
ª2016 Royal Swedish Academy of Sciences 3
Phylogeny of Platypezidae
The goal of this study was to re-evaluate phylogenetic
relationships of Opetiidae and Platypezidae and to confront
the recent hypotheses with current knowledge. Molecular
data provide a new source of phylogenetic evidence and
can help to solve the following key questions: (i) the mono-
phyly of the family, (ii) the validity of the subfamily classifi-
cation and monophyly of subfamilies and genera and
(iii) the validity of the above listed morphological
synapomorphies.
Material and methods
Sampled taxa
We analysed 42 species of 18 genera of Platypezidae, which
represent all extant genera of the family except for Meta-
clythia. All included insect material was identified using
contemporary morphological terminology and synonymies
following Chandler (2001). Lindneromyia is thus treated as
including the synonyms Grossovena, Penesymmetria,Plesio-
clythia and Symmetricella (sensu Chandler 2001).
Table 1 Worldwide distribution of Platypezidae genera and genus Microsania. Assembled following Smith 1980; Kessel 1987; Chandler
1989, 1994, 2010; Chandler & Shatalkin 1998. (*Including as a synonym Chydaeopeza Shatalkin 1992. **including as synonyms Grossovena
Kessel & Maggioncalda 1968; Penesymmetria Kessel & Maggioncalda 1968; Plesioclythia Kessel and Maggioncalda, 1966 and Symmetricella
Kessel, 1965).
Subfamily Genus Author Neotropical Nearctic Palaearctic Oriental Afrotropical Australasian
Microsania
Zetterstedt, 1837 ++++++
Melanderomyiinae
Melanderomyia
Kessel, 1960 –+––– –
Callomyiinae
Grossoseta
Kessel and Kirby, 1968 –+––– –
Platypezina
Wahlgren, 1910 –++ –– –
Agathomyia*
Verrall, 1901 ++++–+
Bertamyia
Kessel, 1970 ++––+–
Callomyia
Meigen, 1804 ++++––
Platypezinae
Calotarsa
Townsend, 1894 ++––– –
Protoclythia
Kessel, 1950 –++ –– –
Seri
Kessel and Kessel, 1966 –++ –– –
Bolopus
Enderlein, 1932 ––+–– –
Polyporivora
Kessel & Maggioncalda, 1968 –++ +––
Kesselimyia
Va
nhara, 1981 ––+–– –
Platypeza
Meigen, 1803 ++++––
Pamelamyia
Kessel and Clopton, 1970 ––––+–
Paraplatypeza
Kessel & Maggioncalda, 1968 –++ +––
Lindneromyia**
Kessel, 1965 ++++++
Metaclythia
Kessel, 1952 –++ –– –
Fig. 1 Summary of generic phylogenetic
relationships of Platypezidae based on
morphological characters. After Chandler
(2001) (mainly adult characters) and
Rotheray et al. (2004) (immature
characters). Long dashed line means
uncertain position of the genus
Metaclythia; green square: Microsaniinae;
red square: Melanderomyiinae.
4ª2016 Royal Swedish Academy of Sciences
Phylogeny of Platypezidae M. Tko
cet al.
In the laboratory work conducted at the Masaryk
University, Brno, Czech Republic, for each species, some
specimens were retained as vouchers and the remainder
was used for DNA extraction. For molecular work, mostly
pinned or alcohol-preserved material not older than
20 years was used. In the molecular work conducted at the
Finnish Museum of Natural History, Helsinki, Finland
(MZH), the DNA was extracted from legs, and the voucher
specimens were labelled accordingly. Pinned or alcohol-
preserved material not older than 3 years was used. The
samples were collected usually by sweeping, Malaise traps
or rearing from fungi in the years 1984–2011, and were
dry-mounted or preserved in 70–96% ethanol; detailed
information on samples examined is provided in Table S1.
The included species with their respective GenBank access
numbers of analysed gene fragments are listed in Table S2.
Outgroups
Several lower Cyclorrhapha outgroup species were included
to cover the related taxa Lonchopteridae, Phoridae, Sciado-
cerinae and Opetiidae. Lonchoptera uniseta (Lonchopteridae)
was chosen to root the trees according to the recently pub-
lished interfamiliar relationships (Wiegmann et al. 2011).
The sequences of Megaselia scalaris (Phoridae), Sciadocera
rufomaculata (Fig. S1) and Lonchoptera uniseta (Lonchopteri-
dae) were obtained from the GenBank (Table S2). Speci-
mens of Opetia nigra (Fig. S2) were sequenced during this
study.
Gene sampling
The regions of mitochondrial genes 12S, 16S, COI, COII,
CytB, and a nuclear gene 28S (D2-D3 expansion region)
were selected for the analyses. These genes have been
widely used in insect molecular systematics, especially in
Diptera (Winterton 2002; Cook et al. 2004; Han & Ro
2004; Petersen et al. 2007; Winterton et al. 2007; Gibson
et al. 2010; Roh
a
cek & T
othov
a 2014). The most com-
monly sequenced regions in insect systematics are mtDNA
and nuclear rDNA (Caterino et al. 2000). Sampling of the
right gene markers is crucial, when building phylogenetic
trees and hypotheses. There is no general rule, which gene
marker is the best choice for species-, genus-, family- and
deeper-levels of phylogenies. The combination of several
different markers is considered as the best practice. In the
Diptera, ribosomal 12S, 16S; protein-encoding COI, COII
and CytB are commonly used in species- and genus-level
phylogenetic analyses (Skevington & Yeates 2000; Spicer &
Bell 2002; Mengual et al. 2008; Roh
a
cek et al. 2009), and
some of them have been successfully used even in the fam-
ily-level phylogenies (Han & Ro 2004). Even though these
mtDNA genes are frequently used in phylogenies of family
level or even deeper phylogenies (interordinal) in some
studies (Wiegmann et al. 2003), usually other markers such
as nuclear markers 28S rDNA, EF1aand CAD and com-
bined analyses with morphological data are used to ensure
higher interpretative value of the results of such analyses
(St
ahls et al. 2003; Winterton et al. 2007; Gibson et al.
2010; Kits et al. 2013; T
othov
aet al. 2013; Roh
a
cek &
T
othov
a 2014; Mengual et al. 2015).
DNA extraction, amplification and sequencing
DNA was extracted using DNeasy Blood and Tissue Kit
(QIAGEN, Venlo, The Netherlands) following the manu-
facturer’s protocol. Individual flies or tissue portions were
rinsed in phosphate-buffered saline (PBS), placed in sterile
Eppendorf tubes and incubated overnight at 56°C with
proteinase K. DNA was also extracted from 1–3fly legs
using the Nucleospin Tissue Kit (Macherey-Nagel, D€uren,
Germany) following manufacturer’s protocol and resus-
pended in 50 lL ultrapure water. PCRs (total vol-
ume =20 lL) were performed using primers published in
Cook et al. (2004) and Roh
a
cek et al. (2009) (ribosomal
12S and 16S), Su et al. (2008) (protein-encoding COI,
COII and CytB) and Belshaw et al. (2001) (ribosomal 28S)
for five mitochondrial genes as well as one nuclear gene.
All primer sequences are given in Table S3. The mito-
chondrial COI was amplified in two fragments. Amplified
products were purified using the QIAquick PCR Purifica-
tion Kit (QIAGEN), and cycle sequenced with BigDye
Terminator ver. 3.1 (Applied Biosystems, Foster City, CA,
USA). Direct sequencing was carried out on ABI 3100 or
ABI 3730 genetic analysers (Perkin Elmer Applied Biosys-
tems, Norwalk, CT, USA). All sequences were assembled
and edited in SEQUENCHER 4.8 (Gene Codes Corporation,
Ann Arbor, MI, USA). GenBank accession numbers for the
sequences are listed in Table S2.
Sequence alignment and analyses
The ribosomal genes 12S, 16S, and 28S and protein-
encoding genes Cytb, COI and COII were aligned using
MAFFT v.7 (Katoh & Standley 2013) with default settings
and manually inspected. The protein-encoding Cytb, COI
and COII sequences were checked based on amino-acid
translations and yielded indel-free nucleotide alignments.
The final concatenated molecular data set consisted of
46 taxa and 4088 characters; 12S –398 bp, 16S –388 bp,
COI –1328 bp, COII –643 bp, CytB –612 bp, 28S –
714 bp. The data set was analysed using maximum
parsimony (MP), maximum likelihood (ML) and Bayesian
inference (BI) to explore the strength of phylogenetic sig-
nal under different optimality criteria.
Each of the partitions was evaluated in MRMODELTEST
v.2.2 (Nylander 2004), using both hierarchical likelihood
ratio tests (hLRTs) and Akaike information criterion (AIC).
ª2016 Royal Swedish Academy of Sciences 5
Phylogeny of Platypezidae
The model GTR +I+Г(Rodriguez et al. 1990) was
favoured for each of the individual gene regions using the
‘unlink’command in MRBAYES.
The partitioned Bayesian inference of 20 million genera-
tions on the concatenated data set was run in MRBAYES v.
3.2.6. (Ronquist et al. 2012) and carried out on the CIPRES
computer cluster (Cyberinfrastructure for Phylogenetic
Research; San Diego Supercomputing Center, Miller et al.
2010). Two independent runs with three heated and one
cold chain and every 1000th tree sampled were performed.
The data were partitioned by gene regions (seven in total,
COI sequenced in two parts was partitioned by these
regions). The tree priors were set to ‘variable’. Node sup-
ports were estimated by posterior probabilities.
Parsimony analyses of the data set were performed using
TNT v.2.0 (Goloboff et al. 2008) with the following parame-
ters: new technology search, level 50, initial addseqs=9, find
minimum tree length five times. Analyses were carried out
with gaps coded as fifth character states and as missing
data. Nodal support was assessed by jackknife resampling
(JK, 1000 replicates with 36.8% character deletion).
The ML analyses were conducted using GARLI v.2.0
(Center for Bioinformatics and Computational Biology,
University of Maryland, MD, USA) (Zwickl 2006). Two
independent runs of 5.0 million generations using the
default automated stopping criterion were carried out.
Nodal support was assessed using a nonparametric boot-
strap with 250 replicates. We ran four identical analyses to
create 1000 bootstrap replicates.
Results and discussion
The results of the data set with all genes (12S, 16S, COI,
COII, CytB and 28S) data are shown in Fig. 2. All the
summarized results of BI, ML and MP are presented on
the Bayesian topology (50% majority-rule tree) with nodal
support values from the Bayesian, ML and MP (indels trea-
ted as fifth character state) analyses.
For the Bayesian analyses, we used 20 million generations,
a burn in of 25%, and the standard deviation of split frequen-
cies was in all cases <0.01. The log likelihood values for the
best tree of the molecular data set were 33 229.63. The
MP analyses of the data set resulted in two most parsimo-
nious trees (TL =7578), of which a strict consensus was cre-
ated with a jackknife resampling of 1000 replications. As no
significant difference was observed during the gap-reading
testing (fifth character vs. missing), we are presenting JK val-
ues for gaps read as fifth character (default setting). We
tested the exclusion of third positions of protein-encoding
genes, and this factor had surprisingly no significant impact
on the topologies from the various analyses.
Concerning the differences between the outputs of differ-
ent analysing methods, the BI tree was most resolved (and
with higher nodal supports) compared with ML and MP anal-
yses. Both ML and MP were congruent on the subfamilial
level with BI, but slightly incongruent or unresolved on the
generic level (Fig. 2). Well-supported relationships were con-
sistent across all the trees obtained. The ML analysis yielded a
tree with low-to-moderate node support values among the
branches representing some of the genera (Fig. 2).
Incongruence between MP and model-based methods
was observed mainly with regard to the well-supported
relationships among genera which are supported also by
morphology, although the terminal nodes were usually well
resolved. That is why we do not present here a separate
figure with MP topology, in concordance with theoretical
arguments indicating the limited performance of the MP
method in molecular phylogenetics (e.g. Gadagkar &
Kumar 2005; Spencer et al. 2005).
Phylogenetic position of Microsania
Microsania was placed outside all other Platypezidae
(PP =1.0, ML =82, MP =93), and it is postulated as a
sister group to Opetiidae with high support (PP =0.96,
ML =87, MP =87). Species of the Microsania clade form
the following well-supported (PP =1.00, ML =100,
MP =100 and PP =0.99, ML =95, MP =94) topology:
M. capnophila +(M. pectipennis +M.collarti).
Our results support the exclusion of Microsania from
Platypezidae and to place Microsania as a genus incertae
sedis within the superfamily Platypezoidea. The strongly
autapomorphic Microsania does not fit into the definition of
any currently defined family. The uncertain position of
Microsania will last until a new morphological evidence of
its familiar classification is found or the former subfamily
Microsaniinae (of Platypezidae) is elevated to family rank.
Detailed morphological revision of adults based on exten-
sive sampling and possible discovery of larval habitat and
description of larval morphology could help to solve this
question. Additionally, new molecular results based on both
mitochondrial and nuclear genes and proper taxon sam-
pling of key taxa of Platypezoidea may reveal the proper
systematic position of Microsania.
The following autapomorphic morphological characters
of Microsania used by Chandler (2001) were supported: (i)
strong costa ending in connection with R
1
, with spinose
setae (Fig. S3); (ii) cross-vein r-m lost or represented only
by a tenuous fold; (iii) pterostigma of unique morphology:
pterostigma of Microsania (Fig. S3) is of different type than
those of Melanderomyia,Grossoseta and Platypezina (Figs S4
and S5), its pterostigma is partly located in the subcostal
wing cell and extends from vein R
1
into the first radial cell,
copying the shape of vein R
1
, the pterostigma is fading
basally in cell r
1
. This is a strong autapomorphous condi-
tion of this character of Microsania.
6ª2016 Royal Swedish Academy of Sciences
Phylogeny of Platypezidae M. Tko
cet al.
Fig. 2 Bayesian hypothesis for Platypezidae relationships based on DNA sequence data –4088 characters (BI –20 mil. gen.); above node or
upper in the frame =posterior probabilities PP over 0.5, below node left or bottom left in the frame =bootstrap support for Garli, below
node right or bottom right in the frame =JK support for MP (indels =5th character state).
ª2016 Royal Swedish Academy of Sciences 7
Phylogeny of Platypezidae
Collart (1938) and Kessel & Maggioncalda (1968) stated
as a probable larval habitat of Microsania, the fungi growing
on fire-killed wood or burnt ground, but this has never
been confirmed and we find it unlikely. Collart (1954) tried
to obtain larvae from such fungi, but with no success, so he
corrected his former hypothesis and considered the larvae
of Microsania to be living in forest soil, where they may
feed on fungal mycelium. Chandler (2001) compiled and
commented on all available records and observations on
Microsania ecology. We believe that both sexes of Microsa-
nia are attracted to smoke only for copulation, using the
smoke as a ‘meeting point’. As most probable, we consider
Microsania larvae to be living in or on soil. The observa-
tions of Ebejer & Andrade (2010) may support this hypo-
thesis. They have documented the habitat of adult
Microsania meridionalis Collart, 1960 on sandy shores in
northern Portugal (Figs S6 and S7). One male of M. merid-
ionalis (Fig. S8) was swept on habitat characterized by ther-
mophilous herbaceous plant species with one small isolated
specimen of Mediterranean pine (Pinus pinaster Ait.) grow-
ing on dunes (R. Andrade, in litt.). There was no fire or
smoke present on the habitat during the observation and
collection of the specimen.
Microsania commonly have clusters of phoretic mites on
the abdomen (Fig. S8), unrecorded in other Platypezidae.
Chandler (2001) summarized all such observations. These
mites were identified as belonging to the genus Parasitus
Latreille, 1795 (Edwards 1934) and Macrocheles muscaedo-
mesticae Scopoli, 1772 (Chandler 2001), which are terres-
trial mites often occurring on fungi, dung and compost,
which indicates possible terrestrial habitat of Microsania
spp. Other material of mites obtained recently from
Microsania was identified as an undescribed species of Pedic-
ulaster Vitzthum, 1931 (Prostigmata: Pygmephoridae) (J.
Ostoj
a-Starzewski, in litt.) and deutonymphs of species of
Dendrolaelaps Halbert, 1915 (Mesostigmata, Digamasellidae)
(V. Huhta, pers. comm.). Members of both genera live in
various terrestrial habitats such as soil, decaying organic
material such as compost, manure, mammal and bird nests,
and nests of social insects.
Relationships of Platypezidae and its subfamilies
The monophyly of the Platypezidae clade was established
with high posterior probability (PP =1.0) in the Bayesian
inference, while the bootstrap values of ML (Garli) and jack-
knife support of MP analyses (TNT) were lower (ML =77,
MP =55), (Fig. 2). Platypezidae consists of three well-sup-
ported monophyletic clades, which are in accordance with
current subfamilial classification of Platypezidae:
Melanderomyiinae (PP =1.0, ML =66, MP =64), Cal-
lomyiinae (PP =1.0, ML =86, MP =87) and Platypezinae
(PP =1.0, ML =100, MP =93). The Melanderomyiinae
clade is sister to the clade Callomyiinae (Fig. 2).
Based on the above presented results, the monophyly of
Platypezidae is highly supported when Microsaniinae
(genus Microsania) is excluded. Chandler (2001) stated that
there are no true synapomorphies defining Platypezidae if
Microsania is included, but suggested several tentative
synapomorphies: uniserial acrostichal setae (lost in Platype-
zinae); compressed hind tarsi (note: the ʻsolesʼ, which are
depressed less sclerotized areas without setulae, are present
only in Callomyiinae and Platypezinae) and M
1+2
forking
beyond cross-vein dm-cu (fork, cross-vein or both are lost
in some genera).
Newly proposed morphological and ecological synapo-
morphies of Platypezidae exclusive of Microsania are as fol-
lows:
1. Bifurcated setae present on femora and tibiae of all legs
of both sexes (Figs 3–6): this newly discovered character
(by SEM observations) is shared by all Platypezidae
genera. Function of these setae is unknown, but in
males, they are probably related to mating –their pres-
ence enables the male to hold the female tighter during
copulation and stimulates the female abdomen with the
hind legs (see Figs 13 and 14). The distribution of these
bifurcated setae on tibiae and tarsi of males corresponds
well with contact surfaces of male legs with the female
body during copulation (Figs 7, 13, 14). The function
of these setae in females is obviously different, probably
facilitating their motion on the surface of the fruiting
bodies of fungi during oviposition.
Fig. 3–12 Morphological characters of adult Platypezidae and Microsania:3–4–Hind leg of Callomyia amoena Meigen, 1804, female,
(3) anterior view of tarsus, arrows indicating tarsal ʻsolesʼ, (4) joint of tibia and first tarsomere in detail, the bifurcated setae represent
a newly discovered morphological synapomorphy defining family Platypezidae; 5–6–Setation of hind legs of Microsania and
Melanderomyia, anterior view of part of tibia and tarsus, (5) Microsania collarti Chandler 2001; female, only regular setae are present,
(6) Melanderomyia kahli Kessel 1960, male, bifurcated setae present; 7 –Distribution of bifurcated setae on hind leg of Platypeza
consobrina Zetterstedt, 1844, male, anterior view, bifurcated setae are present mainly on distal half of tibia and first two tarsomeres,
which are touching the female during copulation (arrows are indicating only some of the bifurcated setae); 8 –Strong bent
posteroventral seta (‘oxhorn’seta) in males of Callomyiinae (missing in Platypezina), posterior view of femur and tibia of fore leg,
Agathomyia elegantula (Zetterstedt, 1819); 9–12 –Female tarsi of hind legs with soles of four different Platypezidae genera (arrows
indicating tarsal soles), (9) Platypezina connexa (Boheman, 1858), (10) Agathomyia falleni (Zetterstedt, 1819), (11) Callomyia amoena
Meigen, 1804, (12) Platypeza consobrina Zetterstedt, 1844.
8ª2016 Royal Swedish Academy of Sciences
Phylogeny of Platypezidae M. Tko
cet al.
ª2016 Royal Swedish Academy of Sciences 9
Phylogeny of Platypezidae
Figs 13–18 Mating position and position of wings ‘in situ’of adult Platypezidae: 13–14 –Pair of Platypeza consobrina Zetterstedt, 1844, (13)
lateral view, (14) anterior view (both photographs by Brian Valentine); 15–18 –Position of wings and overlap of its surface ‘in situ’in
normal resting position, (15) Callomyiinae, (16) Platypezinae, (17) Agathomyia setipes Oldenberg, 1916, female (photograph by Jind
rich
Roh
a
cek), (18) Polyporivora ornata (Meigen, 1838), male (photograph by Dmitry Gavryushin).
10 ª2016 Royal Swedish Academy of Sciences
Phylogeny of Platypezidae M. Tko
cet al.
2. Compressed hind tarsi with ʻsolesʼ(Figs 9–12): this
character condition, obviously related to the mycopha-
gous association of early stages (especially developed
in females, helping them to adhere to the fungus
fruiting body during oviposition and to adhere to the
gills of agarics in case of Platypezinae) is not devel-
oped or is lost in Melanderomyia. The latter genus is
distinguished by many plesiomorphic morphological
conditions (when compared to other Platypezidae) and
is also mycophagous (Kessel 1960). The only known
species M. kahli is known to feed on stinkhorns, that
is Phallus ravenelii (Berkeley and Curtis), Phallus impu-
dicus Linnaeus and Phallus duplicatus Bosc (Kessel
1961).
3. Larva of typical cylindrical or flattened form (Figs S9–
S11): as the larva of possible sister taxa is unknown
(Opetia,Microsania), this might be supported or refuted
by future studies. At the moment, it is only possible to
compare larval morphology with Phoridae or Lon-
chopteridae.
4. Mycophagous larvae (unknown in Grossoseta,Platypezina
and Metaclythia): larval nourishment of possible sister
taxa is unknown (Opetia,Microsania). Larvae of Lon-
choptera feed on rotting vegetable matter.
Our molecular results do not support sister group rela-
tionships of Melanderomyia and Microsania. The validity of
formerly suggested synapomorphies of these two genera
(see Introduction and Chandler 2001) is at least arguable
and needs re-evaluation: (i) dm-cu cross-vein absent (also
missing in Opetia): this character is most probably a
retained primitive condition of the common ancestor or
dm-cu was secondarily lost in Melanderomyia. All other
Platypezidae have this cross-vein developed. (ii) Vein R
1
short, reaching only two-thirds of wing length (long in
Opetia): scoring of this character could be very difficult, as
its definition is based on relative lengths. Relative length
ratio of these two veins varies markedly among genera and
among species within particular genera of Platypezidae and
Microsania as well. As no detailed study of Platypezidae
wing veins morphometry is published, we find this charac-
ter dubious and probably not phylogenetically informative.
(iii) Setae on anal lobe and alula thickened: the appearance
of this character is affected by length of the wing of partic-
ular species, and thus, most probably this represents an
adaptive apomorphy. Both Microsania and Melanderomyia
have generally smaller wing length (1.4–2.7 mm in Euro-
pean Microsania, and about 2.0–2.5 mm in Nearctic Melan-
deromyia), when compared to other genera of European
Platypezidae (Platypezina 3.2–4.1 mm, Agathomyia 2.3–
5.0 mm, Callomyia 3.2–4.6 mm, Platypezinae genera 2.5–
6.3 mm). However, this character can be used as a
synapomorphy, but with limited phylogenetic value. (iv)
Sides of prothorax bare: this is probably a retained primi-
tive condition, thus symplesiomorphy with limited phyloge-
netic value. (v) Medially produced mouth margin: based on
morphological shape, similarly like short vein R
1
based on
relative morphological shape and thus of limited phyloge-
netic value. (vi) Scape bare: this character is also present as
homoplasy in some genera of Platypezinae (viz. Seri,Bolo-
pus,Polyporivora,Metaclythia,Kesselimyia,Paraplatypeza and
Lindneromyia) and thus of limited phylogenetic value for
the clade Microsania +Melanderomyia. Additionally, Chan-
dler & Shatalkin (1998) stated that the above-mentioned
apomorphies are with respect to the ground plan of Mus-
comorpha and that Microsaniinae and Melanderomyia could
be in fact paraphyletic. This paraphyly is here confirmed
by molecular data on the six analysed gene fragments. The
Callomyiinae clade was resolved as monophyletic in all
conducted analyses with high support (PP =1.0, ML =86,
MP =87). The following synapomorphic morphological
characters of Callomyiinae used by Chandler (2001) were
supported: (i) the presence of uniformly dark setulae on
tibia and hind tarsus: coloration of these setulae has only
limited phylogenetic value, and the character and its condi-
tions should be redefined or avoided in future cladistic
analyses. (ii) The presence of strong bent posteroventral
seta (‘oxhorn’seta) in fore femur in males (Fig. 8): missing
in Platypezina, but it is evident that this must be a sec-
ondary loss, because its sister genus Grossoseta has this char-
acter well developed. (iii) The presence of strong
posteroventral seta near the base of hind femur. (iv) Eight
processes (=lappets) on each larval segment (Figs S9 and
S10; larvae of Platypezina and Grossoseta are not known).
The Platypezinae clade was resolved to be monophyletic
in all conducted analyses with very high support (PP =1.0,
ML =100, MP =93). The following synapomorphic mor-
phological characters of Platypezinae used by Chandler
(2001) were supported: (i) first tarsomere of hind tarsus
shorter than second–fourth tarsomeres together (Fig. 12);
(ii) acrostichal setae absent; (iii) only short scattered frontal
setae present in females.
As a fourth newly proposed synapomorphic character
defining subfamily Platypezinae, we here suggest the posi-
tion of wings and their overlap ratio of surfaces ‘in situ’in
the normal resting position. This condition is probably
linked to thoracic and wing musculature, but it is only
observable on live specimens of flat-footed flies. While
members of Melanderomyiinae and Callomyiinae have
‘in situ’wing overlap almost completely to about 50% of
each wing surface (Figs 15 and 17), members of Platypezi-
nae have their wings more outstretched and they overlap
only by 30% at most (Figs 16 and 18).
ª2016 Royal Swedish Academy of Sciences 11
Phylogeny of Platypezidae
Circumversion of the genitalia and mating position
The occurrence of flexion and rotation of Diptera genitalia
was summarized by McAlpine (1981) and Cumming et al.
(1995). Popham (1995) repeated experiments of earlier
authors investigating the circumversion hypothesis and sum-
marized the phenomenon of circumversion in Diptera based
on the observation of Calliphora vicina (Calliphoridae).
The circumversion of the genitalia in Platypezidae was
debated repeatedly by Kessel himself and with his co-work-
ers (Kessel & Kessel 1961, 1962; Kessel 1968; Kessel &
Maggioncalda 1968) and analysed in detail by Griffiths
(1972, pp. 40–66). Chandler commented on Hennig’s,
Griffiths’and Kessel’s view (Chandler 2001) and stated that
completion of rotation after emergence from the puparium
is a significant feature affecting copulation, and it is not
found outside the Platypezidae. However, Cumming et al.
(1995) indicated that completion of rotation after emer-
gence also occurs in Syrphidae, Phoridae and probably
Opetiidae. The genital circumversion in Platypezidae is
characterized by rotating to inverse position of male genital
capsule (hypopygium) within the puparium, which is subse-
quently rotated through a further 180°to the circumverse
rest position soon after emergence. The eighth abdominal
segment is rotated through half the angle of hypopygial
rotation (through 90°within the puparium and then
through a further 90°to inverse position after emergence).
The mating position of Platypezidae was observed sev-
eral times by Kessel & Kessel (1962) and Kessel (1968),
but it has never been photographed. Here, mating of
Platypezidae is documented for the first time (Figs 13 and
14). We expect that all the members of Platypezinae have
the same or very similar mating position as in the case of
the species Platypeza consobrina Zetterstedt, 1844 (Figs 13
and 14) which is the only species so far photographed dur-
ing copulation. The male is standing on the female with
his front tarsi on her eyes, tarsi of mid legs are on anterior
sides of the female thorax and hind legs are gripping the
sides of the abdomen of the female between the fourth and
fifth abdominal segments. The female abdomen is strongly
bent upwards (dorsally), and female genitalia are connected
to the genitalia of male. We are convinced that the bifur-
cated setae on male legs (Fig. 7) are facilitating the male to
adhere to the female body, and thus enabling the successful
course of copulation.
Relationships within Callomyiinae
The clade of Grossoseta +Platypezina is placed in a sister
relationship with high support (PP =1.0, ML =86,
MP =87) to the rest of the subfamily, and their mutual
relationship is of high support (PP =1.0, ML =100,
MP =100). The following synapomorphic morphological
characters of Grossoseta +Platypezina used by Chandler
(2001) were supported: (i) first tarsomere of hind leg
enlarged and with long dorsal setae, (ii) hind femur with
medial posteroventral seta and (iii) long and coiled aedea-
gus of male genitalia.
All remaining taxa from the Callomyiinae clade are sister
to Agathomyia viduella, which would make the genus
Agathomyia paraphyletic. This clade is composed of a
branch of very low support (PP =0.74, ML =NA,
MP =70): Agathomyia + Chydaeopeza +Callomyia, which
formed a moderately supported sister clade to Bertamyia
notata (PP =0.97, ML =58, MP =<50). The genera
Agathomyia, Bertamyia, Chydaeopeza and Callomyia form a
paraphyletic composition; however, the analysed Agath-
omyia species represent four clades which fully agree with
the species groups morphologically defined by Chandler
(2001). The genus Agathomyia was not resolved as a mono-
phyletic group in the analysis, and the formed cluster is of
poor support (PP =0.62, ML =55, MP =<50). The
A. viduella group covers the single species A. viduella and is
the earliest branched clade of the mentioned paraphyletic
group, so its position might show a different generic
affiliation. The A. elegantula group is highly supported
(PP =1.0, ML =90, MP =NA) and includes the analysed
species A. elegantula,A. lundbecki,A. woodella and A. zetter-
stedti. The A. antennata group (PP =0.98, ML =61,
MP =51) includes A. sexmaculata and covers also the clo-
sely related species Chydaeopeza tibialis, excluded from the
genus Agathomyia by Shatalkin (1985, 1992). Based on
these results, we propose the genus Chydaeopeza as a new
synonym of Agathomyia. The genus Chydaeopeza was
erected by Shatalkin (1992) to accommodate one species –
Ch. tibialis. Its definition was mainly based on the follow-
ing characters: positions and directions of cross-veins r-m
and dm-cu, not differentiated orbital setae and the pres-
ence of additional lobes on each side of epandrium. We
do not consider these characters to be significant enough
to diagnose a separate genus because they can fit within
morphological plasticity of Agathomyia as currently recog-
nized.
The A. falleni group (PP =1.0, ML =95, MP =59)
consists of two analysed species A. falleni and A. unicolor
and shows sister relationship of low support (PP =0.86,
ML =NA, MP =<50) to the A. sexmaculata +Chydaeopeza
tibialis pair.
Despite our results showing genus Agathomyia as para-
phyletic, we do not propose a new systematic concept for
its members. First, most of its clades have low support or
are unresolved, and second, we do not have satisfactory
morphological evidence at this moment to define new taxa.
A specialized study is needed to solve this problem, focus-
ing on Agathomyia phylogeny and on its morphology in
particular.
12 ª2016 Royal Swedish Academy of Sciences
Phylogeny of Platypezidae M. Tko
cet al.
The well-supported Callomyia clade (PP =1.0, ML =
100, MP =99) is in the analysis represented by three spe-
cies with the following composition: C. dives +(C. amoena
+C. admirabilis). Callomyia is a morphologically very well-
defined genus characterized mainly by its typical flattened
larva and puparium with sharply pointed processes (=lap-
pets) (Fig. S10) and by the presence of spines on the first
radial vein –R
1
. Its relationships to other taxa within
Agathomyia clade remain unclear (its sister group has not
been recognized). A comprehensive morphological analysis
needs to be performed to confirm whether Callomyia really
is only a subgroup of Agathomyia as our molecular analysis
revealed.
Relationships within Platypezinae
All the included genera form monophyletic clades –the
subfamily Platypezinae were represented by
(Calotarsa +Protoclythia)+(Seri +(Bolopus +Polyporivora))
+(Kesselimyia +(Platypeza +(Pamelamyia +(Paraplatypeza
+Lindneromyia)))) (Fig. 2).
A polytomy with three clades, comprising two (1: Calo-
tarsa,Protoclythia;PP=1.0, ML =100, MP =99), three
(2: Seri,Bolopus,Polyporivora;PP=0.8, ML =NA,
MP =<50) and five (3: Kesselimyia,Platypeza,Pamelamyia,
Paraplatypeza and Lindneromyia;PP=0.94, ML =55,
MP =<50) genera form monophyletic clusters with com-
positions similar to the phylogenetic hypothesis by Chan-
dler (2001) (see also Fig. 1). This is in agreement with the
cladistic scheme of Chandler (2001), supported by the fol-
lowing morphological features: first clade: (1) the four dor-
sal processes (=lappets) of larval penultimate segment
approximated in pairs rather than equidistant (2) sternites
narrowed leaving the membrane between them visible, (3)
two notopleural setae; second clade: (1) scape bare, (2) gena
and parafacial bare, (3) anteroventral apical seta (spur) on
middle tibia lost, (4) cylindrical shape of larva; third clade:
(1) median fork close to wing margin and cell cup long, (2)
mesothorax of larva with processes reduced to four. These
three clusters are also in agreement with the hypothesis of
Rotheray et al. (2004) based on larval characters (see also
Fig. 1).
Conclusions
Forty-two species of Platypezidae and four species from
four related taxa (Opetiidae, Sciadocerinae, Phoridae and
Lonchopteridae) of superfamily Platypezoidea are here sub-
jected to a multigene phylogenetic molecular analysis, with
the following main resulting hypotheses:
1. Family Platypezidae was not resolved as monophyletic
in its current systematic concept. However, when the
genus Microsania is excluded, the family Platypezidae is
monophyletic and this monophyly is also supported by
a new morphological synapomorphy.
2. The monophyly of the ingroup taxa of Platypezidae
(comprising the subfamilies Melanderomyiinae, Cal-
lomyiinae and Platypezinae) is demonstrated with high
support.
3. The position of Microsania is here shown as a sister
group to Opetiidae. Thus, we propose to exclude the
genus Microsania from Platypezidae and consider it as
separate taxon within superfamily Platypezoidea.
4. The majority of the resulting relationships among genera
(except Agathomyia) are in agreement with the previous
morphological analyses. The genus Chydaeopeza is sug-
gested to be a new subjective synonym of Agathomyia
because its type species is closely related to A. sexmaculata.
5. Although the genus Agathomyia is found to be para-
phyletic, its clustering into species groups corresponds
rather well with the recent morphological approach and
with the former definition of the species groups.
Acknowledgements
Our thanks are due to colleagues who provided fresh
material to the molecular analyses, especially J. M. Cum-
ming and J. H. Skevington (Canadian National Collection
of Insects, Ottawa, ON, Canada), S. A. Marshall
(University of Guelph, BC, Canada) and A. I. Shatalkin
(Lomonosov Moscow State University, Russia). J.
Sev
c
ık
(University of Ostrava, Czech Republic), J. Roh
a
cek (Sile-
sian Museum, Opava, Czech Republic) and P. Voni
cka
(North Bohemian Museum, Liberec, Czech Republic) are
acknowledged for providing additional samples. We thank
B. Valentine, R. Andrade, Z. Solt
esz, D. Gavryushin, E.
R€
attel and J. Roh
a
cek for providing photographs used in
the article. For many consultations of earlier drafts of this
study, we are obliged to M. Fik
a
cek (National Museum,
Praha, Czech Republic) and J. Roh
a
cek. We are grateful
for the sponsorship of the Willi Hennig Society, which
made the TNT v.2.0 being available. CIPRES, an NSF-
funded resource for phyloinformatics and computational
phylogenetics, was used to conduct some of the analyses.
We acknowledge financial support from the Masaryk
University, Brno (Czech Republic), European Social Fund
and the Ministry of Education of the Czech Republic
(CETPO project no. CZ.1.07/2.3.00/20.0166). The first
author was supported by the Charles University in Pra-
gue, project GA UK No. 1294214, by the Ministry of
Culture of the Czech Republic (DKRVO 2014/13, 2015/
14 and 2016/14, National Museum, Prague, 00023272)
and by the Institutional Research Support grant of the
Charles University, Prague (No. SVV 260 313/2016). The
authors declare no conflict of interest.
ª2016 Royal Swedish Academy of Sciences 13
Phylogeny of Platypezidae
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Taxa used for DNA analyses (AUS –Aus-
tralia, CZ –Czech Republic; CA –Canada; CR –Costa
Rica; ES –Spain; FI –Finland; FR –France; GB –Great
Britain; NA –Namibia; SK –Slovakia; RU –Russia; BR –
Biosphere reserve; NPR –National Natural Reserve; PR –
Natural Reserve; NP –National Park; ex –reared from.
Table S2. List of GenBank access numbers of analyzed
gene fragments - 12S (398 bp), 16S (389 bp), COIa (571
bp), COIb (758 bp), COII (644 bp), CytB (613 bp) and
28S (715 bp) (sequences of outgroup species with access
numbers in italics used in earlier studies).
Table S3. PCR primers used and their sequences and
sources.
Fig. S1–S11. S1–S2 –Examples of species used in outgroup,
(S1) Sciadocera rufomaculata White, 1916 (photograph by
Zolt
an Solt
esz), (S2) Opetia nigra Meigen, 1830 (photograph
by Jind
rich Roh
a
cek); S3–S5 –Pterostigma of males, arrows
indicating the pterostigma, (S3) Microsania pectipennis (Mei-
gen, 1830), (S4) Melanderomyia kahli Kessel, 1960; (S5)
Platypezina connexa (Boheman, 1858); S6–S8 –Habitat of
Microsania meridionalis, (S6) sandy shores of northern Portu-
gal, (S7) sandy biotope with thermophilous vegetation, (S8)
Microsania meridionalis Collart, 1960, male (all three pho-
tographs by Rui Andrade); S9–S11 –Larvae of Platypezidae,
(S9) Agathomyia lundbecki Chandler in Shatalkin, 1985 (pho-
tograph by Elvira R€
attel), (S10) Callomyia dives Zetterstedt,
1838, (S11) Paraplatypeza atra (Meigen, 1804).
16 ª2016 Royal Swedish Academy of Sciences
Phylogeny of Platypezidae M. Tko
cet al.
