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The Muscoidea (Diptera: Calyptratae) are paraphyletic: Evidence from four mitochondrial and four nuclear genes

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Approximately 5% of the known species-level diversity of Diptera belongs to the Muscoidea with its approximately 7000 described species. Despite including some of the most abundant and well known flies, the phylogenetic relationships within this superfamily are poorly understood. Previous attempts at reconstructing the relationships based on morphology and relatively small molecular data sets were only moderately successful. Here, we use molecular data for 127 exemplar species of the Muscoidea, two species from the Hippoboscoidea, ten species representing the Oestroidea and seven outgroup species from four acalyptrate superfamilies. Four mitochondrial genes 12S, 16S, COI, and Cytb, and four nuclear genes 18S, 28S, Ef1a, and CAD are used to reconstruct the relationships within the Muscoidea. The length-variable genes were aligned using a guide tree that was based on the protein-encoding genes and the indel-free sections of the ribosomal genes. We found that, based on topological considerations, this guide tree was a significant improvement over the default guide trees generated by ClustalX. The data matrix was analyzed using maximum parsimony (MP) and maximum likelihood (ML) and yielded very similar tree topologies. The Calyptratae are monophyletic and the Hippoboscoidea are the sister group to the remaining calyptrates (MP). The Muscoidea are paraphyletic with a monophyletic Oestroidea nested within the Muscoidea as sister group to Anthomyiidae+Scathophagidae. The monophyly of three of the four recognized families in the Muscoidea is confirmed: the Fanniidae, Muscidae, and Scathophagidae. However, the Anthomyiidae are possibly paraphyletic. Within the Oestroidea, the Sarcophagidae and Tachinidae are sister groups and the Calliphoridae are paraphyletic.
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The Muscoidea (Diptera: Calyptratae) are paraphyletic: Evidence from four
mitochondrial and four nuclear genes
Sujatha Narayanan Kutty
a
, Thomas Pape
b
, Adrian Pont
c
, Brian M. Wiegmann
d
, Rudolf Meier
a,*
a
Department of Biological Sciences and University Scholars Programme, National University of Singapore, 14 Science Dr 4, Singapore 117543, Singapore
b
Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen Ø, Denmark
c
Oxford University Museum of Natural History, Parks Road, Oxford, OX1 3PW, UK
d
Department of Entomology, North Carolina State University, Raleigh, NC 27695, USA
article info
Article history:
Received 26 June 2008
Revised 20 August 2008
Accepted 21 August 2008
Available online 29 August 2008
Keywords:
Muscoidea
Calyptratae
Molecular phylogeny
Guide tree
abstract
Approximately 5% of the known species-level diversity of Diptera belongs to the Muscoidea with its
approximately 7000 described species. Despite including some of the most abundant and well known
flies, the phylogenetic relationships within this superfamily are poorly understood. Previous attempts
at reconstructing the relationships based on morphology and relatively small molecular data sets were
only moderately successful. Here, we use molecular data for 127 exemplar species of the Muscoidea,
two species from the Hippoboscoidea, ten species representing the Oestroidea and seven outgroup spe-
cies from four acalyptrate superfamilies. Four mitochondrial genes 12S, 16S, COI, and Cytb, and four
nuclear genes 18S, 28S, Ef1a, and CAD are used to reconstruct the relationships within the Muscoidea.
The length-variable genes were aligned using a guide tree that was based on the protein-encoding genes
and the indel-free sections of the ribosomal genes. We found that, based on topological considerations,
this guide tree was a significant improvement over the default guide trees generated by ClustalX. The
data matrix was analyzed using maximum parsimony (MP) and maximum likelihood (ML) and yielded
very similar tree topologies. The Calyptratae are monophyletic and the Hippoboscoidea are the sister
group to the remaining calyptrates (MP). The Muscoidea are paraphyletic with a monophyletic Oestroi-
dea nested within the Muscoidea as sister group to Anthomyiidae + Scathophagidae. The monophyly of
three of the four recognized families in the Muscoidea is confirmed: the Fanniidae, Muscidae, and Scath-
ophagidae. However, the Anthomyiidae are possibly paraphyletic. Within the Oestroidea, the Sarcophag-
idae and Tachinidae are sister groups and the Calliphoridae are paraphyletic.
Ó2008 Elsevier Inc. All rights reserved.
1. Introduction
With approximately 7000 species in four families, the Muscoi-
dea constitute approximately 5% of the described dipteran diver-
sity. Many of the muscoids are familiar flies that we encounter
on a daily basis. For example, the most speciose family, the Musci-
dae, includes the housefly (Musca domestica) and the stable fly (Sto-
moxys calcitrans). The best known scathophagid is the yellow dung
fly (Scathophaga stercoraria), which is widely used as a model
organism in behavioral biology, and some species of Anthomyiidae
are important agricultural pests as larvae with the best-known
examples being the onion fly (Delia antiqua) and the cabbage root
fly (Delia radicum). The best-studied species from the relatively
small family Fanniidae is the lesser housefly (Fannia canicularis),
and some Fannia species play an important role in forensic
entomology.
The Muscoidea are one of the three superfamilies in the Calyp-
tratae, but due to the lack of a unique autapomorphy that would
support the monophyly of Muscoidea, the taxon has often been
considered a group of convenience or a potentially paraphyletic
residual. For example, Michelsen (1991) characterized the Muscoi-
dea as ‘‘the Calyptratae less the Hippoboscoidea and Oestroidea”.
However, the non-monophyly of the Muscoidea is far from univer-
sally accepted. For example, the Muscoidea were considered
monophyletic by McAlpine (1989), who argued for the monophyly
based on a combination of morphological character states such as
the male anus being situated above the cerci, the male sternite ten
forming bacilliform sclerites, and the female abdominal spiracle
seven being located on tergite six (Hennig, 1973; McAlpine,
1989). However, as some authors have pointed out, these character
states may be plesiomorphic with respect to the Oestroidea
(Michelsen, 1991). With regard to the position of Muscoidea within
Calyptratae, McAlpine (1989) proposed a sister group relationship
between Muscoidea and Oestroidea based on the reduction of the
male sternite 6, the female abdominal segments 6 and 7 being
1055-7903/$ - see front matter Ó2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2008.08.012
* Corresponding author.
E-mail address: dbsmr@nus.edu.sg (R. Meier).
Molecular Phylogenetics and Evolution 49 (2008) 639–652
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
modified for oviposition, strongly developed vibrissae, a close
connection between surstyli and cerci, and a female hypoproct
with lingulae.
Despite being speciose and receiving considerable attention
from applied entomologists, the phylogenetic relationships within
the Muscoidea and its position within the Calyptratae have rarely
been addressed. The constituent families of the Muscoidea are
generally considered monophyletic but the phylogenetic relation-
ships between these families are far from understood and addi-
tional research based on molecular, morphological, and other
data is necessary before this significant portion of Diptera diver-
sity can be reliably placed on the tree of life (Bernasconi et al.,
2000b).
1.1. Fanniidae
The smallest family in the Muscoidea is the Fanniidae with
about 335 described species in four genera that are mostly found
in the Holarctic and Neotropical regions. As larvae, almost all spe-
cies feed on a wide variety of decaying organic matter and a few
can cause human myiasis. The monophyly of this family has been
supported by morphological character states such as the shape of
the apical part of the subcosta that curves evenly towards the costa
and a strongly curved vein A
2
. Fanniid larvae are furthermore char-
acterized by lateral fleshy projections. While the monophyly of the
Fanniidae may seem strongly corroborated, the phylogenetic rela-
tionships within the family are still poorly understood. The mono-
typic Australofannia Pont is currently considered the sister group to
the remaining members of the family (Pont, 1977) because it re-
tains the ejaculatory apodeme that apparently has been lost in
all other Fanniidae.
1.2. Muscidae
There are approximately 5000 described muscid species in
some 170 genera and the family is amply represented in all biogeo-
graphical regions. The larvae are usually saprophagous while
adults can be saprophagous, predacious, hematophagous, or feed-
ers on nectar and pollen. Many muscids are vectors of disease. Pre-
sumably because of the large number of species, genera, and
subfamilies, many different and often conflicting classifications
and phylogenetic hypotheses have been proposed for this group
(Malloch, 1934; Se
´guy, 1937; Roback, 1951; Hennig, 1955–1964,
1965; Couri and Pont, 2000; Carvalho and Couri, 2002; Couri and
Carvalho, 2003; Savage et al., 2004). Muscid monophyly is gener-
ally considered uncontroversial, although it is supported by only
a few morphological character states. These include the loss of
both the female abdominal spiracles 6–7 and the male accessory
glands (Se
´guy, 1937; Roback, 1951; Hennig, 1965, 1973; McAlpine,
1989; Michelsen, 1991; Carvalho and Couri, 2002). Species of the
Palaearctic Achanthiptera Lioy and the Neotropical Cariocamyia
Snyder have been stated to have independently re-acquired spira-
cle 6 (Carvalho et al., 2005), and the absence of male accessory
glands (or rather: glandular tissue continuous with vasa deferentia;
see Riemann, 1973), which has been confirmed for only a minority
of the species, is shared with the Scathophagidae. Muscid mono-
phyly was recently corroborated using molecular data (Schuehli
et al., 2007). Currently, eight subfamilies are recognized (Achan-
thipterinae, Atherigoninae, Azeliinae, Cyrtoneurininae, Coenosii-
nae, Muscinae, Mydaeinae, and Phaoniinae), but the subfamilies
and tribes in this family have undergone many classificatory
changes and various hypotheses of relationships have been pro-
posed (Couri and Pont, 2000; Couri and Carvalho, 2003; Savage
and Wheeler, 2004; Nihei and de Carvalho, 2007; Schuehli et al.,
2007).
1.3. Anthomyiidae
The Anthomyiidae has more than 2000 described species in
approximately 50 genera and are most diverse in the Holarctic re-
gion. These flies are mostly found in wooded and moist habitats,
and they are also very abundant in subarctic and mountainous
areas. The best known anthomyiid species are the onion fly and
cabbage root fly that are major agricultural pests because their lar-
vae are phytophagous as root/shoot miners on many economically
important crops. As adults, anthomyiids feed on different types of
rotting media like dung and decaying plant material, on nectar in
flowers, or they are predacious on small insects. The most common
larval breeding habits include phytophagy and saprophagy on
decaying plant matter, but the family also includes several
mycophagous species. Larvae of certain species are known to be
internal parasites of grasshoppers (Acridomyia spp.), others are
kleptoparasites in solitary bees (Leucophora spp.), and Coenosopsia
spp. are dung breeders.
The oldest confirmed fossil of a calyptrate fly belongs to the
Anthomyiidae (Michelsen, 2000). It comes from Baltic amber,
which has been dated as 40 mya old. With regard to anthomyiid
monophyly, Griffiths (1972:144) stated that ‘‘The limits of the
Anthomyiidae require clarification since no autapomorphous con-
ditions can be put forward to demonstrate that the family, as pres-
ently delimited, is a probable monophyletic group”. Hennig (1973)
also noted the absence of derived ground plan features. However,
in other publications (Hennig, 1976; Michelsen, 1991, 1996), the
Anthomyiidae have been regarded as monophyletic and supported
by many morphological character states. The main ones are the
presence of a strong ventro-basal seta on hind tarsomere 1, hair-
like setulae subapically on the underside of scutellum, a surstylus
with a sclerotised connection to the cercus, a surstylus that is dis-
tally biramous (but often secondarily simplified), and fused male
cerci.
The Anthomyiidae were previously classified as a subfamily of a
Muscidae sensu lato (even including the Fanniidae) and were fur-
ther split into two or more subfamilies. Hucket (1965) considered
the genera Fucellia Robineau-Desvoidy and Circia Malloch [=Alliop-
sis Schnabl & Dziedzicki] a separate subfamily (Fucelliinae) while
the remaining anthomyiids were in his subfamily Anthomyiinae,
which was divided into two tribes, viz. the Anthomyiini and Myop-
inini. This classification has since been completely abandoned, and
the family now stands without any subfamilies (e.g. Suwa and Dar-
vas, 1998) or is tentatively subdivided into four major subgroups:
the Phaonantho Albuquerque genus-group and the subfamilies
Myopininae, Pegomyinae, and Anthomyiinae (Michelsen, 2000). A
controversial issue is whether the New World genera Coenosopsia
and Phaonantho together constitute the extant sister group of the
remaining family (Michelsen, 1991, 2000), or whether these two
genera are not sister groups and neither is a basal anthomyiid tax-
on (Nihei and de Carvalho, 2004).
1.4. Scathophagidae
The Scathophagidae are another relatively small muscoid family
with about 400 described species. This family exhibits an unusu-
ally varied natural history ranging from saprophagy over phyto-
phagy to predation: some species breed in different types of
dung or other decaying organic matter such as rotting seaweed;
others mine in leaves, bore in culms, and/or feed on immature
flower heads or seed capsules, and ovules. Larvae of a few species
are also known to be predators of small invertebrates or caddis fly
egg masses. The monophyly of the Scathophagidae has found no
(Griffiths, 1972) or only little (Hennig, 1973) support from mor-
phological characters, but two recent molecular studies have
brought considerable progress with the result that phylogenetic
640 S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652
relationships in this family are now comparatively well understood
(Bernasconi et al., 2000a; Kutty et al., 2007). Kutty et al. (2007)
reconstructed the relationships in the Scathophagidae from 63 spe-
cies (representing 22 genera) based on the mitochondrial genes
12S rRNA, 16S rRNA, COI, Cytb, and the nuclear genes 28S rRNA,
Ef1a, and RNA polymerase II (Pol II). The monophyly of the Scath-
ophagidae was corroborated with strong support. The two recog-
nized subfamilies of the Scathophagidae, Scathophaginae, and
Delininae, emerged as monophyletic sister groups. The monophyly
of most genera, including Cordilura,Nanna,Norellia,Gimnomera,
Hydromyza, and Spaziphora, was also confirmed.
1.5. Interfamilial relationships
The phylogenetic relationships between the families of Muscoi-
dea are controversial. This is well illustrated by the different
hypotheses that exist for the position of the Fanniidae within the
Muscoidea. A sister group relationship has been suggested between
the Fanniidae and Muscidae (Hennig, 1965, 1973) and the Fannii-
dae were also considered a subfamily of the Muscidae (Huckett
and Vockeroth, 1987). Alternatively, the Fanniidae were proposed
to be the sister group of the remaining Muscoidea (Pont, 1977).
The other families have similarly generated conflicting hypotheses.
Based on morphological characters, the Muscidae and Anthomyii-
dae have been proposed as sister groups (Michelsen, 1991),
whereas the Scathophagidae have been regarded as the sister group
to the Anthomyiidae on the basis of molecular data (Bernasconi
et al., 2000a; Bernasconi et al., 2000b; Kutty et al., 2007). McAlpine
(1989) concluded, based on several allegedly autapomorphic char-
acter states, that a taxon composed of Anthomyiidae, Muscidae, and
Fanniidae is monophyletic, which suggested that the Scathophagi-
dae are the sister group of the remaining Muscoidea.
Much taxonomic and systematic research on the various taxa
within the Muscoidea has been carried out, but these studies mostly
addressed issues at the species and genus level. Comparatively few
studies also addressed relationships across families and even fewer
studies explicitly targeted the interfamilial relationships within the
Muscoidea. Exceptions include McAlpine (1989) and Hennig (1973),
who both utilized morphological characters but nevertheless ob-
tained conflicting results. Therefore, it appears timely to use a differ-
ent source of data; i.e. DNA sequences. A small-scale phylogenetic
study using the genes Cytochrome oxidase subunit I and II was car-
ried out by Bernasconi et al. (2000b), but the authors had to con-
clude that ‘‘the exact relationships among the Muscoidea still
remain unclear” and they stressed the need for further research. In
our study, we test the monophyly of Muscoidea and address the po-
sition of the superfamily and its constituent families within the
Calyptratae. In particular, we focus on the relationship between
the Muscoidea and the Oestroidea and the phylogenetic relationship
between the four families of Muscoidea. To this end we use DNA se-
quence data from eight genes (12S, 16S, COI, Cytb, 18S, 28S, Ef1a, and
CAD) and 127 species from all four muscoid families, ten species
from three families of Oestroidea, two species of Hippoboscoidea,
and seven outgroups from the Acalyptratae. This study of the Mus-
coidea is our third contribution to a better understanding of calyp-
trate relationships. Previous studies addressed the intrafamilial
relationships of the Scathophagidae (Kutty et al., 2007) and the
Hippoboscoidea (Petersen et al., 2007), respectively.
2. Materials and methods
2.1. Taxa and DNA extraction
The Muscoidea are here represented by 127 exemplar species
from the four constituent families (Table 1). With regard to the
remaining two calyptrate superfamilies, we included two species
from the Hippoboscoidea (Glossinidae and Hippoboscidae, respec-
tively), while the Oestroidea are represented by ten species from
the four major families (Calliphoridae, Rhinophoridae, Sarcophagi-
dae, and Tachinidae). The probable calliphorid non-monophyly as
shown by Rognes (1997) has not been an issue of the present study
and will be addressed in a forthcoming publication. As outgroups
we included seven acalyptrate species representing four different
superfamilies: Carnoidea (Hemeromyia anthracina), Lauxanoidea
(Celyphidae sp.), Sciomyzoidea (Lopa convexa, Gluma nitida), and
Tephritoidea (Ceratitis capitata,Bactrocera dorsalis, and Bactrocera
oleae). Most of the DNA extractions utilized a CTAB extraction pro-
tocol as described in Kutty et al. (2007). DNA extractions for some
species were also carried out according to manufacturer’s instruc-
tions using the QIAamp tissue kit (QIAGEN, Santa Clara, CA).
2.2. DNA amplification
Standard PCR amplifications were carried out using either Taka-
ra Ex-Taq or Bioline Taq on 1–5
l
l of template DNA. Nine different
gene regions were amplified which included the mitochondrial
genes 12S ribosomal RNA, 16S ribosomal RNA, Cytochrome oxidase
I (in two parts), Cytochrome b, and the nuclear genes 18S ribo-
somal RNA, 28S ribosomal RNA, Elongation factor 1-
a
, and a frag-
ment of the carbamolyphosphate synthetase (CPS) region of the
CAD gene. Due to the variable quality of the extracted DNA and
the large phylogenetic distances between the exemplar species,
not all genes successfully amplified. Furthermore, due to the con-
servative nature of the 18S sequences, we only amplified this gene
for 20 species representing the major taxa in the analysis (see Ta-
ble 1). The PCR cycles for all amplifications except CAD consisted of
an initial denaturation step at 95 °C for 7 min, followed by 95 °C for
1.5 min, annealing at temperatures ranging from 44–50 °C, and
extension at 72 °C for 1.5 min. A final extension at 72 °C for approx-
imately 5 min was also added. The amplified gene products were
purified using Bioline Sure-Clean solution following the manufac-
turer’s protocol. For CAD, the PCR protocol described by Moulton
and Wiegmann (2004) was used for the amplification, and gel
extraction was carried out on the amplified product using QIAquick
Gel extraction kit following the manufacturer’s protocol. Cycle
sequencing was performed on the purified products using BigDye
Terminator v3.1 and direct sequencing was carried out on an ABI
3100 genetic analyser (Perkin Elmer). The sequences were edited
and assembled in Sequencher 4.0 (Gene Codes Corp., Ann Arbor, MI).
2.3. Alignments
The protein encoding genes COI, Cytb, Ef1a, and CAD were
aligned based on amino acid translations in AlignmentHelper
(McClellan and Woolley, 2004), which uses ClustalW (Thompson
et al., 1994) for the amino acid alignment. The nucleotide align-
ments were indel-free for most genes except for CAD where codon
insertions were found in two muscid species. Due to the large size
of the data set, aligning the ribosomal genes was challenging. We
rejected a manual alignment because it would yield non-repeat-
able results, while a visual inspection of the default alignments
in ClustalX revealed unconvincing homology hypotheses. Given
that the quality of alignments is heavily dependent on the guide
trees used during the alignment (Kumar and Filipski, 2007), we in-
spected the default guide trees. These guide trees were in conflict
with regard to many of the well supported monophyletic groups.
For example, in the 12S guide tree we find that the muscids,
scathophagids, and anthomyiids scattered across the tree and the
same pattern was seen in the other guide trees for ribosomal genes
(see Supplementary material). We thus decided to improve the
guide tree using the following steps. We determined a preliminary
S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652 641
Table 1
List of taxa used in study with GenBank Accession Numbers
Taxa Voucher
number
RMBR #
Author name GenBank Accession Numbers
12S 16S 28S COI CYTB EF1a CAD4 18S
Carnidae
Hemeromyia anthracina 102770 Collin (1949) FJ025402 FJ025464 FJ025553 FJ025644 N/A N/A N/A N/A
Celyphidae
Celyphidae sp. 102769 FJ025401 FJ025463 FJ025552 FJ025643 N/A N/A FJ025568 N/A
Coelopidae
Lopa convexa 102737 McAlpine (1991) N/A AF403450 FJ025535 EU435768 EU435900 AY048515 N/A EU435620
Gluma nitida 102710 McAlpine (1991) N/A AF403468 FJ025517 EU435770 EU435902 AY048533 N/A EU435622
Tephritidae
Ceratitis capitata 102676 Wiedemann (1824) AJ242872 AJ242872 N/A AJ242872 AJ242874 N/A N/A DQ490237
Bactrocera dorsalis 102670 Hendel (1912) DQ845759 DQ845759 N/A DQ845759 DQ845759 N/A N/A N/A
Bactrocera oleae 102669 Rossi (1790) AY210702 AY210702 N/A AY210702 AY210702 N/A N/A N/A
Glossinidae
Glossina pallidipes 102712 Austen (1903) N/A EF531111 EF531136 EF531201 N/A N/A EF531179 N/A
Hippoboscidae
Ornithomya biloba 102768 Dufour (1827) N/A EF531119 EF531147 EF531212 N/A N/A EF531169 N/A
Fanniidae
Fannia canicularis 102705 Linnaeus (1761) DQ656884 DQ648647 DQ656961 DQ657037 DQ657051 N/A EF531184 FJ025489
Fannia manicata 102706 Meigen (1826) DQ656885 N/A DQ656962 DQ657038 DQ657052 N/A N/A FJ025490
Muscidae
Musca domestica 102741 Linnaeus (1758) DQ656896 DQ648650 DQ656974 AF104622 DQ657064 DQ657113 FJ025591 N/A
Mesembrina mystacea 102745 Linnaeus (1758) DQ656895 FJ025453 DQ656973 DQ657046 DQ657063 N/A N/A FJ025493
Stomoxys calcitrans 102784 Linnaeus (1758) DQ656886 EF531122 DQ656963 DQ657039 DQ657053 FJ025698 EF531173 FJ025499
Coenosia testacea 102690 Robineau-Desvoidy (1830) FJ025367 FJ025426 N/A FJ025605 FJ025707 N/A FJ025569 N/A
Coenosia tigrina 102691 Fabricius (1775) FJ025368 FJ025427 FJ025503 FJ025606 FJ025708 N/A FJ025570 N/A
Drymeia alpicola 102697 Rondani (1871) FJ025370 FJ025430 FJ025508 FJ025608 FJ025710 FJ025669 FJ025572 N/A
Drymeia hamata 102698 Fallén (1823) FJ025371 FJ025431 FJ025509 FJ025609 FJ025711 FJ025670 FJ025573 N/A
Eudasyphora cyanella 102701 Meigen (1826) FJ025373 FJ025433 FJ025511 FJ025611 N/A FJ025671 FJ025574 N/A
Haematobosca stimulans 102722 Meigen (1824) FJ025375 FJ025437 FJ025518 FJ025615 FJ025716 FJ025673 FJ025576 N/A
Phaonia pallida 102779 Fabricius (1787) FJ025409 FJ025469 FJ025555 FJ025651 FJ025747 N/A FJ025596 N/A
Haematobia irritans 102727 Linnaeus (1758) NC007102 FJ025436 N/A DQ029097 FJ025715 N/A N/A N/A
Hebecnema fumosa 102717 Meigen (1826) N/A N/A FJ025519 FJ025616 N/A N/A N/A N/A
Hebecnema umbratica 102723 Meigen (1826) N/A N/A FJ025520 FJ025617 FJ025717 N/A N/A N/A
Helina celsa 102713 Harris (1780) FJ025376 FJ025438 FJ025521 FJ025618 FJ025718 FJ025674 N/A N/A
Helina evecta 102726 Harris (1780) FJ025377 FJ025439 FJ025522 FJ025619 FJ025719 FJ025675 N/A N/A
Helina impuncta 102718 Fallén (1825) FJ025378 FJ025440 FJ025523 FJ025620 N/A FJ025676 N/A N/A
Helina lasiophthalma 102721 Macquart (1835) FJ025379 N/A FJ025524 FJ025621 FJ025720 N/A FJ025577 N/A
Hydrotaea cyrtoneurina 102715 Zetterstedt (1845) FJ025380 FJ025441 FJ025526 FJ025622 FJ025721 FJ025678 FJ025578 N/A
Hydrotaea dentipes 102716 Fabricius (1805) FJ025381 FJ025442 FJ025527 FJ025623 FJ025722 FJ025679 FJ025579 N/A
Hydrotaea irritans 102719 Fallén (1823) N/A FJ025443 N/A FJ025624 FJ025723 FJ025680 FJ025580 N/A
Limnophora exuta 102730 Kowarz (1893) FJ025384 FJ025446 FJ025530 FJ025626 FJ025725 FJ025684 FJ025581 N/A
Limnophora maculosa 102732 Meigen (1826) FJ025385 FJ025447 FJ025531 FJ025627 FJ025726 FJ025685 FJ025582 N/A
Limnophora olympiae 102733 Lyneborg (1965) FJ025386 FJ025448 FJ025532 FJ025628 FJ025727 FJ025686 FJ025583 N/A
Limnophora riparia 102734 Fallén (1824) FJ025387 FJ025449 FJ025533 FJ025629 FJ025728 N/A FJ025584 N/A
Lispe tentaculata 102736 De Geer (1776) FJ025388 FJ025450 FJ025534 FJ025630 FJ025729 FJ025687 FJ025585 N/A
Mesembrina meridiana 102744 Linnaeus (1758) FJ025390 FJ025452 FJ025537 FJ025633 N/A N/A FJ025586 N/A
Morellia aenescens 102738 Robineau-Desvoidy (1830) FJ025391 FJ025454 FJ025539 FJ025634 FJ025731 N/A FJ025587 N/A
Morellia hortorum 102742 Fallén (1817) FJ025392 FJ025455 FJ025540 FJ025635 FJ025732 N/A FJ025588 N/A
Morellia simplex 102748 Loew (1857) FJ025393 FJ025456 FJ025541 FJ025636 FJ025733 N/A FJ025589 N/A
Musca autumnalis 102740 De Geer (1776) FJ025394 FJ025457 FJ025542 FJ025637 FJ025734 N/A FJ025590 N/A
Muscina levida 102743 Harris (1780) FJ025395 FJ025458 FJ025544 FJ025638 FJ025735 FJ025688 N/A N/A
Muscina stabulans 102749 Fallén (1817) FJ025396 EF531117 EF531145 EF531210 FJ025736 FJ025689 EF531167 N/A
Mydaea ancilla 102739 Meigen (1826) FJ025398 FJ025460 FJ025547 FJ025639 FJ025737 FJ025690 FJ025592 N/A
Mydaea rufinervis 102747 Pokorny (1889) N/A FJ025461 N/A FJ025640 FJ025738 N/A N/A N/A
Mydaea urbana 102752 Meigen (1826) FJ025399 N/A FJ025548 FJ025641 FJ025739 FJ025691 FJ025593 N/A
Myospila meditabunda 102751 Fabricius (1781) FJ025400 FJ025462 FJ025549 FJ025642 N/A FJ025692 FJ025594 N/A
Phaonia subventa 102781 Harris (1780) FJ025410 FJ025470 N/A FJ025652 FJ025748 N/A N/A N/A
Polietes lardarius 102776 Fabricius (1781) FJ025411 FJ025471 FJ025557 FJ025653 N/A FJ025695 FJ025597 N/A
Potamia littoralis 102777 Robineau-Desvoidy (1830) FJ025412 FJ025472 FJ025558 FJ025654 N/A N/A FJ025598 N/A
Spilogona caliginosa 102785 Stein (1916)) FJ025414 FJ025474 N/A FJ025657 FJ025750 N/A N/A N/A
Spilogona dispar 102787 Fallén (1823) FJ025415 FJ025475 FJ025560 FJ025658 FJ025751 N/A FJ025599 N/A
Thricops aculeipes 102805 Zetterstedt (1838) FJ025417 FJ025477 N/A FJ025660 FJ025752 FJ025699 N/A N/A
Thricops cunctans 102806 Meigen (1826) FJ025418 FJ025478 FJ025564 FJ025661 FJ025753 FJ025700 FJ025600 N/A
Thricops genarum 102809 Zetterstedt (1838) FJ025419 FJ025479 N/A FJ025662 FJ025754 FJ025701 N/A N/A
Thricops nigritellus 102810 Zetterstedt (1838) FJ025420 FJ025480 N/A FJ025663 FJ025755 FJ025702 N/A N/A
Villeneuvia aestuum 102811 Villeneuve (1902) FJ025421 FJ025481 N/A FJ025664 FJ025756 FJ025703 N/A N/A
Anthomyiidae
Botanophila fugax 102671 Meigen (1826) DQ656890 N/A DQ656967 DQ657042 DQ657057 FJ025665 N/A N/A
Botanophila rubrifrons 102673 Ringdahl (1933) FJ025364 FJ025423 N/A FJ025602 N/A N/A N/A N/A
Delia platura 102700 Meigen (1826) DQ656894 N/A DQ656972 DQ657045 DQ657062 N/A N/A FJ025486
Emmesomyia grisea 102702 Robineau-Desvoidy (1830) FJ025372 N/A FJ025510 FJ025610 FJ025712 N/A N/A FJ025487
Eutrichota frigida 102704 Zetterstedt (1845) N/A N/A FJ025513 FJ025613 FJ025714 N/A N/A N/A
Hydrophoria lancifer 102720 Harris (1780) DQ656891 N/A DQ656968 DQ657043 DQ657058 N/A EF531164 N/A
Hylemya vagans 102724 Panzer (1798) FJ025382 N/A N/A FJ025625 N/A N/A N/A N/A
Hylemya variata 102725 Fallén (1823) FJ025383 FJ025444 N/A N/A FJ025724 N/A N/A N/A
Lasiomma latipenne 102731 Zetterstedt (1838) DQ656892 FJ025445 DQ656970 DQ657044 DQ657060 FJ025683 N/A FJ025491
Lasiomma seminitidum 102735 Zetterstedt (1845) DQ656893 DQ648649 DQ656971 AF104624 DQ657061 DQ657112 N/A N/A
Mycophaga testacea 102750 Gimmerthal (1834) DQ656890 N/A DQ656967 DQ657042 DQ657057 N/A N/A N/A
Paregle coerulescens 102774 Strobl (1893) FJ025403 N/A N/A FJ025645 FJ025741 FJ025693 N/A N/A
Pegomya winthemi 102782 Meigen (1826) FJ025406 N/A N/A FJ025648 FJ025744 N/A N/A N/A
Pegoplata aestiva 102771 Meigen (1826) FJ025407 N/A N/A FJ025649 FJ025745 N/A N/A N/A
Pegoplata infirma 102775 Meigen (1826) FJ025408 N/A FJ025554 FJ025650 FJ025746 N/A N/A FJ025497
Scathophagidae
Acanthocnema glaucescens 102667 Loew (1864) DQ656897 DQ648651 DQ656975 AF181023 DQ657065 DQ657114 N/A N/A
Acerocnema macrocera 102668 Meigen (1826) DQ656898 DQ648652 DQ656976 AF181025 DQ657066 N/A N/A N/A
642 S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652
alignment with ClustalX 2.0 (Thompson et al., 1997) for the ribo-
somal genes 12S, 16S, 18S, and 28S (gap opening and extension
cost: 15:6.66). We then identified the indel-free fragments of the
ribosomal genes that are likely to correspond to stem regions of
the rRNAs and then concatenated the indel-free rDNA sequences
with the aligned sequences for the protein-encoding genes. We
then estimated a guide tree from this dataset by analyzing it with
parsimony (TNT v2.0: Goloboff et al., 2000: new technology search
at level 50, initial addseqs = 9, find minimum tree length 5 times).
This analysis yielded ten most parsimonious trees. Each of these
topologies was then used as guide trees for aligning the full-length
DNA sequence data for the ribosomal genes in ClustalX. We thus
obtained ten different alignments. Using tree length as an optimal-
ity criterion, the alignment that yielded the shortest tree was used
Table 1 (continued)
Taxa Voucher number
RMBR #
Author name GenBank Accession Numbers
12S 16S 28S COI CYTB EF1a CAD4 18S
Americina adusta 102666 Loew (1863) DQ656899 DQ648653 DQ656977 AF181030 DQ657067 N/A N/A N/A
Ceratinostoma ostiorum 102684 Curtis (1832) DQ656914 DQ648668 DQ656992 AF180792 AF180986 DQ657123 N/A N/A
Chaetosa (Opsiomyia)palpalis 102685 Coquillett DQ656915 DQ648669 DQ656993 AF181017 DQ657082 DQ657124 N/A N/A
Chaetosa punctipes 102689 Meigen (1826) DQ656916 DQ648670 DQ656994 AF181016 DQ657083 N/A N/A N/A
Chylizosoma vittatum 102694 Meigen (1826) DQ656900 DQ648654 DQ656978 AF181031 DQ657068 N/A N/A N/A
Cleigastra apicalis 102696 Meigen (1826) DQ656901 DQ648655 DQ656979 AF181024 DQ657069 DQ657115 N/A N/A
Cordilura (Achaetella)varipes 102693 Walker (1849) DQ656913 DQ648667 DQ656991 AF180996 DQ657081 FJ025668 N/A N/A
Cordilura (Cordilura)carbonaria 102677 Walker (1849) DQ656904 DQ648658 DQ656982 AF180988 DQ657072 DQ657117 N/A N/A
Cordilura (Cordilura)ciliata 102678 Meigen (1826) DQ656905 DQ648659 DQ656983 AF180989 DQ657073 N/A EF531159 N/A
Cordilura (Cordilura)ontario 102683 Curran (1929) DQ656908 DQ648662 DQ656986 AF180992 DQ657076 DQ657120 N/A N/A
Cordilura (Cordilura)pudica 102688 Meigen (1826) DQ656911 DQ648665 DQ656989 AF180991 DQ657079 N/A N/A N/A
Cordilura (Cordilura)umbrosa 102692 Loew (1873) DQ656912 DQ648666 DQ656990 AF180990 DQ657080 N/A N/A N/A
Cordilura (Cordilurina)albipes 102674 Fallén (1819) DQ656902 DQ648656 DQ656980 AF180995 DQ657070 N/A N/A N/A
Cordilura (Parallelomma) dimidiata 102679 Cresson (1918) DQ656906 DQ648660 DQ656984 AF180993 DQ657074 DQ657118 N/A N/A
Cordilura (Parallelomma)pleuritica 102686 Loew (1863) DQ656909 DQ648663 DQ656987 AF180994 DQ657077 DQ657121 N/A N/A
Cordilura atrata 102675 Zetterstedt (1846) DQ656903 DQ648657 DQ656981 DQ657047 DQ657071 DQ657116 N/A N/A
Cordilura latifrons 102681 Loew (1869) DQ656907 DQ648661 DQ656985 AF180997 DQ657075 DQ657119 N/A N/A
Cordilura pubera 102687 Linnaeus (1758) DQ656910 DQ648664 DQ656988 AF180987 DQ657078 DQ657122 N/A FJ025485
Delina nigrita 102699 Fallén (1819) DQ656889 DQ648648 DQ656966 AF181029 DQ657056 N/A N/A N/A
Gimnomera cerea 102707 Coquillett (1908) DQ656917 DQ648671 DQ656995 AF181009 DQ657084 DQ657125 N/A N/A
Gimnomera dorsata 102709 Zetterstedt (1838) DQ656919 DQ648673 DQ656997 AF181008 DQ657086 N/A N/A N/A
Gymnomera cuneiventris 102708 Zetterstedt (1846) DQ656918 DQ648672 DQ656996 AF181011 DQ657085 N/A N/A N/A
Gymnomera tarsea 102711 Fallén (1819) DQ656920 DQ648674 DQ656998 AF181010 DQ657087 N/A N/A N/A
Hydromyza confluens 102714 Loew (1863) DQ656921 DQ648675 N/A AF181015 DQ657088 DQ657126 N/A N/A
Hydromyza livens 102728 Fabricius (1794) DQ656922 DQ648676 DQ656999 AF181014 DQ657089 N/A N/A N/A
Microprosopa pallidicauda 102746 Zetterstedt (1838) DQ656923 DQ648677 DQ657000 AF181021 DQ657090 N/A N/A N/A
Nanna articulata 102754 Becker (1894) DQ656924 DQ648678 DQ657001 DQ657048 DQ657091 N/A N/A N/A
Nanna brunneicosta 102755 Johnson (1927) DQ656925 DQ648679 DQ657002 AF181006 DQ657092 DQ657127 N/A N/A
Nanna fasciata 102756 Meigen (1826) DQ656926 DQ648680 DQ657003 AF181003 DQ657093 N/A N/A N/A
Nanna flavipes 102757 Fallén (1819) DQ656927 DQ648681 DQ657004 AF181005 DQ657094 N/A N/A N/A
Nanna inermis 102758 Becker (1894) DQ656928 DQ648682 DQ657005 AF181004 DQ657095 N/A N/A N/A
Nanna tibiella 102759 Zetterstedt (1838) DQ656929 DQ648683 DQ657006 AF181007 DQ657096 DQ657128 N/A N/A
Neorthacheta dissimilis 102753 Malloch (1924) DQ656930 DQ648684 DQ657007 AF181027 DQ657097 DQ657129 N/A N/A
Norellia (Norellia)tipularia 102765 Fabricius (1794) DQ656936 DQ648690 DQ657013 AF180998 DQ657103 DQ657133 N/A N/A
Norellia (Norellisoma)flavicorne 102760 Collin (1958) DQ656931 DQ648685 DQ657008 DQ657049 DQ657098 DQ657130 N/A N/A
Norellia (Norellisoma)liturata 102761 Wiedemann (1826) DQ656932 DQ648686 DQ657009 AF181001 DQ657099 DQ657131 N/A N/A
Norellia (Norellisoma)mirusae 102762 Šifner (1974) DQ656933 DQ648687 DQ657010 AF181002 DQ657100 DQ657132 N/A N/A
Norellia (Norellisoma)spinimana 102763 Fallén (1819) DQ656934 DQ648688 DQ657011 AF180999 DQ657101 N/A N/A N/A
Norellia (Norellisoma)striolata 102764 Meigen (1826) DQ656935 DQ648689 DQ657012 AF181000 DQ657102 N/A N/A N/A
Okeniella caudata 102766 Zetterstedt (1838) DQ656937 DQ648691 DQ657014 AF181020 DQ657104 N/A N/A N/A
Orthacheta cornuta 102767 Loew (1863) DQ656938 DQ648692 DQ657015 AF181026 DQ657105 DQ657134 N/A N/A
Phrosia albilabris 102772 Fabricius (1805) DQ656939 DQ648693 DQ657016 AF181028 DQ657106 N/A N/A N/A
Pogonota (Lasioscelus)sahlbergi 102780 Becker (1900) DQ656941 DQ648695 DQ657018 AF181018 DQ657108 N/A N/A N/A
Pogonota (Pogonota)barbata 102773 Zetterstedt (1838) DQ656940 DQ648694 DQ657017 AF181019 DQ657107 N/A N/A N/A
Scathophaga analis 102790 Meigen (1826) DQ656942 DQ648696 DQ657019 AF180783 AF180977 DQ657135 N/A N/A
Scathophaga calida 102791 Curtis (1832) DQ656943 DQ648697 DQ657020 AF181003 AF180981 DQ657136 N/A N/A
Scathophaga cineraria 102792 Meigen (1826) DQ656944 DQ648698 DQ657021 AF180784 AF180978 DQ657137 N/A N/A
Scathophaga furcata 102793 Say (1823) DQ656945 DQ648699 DQ657022 AF180777 AF180974 DQ657138 N/A N/A
Scathophaga incola 102794 Becker (1900) DQ656946 DQ648700 DQ657023 AF180786 AF180980 N/A N/A N/A
Scathophaga inquinata 102795 Meigen (1826) DQ656947 DQ648701 DQ657024 AF180781 AF180976 N/A N/A N/A
Scathophaga litorea 102796 Fallén (1819) DQ656948 DQ648702 DQ657025 AF180789 AF180983 DQ657139 N/A N/A
Scathophaga lutaria 102797 Fabricius (1794) DQ656949 DQ648703 DQ657026 AF180779 AF180975 DQ657140 N/A N/A
Scathophaga obscura 102798 Fallén (1819) DQ656950 DQ648704 DQ657027 AF180790 AF180984 N/A N/A N/A
Scathophaga pictipennis 102799 Oldenberg (1923) DQ656951 DQ648705 DQ657028 AF180785 AF180979 N/A N/A N/A
Scathophaga stercoraria 102800 Linnaeus (1758) DQ656952 DQ648706 DQ657029 AF180759 AF180971 DQ657141 N/A N/A
Scathophaga suilla 102801 Fabricius (1794) DQ656953 DQ648707 DQ657030 AF180773 AF180973 DQ657142 N/A N/A
Scathophaga taeniopa 102802 Rondani (1866) DQ656954 DQ648708 DQ657031 AF180768 AF180972 N/A N/A N/A
Scathophaga tinctinervis 102803 Becker (1894) DQ656955 DQ648709 DQ657032 AF180079 AF180985 FJ025697 N/A N/A
Scathophaga tropicalis 102804 Malloch (1931) DQ656956 DQ648710 DQ657033 AF180788 AF180982 DQ657143 N/A N/A
Spaziphora cincta 102786 Loew (1863) DQ656957 DQ648711 DQ657034 AF181012 DQ657109 N/A N/A N/A
Spaziphora hydromyzina 102788 Fallén (1819) DQ656958 DQ648712 DQ657035 AF181013 DQ657110 N/A N/A N/A
Trichopalpus fraterna 102808 Meigen (1826) DQ656959 DQ648713 DQ657036 AF181022 DQ657111 N/A N/A N/A
Sarcophagidae
Peckia gulo 102783 Fabricius (1805) FJ025405 FJ025467 N/A FJ025647 FJ025743 N/A N/A N/A
Sarcophaga arizonica 102789 Townsend (1919) FJ025413 FJ025473 FJ025559 FJ025655 FJ025749 FJ025696 N/A FJ025498
Tachinidae
Tachina ferox 102807 Panzer (1809) FJ025416 FJ025476 FJ025562 FJ025659 N/A N/A N/A FJ025500
Calliphoridae
Bengalia peuhi 102672 Villeneuve (1914 FJ025363 FJ025422 FJ025501 FJ025601 FJ025704 N/A FJ025566 N/A
Calliphora vomitoria 102695 Linnaeus (1758) FJ025365 FJ025424 FJ025502 FJ025603 FJ025705 FJ025666 FJ025567 FJ025482
Compsomyiops fulvicrura 102680 Robineau-Desvoidy (1830) FJ025369 FJ025428 FJ025504 FJ025607 FJ025709 FJ025667 FJ025571 FJ025484
Chrysomya megacephala 102682 Fabricius (1794) FJ025366 FJ025425 N/A FJ025604 FJ025706 N/A N/A FJ025483
Eurychaeta palpalis 102703 Robineau-Desvoidy (1830) FJ025374 FJ025434 FJ025512 FJ025612 FJ025713 FJ025672 FJ025575 N/A
Lucilia caesar 102729 Linnaeus (1758) FJ025389 FJ025451 N/A FJ025632 FJ025730 N/A N/A FJ025492
Rhinophoridae
Paykullia maculata 102778 Fallén (1815) FJ025404 FJ025466 N/A FJ025646 FJ025742 FJ025694 FJ025595 FJ025496
S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652 643
in all subsequent analyses, but we also confirmed that the trees
based on the remaining alignments were very similar (see Supple-
mentary materials).
2.4. Tree search strategies
The aligned Muscoidea dataset had 146 taxa and 7202 charac-
ters. It was analyzed using both maximum parsimony (MP) and
maximum likelihood (ML). The tree was rooted using the acalyp-
trate Hemeromyia anthracina (Carnidae), although any other aca-
lyptrate family could have been used as outgroup given that we
currently do not have a viable hypothesis as to which acalyptrate
taxon may be the sister group to the Calyptratae. Maximum parsi-
mony analyses were carried out in TNT v2.0 (Goloboff et al., 2000:
new technology search at level 50, initial addseqs = 9, find mini-
mum tree length 5 times), with indels coded once as missing data
and once as fifth character states. Node support was assessed by
jackknife resampling percentiles (250 replicates, same search op-
tions as above) obtained at 36% deletion as recommended by Farris
et al. (1996). For the likelihood analyses, we used MrModeltest ver-
sion 2.2 (Nylander, 2004) for identifying the best fit model
(GTR + I + V) based on the Akaike Information Criterion (AIC). The
Fig. 1. Strict consensus of three most parsimonious trees (indel = 5th character); above node jackknife support for indel = 5th character state; below node indel = missing;
nodes shared with ML tree indicated by H.
644 S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652
likelihood analyses were conducted with Garli v0.951 (Zwickl,
2006). Three independent runs were carried out and node support
was assessed using a non-parametric bootstrap with 250 replicates
using the automated stopping criterion set at 10,000 generations
for each replicate.
3. Results
After the alignment and concatenation of the eight genes 12S,
16S, COI, Cytb, 18S, 28S, Ef1a, and CAD, 2437 sites of the 7202 base
pairs were parsimony informative. The parsimony analysis with in-
dels coded as a fifth character state yielded three most parsimoni-
ous trees with a tree length of 24,267, while the analysis with
indels coded as missing data yielded 23 most parsimonious tress
with a tree length of 23,201. Parsimony analysis coding indels as
missing data (see Supplementary material) and as a fifth character
resulted in trees with identical family-level relationships and shar-
ing approximately 85% of the nodes, which suggests that indel cod-
ing has only a minor influence on the tree topology (Fig. 1). Since
the parsimony analyses for indels coded as missing data and as a
fifth character state respectively result in topologies that are con-
gruent for higher level relationships, other indel coding methods
like simple indel coding (SIC) and modified complex indel coding
(MCIC; Simmons et al., 2007; Simmons and Ochoterena, 2000)
Fig. 1 (continued)
S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652 645
were not tested. The maximum likelihood tree is shown in Fig. 2.
The overall tree topologies of the MP and ML trees are also very
similar and recover the same family-level relationships for the
Muscoidea. However, there is a conflict with regard to the Hipp-
oboscoidea, which are polyphyletic on the ML tree where the Glos-
sinidae emerge as the sister group of a clade consisting of the non-
fanniid Muscoidea plus the Oestroidea. There are also other topo-
logical differences within the muscoid and oestroid families (com-
pare Figs. 1 and 2).
The Calyptratae are corroborated as monophyletic, with modest
support in the maximum parsimony analysis and high support in
the maximum likelihood analysis. On the strict consensus of the
most parsimonious trees, the Hippoboscoidea are monophyletic
and placed as the sister group to the remaining calyptrates,
although with very modest support. The Muscoidea are the only
calyptrate superfamily that is paraphyletic and this paraphyly is
found in all the different analyses regardless of indel coding and
the use of maximum parsimony or maximum likelihood. The
monophyly of the Oestroidea is well supported and this superfam-
ily is nested within a paraphyletic Muscoidea. The Fanniidae are
the sister group to the remaining Muscoidea plus the Oestroidea.
The Muscidae are monophyletic and sister group to a clade com-
posed of Anthomyiidae, Scathophagidae, and Oestroidea. The
Anthomyiidae + Scathophagidae form a moderately supported
clade and the Oestroidea are well supported as the sister group
to this. In all analyses, we find that the Scathophagidae are nested
within a paraphyletic Anthomyiidae.
Most genera of the Musicdae are monophyletic: Coenosia,
Helina,Hebecnema,Limnophora,Mydaea,Mesembrina,Morellia,
Musca,Muscina,Phaonia,Spilogona, and Thricops. Only Hydrotaea
Fig. 2. Likelihood tree from ML analysis (Garli) indicating bootstrap support, nodes shared with selected guide tree indicated by Nand majority rule consensus of all ten guide
trees indicated by r.
646 S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652
does not emerge as monophyletic in the maximum parsimony
analysis, but the resampling analysis does not provide a conclusive
result. The maximum likelihood analysis has a highly corroborated
monophyletic Hydrotaea. At the subfamily level, we only recover a
monophyletic Coenosiinae, while the remaining subfamilies are
either para- or polyphyletic on the most parsimonious tree
(Mydaeinae, Phaoniinae, Azeliinae, and Muscinae). However, on
the maximum likelihood tree, the Muscinae are also monophyletic.
The Scathophagidae are monophyletic as are most genera
including Nanna,Gimnomera,Norellia, and Cordilura. The subfamily
Delininae is monophyletic but nested within the Scathophaginae.
In all analyses, the Anthomyiidae are paraphyletic. The genera
Botanophila,Hylemya, and Pegoplata are monophyletic, while Las-
iomma is paraphyletic. The two subfamilies Anthomyiinae and Peg-
omyinae are para- or poly-phyletic on the MP tree while the
subfamily Anthomyiinae is monophyletic on the ML tree. The Myo-
pininae are monophyletic on both the ML and MP tree. Within the
oestroids, the Sarcophagidae are monophyletic with high node
support. Tachina ferox and the Sarcophagidae are sister groups.
The calliphorid exemplars form a paraphyletic grade, with Paykul-
lia maculata (Rhinophoridae) being the sister group to Eurychaeta
palpalis.
Fig. 2 (continued)
S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652 647
4. Discussion
The Muscoidea are of economic importance, because many of its
species are pests on both agricultural crops and livestock, while oth-
ers are of medical importance with some species being vectors of dis-
ease. Muscoid flies are also among the most common insects and
many species live in close association with humans. However, the
relationships among the main clades of the Muscoidea have re-
mained poorlyunderstood, and past analyses yielded veryconflicting
hypotheses.Even the taxonomic composition of the Muscoidea with-
in Diptera has been controversial, and as mentioned by McAlpine
(1989): ‘‘The name Muscoidea has probably been used in a wider
variety of senses than any other suprageneric name in Diptera” (p.
1496). The usages range from encompassing all of Schizophora
(Coquillett, 1901) to being a subgroup of the Schizophora (Griffiths,
1972), to being a subgroup of the Calyptratae (Roback, 1951; Hennig,
1973; McAlpine, 1989). However, the most commonly used concept
of Muscoidea is that of Hennig (1973) and McAlpine (1989),who
classified the Anthomyiidae, Fanniidae, Scathophagidae, and Musci-
dae in the superfamily Muscoidea. In the absence of convincing evi-
dence to the contrary, Hennig (1973) used the monophyly of
Muscoidea as a working hypothesis, but kept the interfamilial rela-
tionships unresolved and considered the relationships to the other
calyptratesuperfamilies (Oestroidea and Hippoboscoidea) unknown.
Michelsen (1991), however, explicitly acknowledged the lack of sup-
port for muscoid monophyly by defining the Muscoidea as ‘‘the
Calyptratae less the Hippoboscoidea and Oestroidea”. Based on our
data we are able to test many of these hypotheses.
4.1. Comparison of tree hypotheses
The tree topologies from the three different analyses, MP with
indels treated as a fifth character state, MP with indels treated as
missing data and ML, are largely congruent. Most high-level rela-
tionships are uncontroversial regardless of which indel treatment
or the analysis strategy is used. The calyptrate monophyly is sup-
ported on all trees. Well supported is the position of the mono-
phyletic Oestroidea, which are always placed as the sister group
to the clade Anthomyiidae + Scathophagidae. The interfamilial
relationship (Fanniidae + (Muscidae + (Anthomyiidae + Scathoph-
agidae) + Oestroidea))) is also recovered irrespective of the ana-
lytical method. Of the approximately 80% nodes shared
between the MP and ML trees, many relationships at the subfam-
ily level are identical, including the monophyly of the subfamilies
Delininae (Scathophagidae) and Coenosiinae (Muscidae) and the
sister group relationship between the Phaoniinae + Mydaeinae
clade and Coenosiinae in the Muscidae. The terminal nodes are
generally supported by high jackknife values on the MP tree
and bootstrap values on the ML tree. However, the node support
for the higher level relationships is generally lower, which is
similar to the findings of many recent phylogenetic analyses of
higher-level phylogenetic relationships in Diptera (e.g. Tephritoi-
dea: Han and Ro, 2005; Empidoidea: Moulton and Wiegmann,
2007; Asiloidea: Holston et al., 2007; Opomyzoidea: Scheffer
et al., 2007). In the Muscoidea analysis the node support for
many higher level relationships is similarly low, despite the use
of large amounts of data and congruence between the tree topol-
ogies obtained using different analysis methods such as parsi-
mony and maximum likelihood. The only major conflict
between our MP and ML trees is the monophyly and position
of Hippoboscoidea. Regardless of indel codings, it is monophy-
letic on the MPTs, which is in agreement with the currently ac-
cepted hypothesis (Hennig, 1973; McAlpine, 1989; Nirmala
et al., 2001; Dittmar et al., 2006; Petersen et al., 2007). However,
the Hippoboscoidea are not monophyletic on the ML tree.
4.2. Calyptrate monophyly
The monophyly of the Calyptratae is well supported by a
large number of morphological characters but molecular data
have consistently suggested that the calyptrates may be para-
phyletic (Vossbrinck and Friedman, 1989; Bernasconi et al.,
2000b) with some acalyptrates being nested within. We believe
that this is due to very sparse taxon sampling in the earlier
molecular analyses, because in our study calyptrate monophyly
is consistently supported despite rigorous testing via the inclu-
sion of acalyptrate outgroups from four different superfamilies.
All remaining molecular studies only included few outgroup
taxa.
4.3. Superfamily monophyly and the relationships between the
calyptrate superfamilies
Among the three calyptrate superfamilies, the monophyly
and phylogeny of the Hippoboscoidea has been well studied
and supported using both morphological and molecular data
(Hennig, 1973; McAlpine, 1989; Nirmala et al., 2001; Dittmar
et al., 2006; Petersen et al., 2007). Due to insufficient gene over-
lap with the Petersen et al. (2007) study, our dataset included
only two representative species from this superfamily, but they
form a monophyletic group in the parsimony analyses. The
monophyly of and relationships among the remaining two
superfamilies, Muscoidea and Oestroidea, has been more open
to discussion. Previous studies suggested a sister group relation-
ship between Hippoboscoidea and the remaining calyptrate flies
(McAlpine, 1989), whereas in Petersen et al. (2007) the Hipp-
oboscoidea were deeply nested within the Calyptratae, although
outgroup sampling was sparse and the support for this hypoth-
esis was weak. Based on our most parsimonious tree, we find
that the Hippoboscoidea are the sister group of the remaining
Calyptratae, and that the Oestroidea are monophyletic. However,
the Muscoidea are likely to be paraphyletic with regard to the
Oestroidea. This confirms Michelsen’s proposal, but is in conflict
with McAlpine’s (1989) hypothesis of monophyly. However, it is
important to remember that McAlpine assessed all synapomor-
phies relative to the (hypothetical) groundplan of the Schizo-
phora; i.e. all the characters proposed as supporting Muscoidea
monophyly could have been plesiomorphic relative to the
groundplan of the Calyptratae. Our study is not the first to sug-
gest that the Muscoidea are a paraphyletic grade (Michelsen,
1991; Bernasconi et al., 2000b; Nirmala et al., 2001), but our
study is based on a much larger gene and taxon sample than
previous analyses, and we can place all muscoid families on
our phylogenetic hypothesis.
Once a group with a well-established name is shown to be
paraphyletic, a new classification and/or new names have to
be proposed. For example, Yeates and Wiegmann (1999) pro-
posed the informal names ‘‘lower Diptera” for Nematocera and
‘‘lower Cyclorrhapha” for Aschiza instead of proposing new
ranks and new names for subgroups within the non-Brachyceran
Diptera and non-Schizophoran Cyclorrhapha. We are in favour of
this approach that was also adopted in a recent review of Dip-
tera classification (Yeates et al., 2008). We thus propose that the
best way of referring to the paraphyletic Muscoidea will be as
the ‘‘muscoid grade”. An alternative would be a new superfam-
ily-level classification that would either require that the Oestroi-
dea are subsumed in the Muscoidea or that separate
superfamilies are recognized for the Fanniidae, Muscidae, and
Anthomyiidae + Scathophagidae, respectively. We consider the
latter as an unnecessary inflation in the number of superfami-
lies, and at least two would contain a single family only and
thereby be redundant.
648 S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652
4.4. Interfamilial relationships within the muscoid grade
The Fanniidae are placed as the sister group to a clade con-
sisting of the Oestroidea plus the remaining families of the mus-
coid grade on the most parsimonious tree (Fig. 1). This position
had been suggested based on molecular characters by Bernasconi
et al. (2000b), but was in conflict with the more traditional
views, which placed the family either as the sister group of the
Muscidae (Hennig, 1973) or as the sister group of Anthomyii-
dae + Muscidae (McAlpine, 1989). On the likelihood tree, the Fan-
niidae are in a similar position, but surprisingly Glossina
pallidipes is the sister group of Muscoidea + Oestroidea. Given
the strong morphological support for a monophyletic Hippobo-
scoidea, we believe that the overall evidence supports the most
parsimonious topology with Fanniidae being sister group to Mus-
coidea + Oestroidea. In any case, Fanniidae are never the sister
group of Muscidae or Anthomyiidae as had been previously
suggested.
With regard to the Muscidae, various authors have corroborated
the monophyly of this family using both morphological and molec-
ular data. This monophyly is further corroborated here. However,
our analysis is the first to address the relative position of Muscidae
within the calyptrates based on a large data set. In our analysis, the
family is the sister group of Oestroidea + (Scathophagidae + Antho-
myiidae). We also consistently find that Anthomyiidae + Scathoph-
agidae form a monophyletic group. This relationship was
suggested by Roback (1951), who included the Scathophagidae
(as Scopeumatinae) as a subfamily of the Anthomyiidae. His
hypothesis was based on vein A
1
+ CuA
2
reaching the wing margin,
which is probably a symplesiomorphy, and on the larval morphol-
ogy of Scathophaga stercoraria, which was stated to be ‘‘distinctly
anthomyoid in all its characteristics” (p. 333). However, in spite
of this, Roback also noted that ‘‘on the basis of the male genitalia,
and the presence of the three sternopleurals the Anthomyiinae can
be considered more advanced than the Scopeumatinae, and closer
to the remainder of the Muscoidea and the Sarcophagoidea” (p.
334). No other author has to our knowledge proposed morpholog-
ical support for Anthomyiidae + Scathophagidae, but it is consis-
tently supported by molecular data (Bernasconi et al., 2000b;
Kutty et al., 2007). The finding that the Oestroidea are the sister
group to the clade Anthomyiidae + Scathophagidae is a new result.
Previously, it has been thought that the Oestroidea are the sister
group to a monophyletic Muscoidea (Hennig, 1973; McAlpine,
1989), or even that the Oestroidea were paraphyletic with regard
to the Muscoidea (Roback, 1951).
4.5. Family monophyly and relationships within families
The Fanniidae here represented by two Fannia species are
monophyletic and this is consistent with morphological studies
on this family. The Muscidae are monophyletic but the support
is not very high, which may be due to a relatively small taxon
sample for this very large family. Although our species-level
sample is small, we do include representatives of most subfam-
ilies so that the test for monophyly is overall quite rigorous.
The monophyly of the subfamily Coenosiinae is corroborated.
On both the ML tree and the MP tree, Spilogona and Villeneuvia
are sister groups and closely related to the Coenosia species,
although Spilogona and Villeneuvia are generally considered more
closely related to the clade Limnophora +Lispe (Hennig, 1965).
The other subfamilies Azeliinae, Muscinae, Mydaeinae, and Phao-
niinae are not monophyletic in the MP tree, although the mono-
phyly of the clade Mydaeinae + Phaoniinae is well supported and
with the genera Helina,Phaonia,Mydaea, and Hebecnema being
monophyletic. The subfamily Coenosiinae and Phaoniinae are
also closely related as suggested by Schuehli et al. (2007) and
the Mydaeinae + Phaoniinae + Coesnosiinae clade has moderate
support. However, it is puzzling that the azeliine Muscina is sis-
ter group to Phaoniinae + Mydaeinae + Coenosiinae with moder-
ate support instead of being placed on the Azeliinae + Muscinae
branch. Similarly, the muscine genera Polietes and Mesembrina
are suprisingly nested within the Azeliinae. The genera Thricops
and Drymeia from the subfamily Azeliinae are monophyletic
and so are the muscine genera Musca and Morellia. The tribe
Stomoxyini within the Muscinae is here represented by Stomoxys
calcitrans and Haematobosca stimulans is monophyletic and the
sister group relationship of these two species was also suggested
in the molecular phylogeny of the Muscidae by Schuehli et al.
(2007) and Carvalho (1989).However, it is clear from some of
the unexpected findings that a more extensive taxon sample
for the Muscidae will be needed in order to resolve tribal and
subfamily level relationships.
In our analysis, the Anthomyiidae are paraphyletic but we find
that none of the nodes that render this family paraphyletic have
jackknife support on the MP tree (>50) or bootstrap support on
the ML tree (>50). Of the three subfamilies recognised by Michel-
sen (2000), only the Myopininae are monophyletic on both the
ML and MP tree. The genera Botanophila,Hylemya and Pegoplata
are monophyletic and only Lasiomma is paraphyletic (remaining
genera being represented by only a single exemplar species). It
should be noted that the Anthomyiidae are the most poorly sam-
pled muscoid family in our dataset and that additional species
are needed for a more rigorous test of anthomyiid monophyly. In
particular, exemplar species from the genera Coenosopsia and Pha-
onantho should be included.
The Scathophagidae and most of its genera are monophyletic.
However, the relationships between some of the genera and spe-
cies differ from the phylogenetic hypothesis in Kutty et al.
(2007). The most significant difference is a change in the position
of the subfamily Delininae which is here nested within the Scatho-
phaginae as opposed to being their sister group. However, it must
be recalled that in Kutty et al. (2007) a sensitivity analysis had
been conducted that revealed that the downweighting of transi-
tions was favoured (Meier and Wiegmann, 2002; Laamanen
et al., 2005), while in this study all character changes are equally
weighted because, due to the large size of the current data set, such
a sensitivity analysis would have been computationally prohibi-
tive. Most scathophagid genera are monophyletic as proposed by
Kutty et al. (2007), but some relationships between the genera dif-
fer on the ML and MP trees. However, these branches have low
node support in the MP and ML analyses.
Within the Oestroidea, the clade Sarcophagidae + Tachinidae is
monophyletic as suggested by Pape (1992) and Rognes (1997)
although these authors presented different morphological evi-
dence corroborating this sister group relationship. Our results
indicate non-monophyly of Calliphoridae as the single exemplar
species of the Rhinophoridae emerges as the sister group of Eury-
chaeta palpalis (Calliphoridae: Helicoboscinae), i.e. as nested
within the calliphorids. This, however, is in conflict with the
analyses of Rognes (1997) and Pape and Arnaud (2001),who
found the Rhinophoridae to be the sister group of either the
clade Sarcophagidae + Tachinidae or of the Rhiniinae (a former
blow fly subfamily, not included in this study and subsequently
raised to family rank by Evenhuis et al. (2008)). Blow fly non-
monophyly as argued by Rognes (1997) and Pape and Arnaud
(2001) is caused by the Rhiniinae falling outside and the Oestri-
dae falling inside the traditional Calliphoridae, but this has not
been tested in the present study. The position of Oestroidea
within the Calyptratae and the relationships between their con-
stituent families are currently under study based on a much lar-
ger set of exemplar species and will be the focus of a future
publication.
S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652 649
4.6. Alignments of the ribosomal genes
The use of ribosomal genes for the reconstruction of phyloge-
netic relationships has its advantages and disadvantages (Hillis
and Dixon, 1991; Simon et al., 1994; Caterino et al., 2000). Ribo-
somal genes vary in length across species of different ages and
the genes can thus provide valuable information for many phyloge-
netic questions. Furthermore, due to the large number of copies,
the genes can often be amplified for degraded templates. However,
one of the major difficulties with ribosomal data is their alignment,
i.e. establishing primary homologies between nucleotide sites from
distantly related taxa. Some of the most commonly used tech-
niques include alignments based on secondary structure of the
respective ribosomal genes (Gutell, 1994; Hickson et al., 1996;
De Rijk et al., 1999; Van de Peer et al., 1999), progressive alignment
with a guide tree as implemented in alignment programs like Clus-
tal (Thompson et al., 1994), Muscle (Edgar, 2004), Malign (with the
evaluation of multiple guide trees: Wheeler and Gladstein, 1994),
and optimization alignment (Wheeler, 1996). Given that alignment
is critical for the results obtained in a phylogenetic analysis (Mor-
rison and Ellis, 1997; Morrison, 2006), there are numerous other
techniques that have been suggested in the literature apart from
the methods mentioned above (Wheeler, 1995, 1996; Giribet and
Wheeler, 1999; Simmons and Ochoterena, 2000; Hickson et al.,
2000; Wheeler, 2003; Danforth et al., 2005; Benavides et al.,
2007; Kumar and Filipski, 2007; Simmons et al., 2007). In studies
utilizing progressive alignment programs, the authors frequently
manually re-adjust the numerical alignments and/or exclude some
parts of the alignment after inspection. However, manual re-align-
ments and data deletion have been criticized for being subjective.
Many authors thus prefer numerical techniques (Giribet and
Wheeler, 1999).
The alignments for the ribosomal genes in this study were
based on an approach that is still rarely used although this tech-
nique has recently been promoted by Benavides et al. (2007) for
the alignment of nuclear introns which faces similar issues related
to length differences and variability. They used the conserved re-
gions of the introns and nuclear coding genes to generate a guide
tree for the alignment of the complete intron sequences. Here,
we similarly use a user-defined ‘guide tree’ estimated based on
conserved sequences instead of relying on the Clustal’s default
guide trees. This user-defined guide tree is then used for aligning
the full-length ribosomal fragments (including the variable re-
gions) while Clustal’s guide trees are calculated from a distance
matrix based on dissimilarity scores between sequence pairs. How-
ever, such dissimilarity scores are unlikely to reflect true evolu-
tionary distances and the guide trees may thus be misleading.
We believe that some of the guide trees generated by ClustalX
for our ribosomal genes 12S, 16S, 18S, and 28S fall into this cate-
gory (see Supplementary material). On these guide trees, we would
expect the species of, for example, the Muscidae to cluster. Instead,
they are scattered over the guide tree (e.g. see 12S guide tree). To
reduce this source of error that can have serious effects on the
downstream phylogenetic inferences (Kumar and Filipski, 2007),
we thus generated a guide tree to improve alignments for the ribo-
somal genes in our dataset. The guide tree used in our alignments
was not based on subjective opinions about calyptrate relation-
ships. Instead, we use the phylogenetic signal from those se-
quences that code for protein-encoding genes and those parts of
the ribosomal genes that likely code for the stem-regions of rRNAs.
The method that we use is thus numerical, repeatable, and compu-
tationally tractable. It also does not require the deletion of se-
quence data. In the guide tree analysis, the protein encoding
mitochondrial genes COI and Cytb may have a heavy influence
on the tree topology due to the comparatively poor sampling for
the nuclear genes. This could be a concern since mitochondrial
genes such as COI tend to perform poorly in some higher level phy-
logenetic studies (Winterton et al., 2007). However, the age of the
Muscoidea is estimated at less than 50 mya and for such a recent
and young group, mitochondrial genes can be expected to be infor-
mative. Furthermore, the guide tree based on a data set including
COI and Cytb yielded hypotheses that were more congruent with
well established taxa in the Muscoidea than the guide tree based
on only the nuclear genes.
A main concern may be that the guide tree may have undue
influence on the downstream phylogenetic results; i.e. that the
trees based on all data merely reflect the relationships on the guide
tree. We therefore compared the guide tree to our phylogenetic
hypotheses based on MP and ML. We find that the two trees have
a surprisingly small number of shared nodes. About 60% of the
nodes are shared between the MP tree and the guide tree while
only 50% nodes are the same between the guide tree and the ML
tree (see Fig. 2.). On examining the ten guide trees we find that
three taxa, two Hippoboscoidea species and Tachina are placed
within the Muscidae (see Supplementary material). This placement
is in stark conflict with the well established monophyly of Musci-
dae, Tachinidae, and Oestroidea. However, this conflict disappears
on the trees that are based on the guide-tree assisted alignment of
all data (see Supplementary material). All three species are now
placed in positions that are consistent with well supported high-
er-level hypotheses for calyptrates. It thus appears that in this case
the phylogenetic signal from the hypervariable regions of the ribo-
somal genes is valuable in that it placed these taxa in positions (on
the most parsimonious tree) that are in agreement with previously
suggested hypotheses.
Alignment techniques based on user-defined guide trees have
been accused of circularity (Benavides et al., 2007), but not only
are the guide tree and phylogenetic trees not identical, they also
have several conflicting nodes. Furthermore, one could also argue
that a strong influence of the guide tree on the downstream phylo-
genetic results is wanted given that it is based on the signal in the
data. Using a user-defined guide tree based on those data that can
be aligned with little ambiguity thus appears to be an interesting
option for those systematists who would like to avoid subjective
manual alignments and data deletions and yet deal with large data
sets that are difficult to analyze using techniques such as second-
ary structure and/or optimization alignment.
5. Conclusion
Our study provides a large amount of novel molecular evidence
for the phylogenetic relationships of the Calyptratae, and in partic-
ular for the paraphyly of, and phylogenetic relationships within,
what we suggest to call the muscoid grade, i.e., the Fanniidae, Mus-
cidae, Anthomyiidae, and Scathophagidae. The position of this
grade within the Calyptratae and the relationships of its constitu-
ent families to the remaining calyptrate superfamilies, viz. Hipp-
oboscoidea and Oestroidea, are resolved. The relationships
between the four muscoid families are established and the mono-
phyly of the Fanniidae, Muscidae, and Scathophagidae is further
corroborated while no molecular support was obtained for antho-
myiid monophyly.
Acknowledgments
This research was funded by the Academic Research Fund
Grants R-154-000-207-112 from the Ministry of Education in Sin-
gapore and the NSF-ATOL Grant EF-0334948. We would like to
thank the following colleagues for their help with providing part
of the identified material and DNA samples: Dr. V. Michelsen, Nat-
650 S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652
ural History Museum of Denmark; Dr. D.M. Ackland, Oxford Uni-
versity Museum of Natural History; Dr J. Ziegler, Zoologisches Mu-
seum der Humboldt-Universität, Berlin; Dr J. Mariluis, ANLIS,
Buenos Aires; Mr C. Dewhurst, EMPRES/CR, Khartoum; Dr. Marco
V. Bernasconi, Zoological Museum, University of Zurich-Irchel.
We would like to thank Mr. B. Cassel of the Wiegmann Lab at the
North Carolina State University and members of the Evolutionary
Biology Lab at the National University of Singapore.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ympev.2008.08.012.
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... The monophyly and the family rank of the Fanniidae have been confirmed based primarily on the morphological characters of the adults, as well as of the larvae [2,17,[22][23][24] and then corroborated by DNA studies [25][26][27]. However, the phylogenetic relationships within the Fanniidae, as well as the systematic position of this family within the muscoid grade, have not been completely established. ...
... Pont [1] suggested that the Fanniidae are the most plesiomorphic lineage of the muscoids and the sister group of the Scathophagidae + Anthomyiidae + Muscidae. Based on molecular data, the monophyletic Fanniidae are considered the sister group of the remaining non-hippoboscoid calyptrates ((Muscidae + (Anthomyiidae + Scathophagidae)) + Oestroidea) and are placed at the base of the muscoid grade [25][26][27]30,31]. The phylogenetic relationships within the muscoid grade thus remain controversial. ...
... The position of the Fanniidae in the calyptrate phylogeny has been further corroborated by the structure of the sclerites and muscles of the male abdominal segments and terminalia. Our results confirmed the molecular data which places the Fanniidae as the sister group to a clade consisting of the Oestroidea plus remaining muscoids [25][26][27]30,31]. ...
Article
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The abdominal and pregenital segments and the genitalia were studied in males of Fannia subpellucens (Zetterstedt, 1845), Fannia canicularis (Linnaeus, 1761) and Fannia incisurata (Zetterstedt, 1838). In comparison with the remaining members of the muscoid grade, in addition to the symmetry of the pregenital segments, significant reductions of the sclerites and musculature of the male terminalia have been observed in Fanniidae. The muscular structure of pregenital segments confirms that the fused pregenital ring is syntergosternite VI + VII + VIII. Symmetry and fusion, as well as the lower number of the sclerites and muscles of the pregenital segments and male genitalia of the Fanniidae, can be considered apomorphic character states. The presence of the lateral bacilliform sclerite, as well as the presence and position of the epandrial muscles M 26, three pairs of muscles M 19 and paired muscles M 18, can be considered as a plesiomorphic character state of the Fanniidae. The structure of the sclerites and muscles of the male abdominal segments and terminalia place the Fanniidae at the base of the muscoid grade and Oestroidea, as has been confirmed by recent molecular studies.
... Additionally, 30 terminals were included as outgroups in the analyses, namely, representatives of Muscidae (11), Oestroidea (8) and Scathophagidae (11). Forty-one sequences of 12 Neotropical species of Anthomyiidae were newly generated for this study and 190 sequences were obtained from GenBank from previous studies (e.g., Kutty et al., 2007Kutty et al., , 2008Kutty et al., , 2010Wiegmann et al., 2011). Outgroups were chosen based on previous phylogenetic treatments for Calyptratae, as provided by Bernasconi et al. (2000), Kutty et al. (2007Kutty et al. ( , 2008Kutty et al. ( , 2010 Laboratory procedures DNA extraction was conducted by a non-destructive method, using the whole specimen with body perforations and the GenElute TM Blood Genomic DNA Kit, following the manufacturer's protocol. ...
... Forty-one sequences of 12 Neotropical species of Anthomyiidae were newly generated for this study and 190 sequences were obtained from GenBank from previous studies (e.g., Kutty et al., 2007Kutty et al., , 2008Kutty et al., , 2010Wiegmann et al., 2011). Outgroups were chosen based on previous phylogenetic treatments for Calyptratae, as provided by Bernasconi et al. (2000), Kutty et al. (2007Kutty et al. ( , 2008Kutty et al. ( , 2010 Laboratory procedures DNA extraction was conducted by a non-destructive method, using the whole specimen with body perforations and the GenElute TM Blood Genomic DNA Kit, following the manufacturer's protocol. Following DNA extraction, each specimen was dry-mounted and labelled Table 1. ...
... We present the first molecular phylogenetic hypothesis for Anthomyiidae including Neotropical taxa. Our First insights into evolution of anthomyiids results corroborate the close relationship between Anthomyiidae and Scathophagidae, as reported by previous molecular-based phylogenetic studies (Bernasconi et al., 2000;Kutty et al., 2007Kutty et al., , 2008Kutty et al., , 2010Kutty et al., , 2019Wiegmann et al., 2011;Ding et al., 2015;Junqueira et al., 2016;Zhang et al., 2016;Cerretti et al., 2017;Buenaventura et al., 2021). Griffiths (1982) indicated that Anthomyiidae could be a non-monophyletic assemblage of primitive subgroups of Calyptratae, and some authors have indicated morphological evidence that supports the different relationships of Anthomyiidae within Muscoidea. ...
Article
Anthomyiidae is a family of flies distributed worldwide comprising approximately 2,000 species, which, based on molecular analyses, belong to the Muscoidea grade. The phylogenetic data for this family are limited and the Neotropical lineages have never been studied at the molecular level. Herein, we present the first phylogenetic analyses of Anthomyiidae including Neotropical taxa. The resulting phylogenetic trees – inferred using maximum-likelihood and Bayesian criteria and based on two nuclear and three mitochondrial genes and 56 terminals, including 13 Neotropical anthomyiid species – confirmed Anthomyiidae as a monophyletic lineage and sister group of Scathophagidae, which is also monophyletic. The clade Coenosopsia + Phaonantho was not recovered as a sister group of the remaining anthomyiids, as previously indicated by the results of morphological analyses. These and other generic relationships are discussed in the light of morphological data from previous studies. The divergence time estimation suggested that after a split from the Oestroidea clade approximately 51.7 Ma, the radiation of Anthomyiidae + Scathophagidae began during the Eocene, with the oldest common ancestor of both families living approximately 40.5 Ma and with the oldest anthomyiid ancestor living approximately 36.8 Ma. The split of the clade Coenosopsia + Phaonantho was dated to the Oligocene (c. 23 Ma). These results provide new insights into the timing and rate of anthomyiid and scathophagid fly evolution.
... Analyses using molecular characters tell us another story, suggesting that Reinwardtiini is a polyphyletic assemblage (Schuehli et al., 2007;Kutty et al., 2014;Haseyama et al., 2015). Only Kutty et al., (2008Kutty et al., ( , 2010 indicated that the tribe is monophyletic. However, in all those analyses Reinwardtiini was poorly sampled: three genera in the analysis of Schuehli et al., (2007), one in Kutty et al. (2008Kutty et al. ( , 2010, two in Kutty et al., (2014) and six in Haseyama et al., (2015). ...
... Only Kutty et al., (2008Kutty et al., ( , 2010 indicated that the tribe is monophyletic. However, in all those analyses Reinwardtiini was poorly sampled: three genera in the analysis of Schuehli et al., (2007), one in Kutty et al. (2008Kutty et al. ( , 2010, two in Kutty et al., (2014) and six in Haseyama et al., (2015). There are 12 Reinwardtiini genera in the Neotropical region (de Savage, 2009). ...
... A key difference between our analysis and all previous studies (de Couri & Carvalho 2003;Schuehli et al., 2007;Kutty et al., 2008Kutty et al., , 2010Kutty et al., , 2014Haseyama et al., 2015) is the sampling. It is well-recognized that different taxa and character sampling affect the results of phylogenetic analyses (see Zhao et al., 2013). ...
Article
Reinwardtia Brauer & Bergenstamm is a monotypic genus, with only R. tachinina described from Venezuela. Here, we revised the genus to include Reinwardtia bicolor sp. nov. from Colombia and Ecuador. To investigate the phylogenetic positioning of Reinwardtia, we present a morphological analysis, including 16 species of the Neotropical Reinwardtiini genera. We used 35 characters from male and female adults. Based on this phylogenetic analysis, Reinwardtia is a monophyletic genus, a sister-group of the monotypic Synthesiomyia Brauer & Bergenstamm. http://zoobank.org:pub:F8495ED0-FBDA-4A91-9A76-40B708FC6997 http://zoobank.org/urn:lsid:zoobank.org:act:8BF649B3-C9F3-4B90-B120-1CE28F051F5A
... The modern concept of Muscidae (Roback, 1951;Pont, 1986;Michelsen, 1991) has been corroborated by multilocus Sanger sequencing (mS-seq) (Kutty et al., 2008(Kutty et al., , 2014Haseyama et al., 2015) as well as transcriptomic data (Kutty et al., 2019), which have also provided major breakthroughs in reconstructing relationships within the family. Several studies have split Muscidae into a basal dichotomy: Muscinae + Azeliini versus all other Muscidae (Schuehli et al., 2007;Kutty et al., 2008Kutty et al., , 2010Kutty et al., , 2014Haseyama et al., 2015). ...
... The modern concept of Muscidae (Roback, 1951;Pont, 1986;Michelsen, 1991) has been corroborated by multilocus Sanger sequencing (mS-seq) (Kutty et al., 2008(Kutty et al., , 2014Haseyama et al., 2015) as well as transcriptomic data (Kutty et al., 2019), which have also provided major breakthroughs in reconstructing relationships within the family. Several studies have split Muscidae into a basal dichotomy: Muscinae + Azeliini versus all other Muscidae (Schuehli et al., 2007;Kutty et al., 2008Kutty et al., , 2010Kutty et al., , 2014Haseyama et al., 2015). However, the use of mS-seq was unable to robustly resolve the deeper splits within these two groups, and some highly supported clades were at odds with classifications based on adult morphology (Kutty et al., 2014;Haseyama et al., 2015;Grzywacz et al., 2017b). ...
Article
Muscidae are a megadiverse dipteran family that exhibits extraordinary diversity in morphology and life history as both immatures and adults. The classification of Muscidae has been long debated, and most higher-level relationships remain unknown. In this study, we used multilocus Sanger sequencing (mS-seq), anchoredhybrid enrichment (AHE) and restriction-site associated DNA sequencing (RAD-seq)approaches to examine relationships within Muscidae. The results from AHE andRAD-seq largely correspond to those obtained from mS-seq in terms of overall topology,yet phylogenomic approaches received much higher nodal support. The results from all molecular approaches contradict the traditional classification based predominantlyon adult morphology, but provide an opportunity to re-interpret the morphology of immature stages. Rearrangements in Muscidae classification are proposed as follows:(i) Mesembrina Meigen and Polietes Rondani are transferred from Muscinae to Azeliinae; (ii) Reinwardtiinae stat. rev.is resurrected as a subfamily distinct from Azeliinae; (iii) Eginia Robineau-Desvoidy, Neohelina Malloch, Syngamoptera Schnabl and Xenotachina Malloch are transferred to Reinwardtiinae stat. rev.
... But little information was available about the natural enemy complex of bumble bees in the United States. Different researchers (Collins and Weigmann, 2002;Kutty et al., 2008;Peterson et al., 2007;Winterton et al., 2007) utilized the DNA sequences in characterization of phylogenetic analysis of dipteran families and subfamilies. It was known that the nuclear ribosomal gene 28S, the only gene present universally in the genome of all dipterans, have a great importance in phylogenetic studies. ...
Article
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Bumble bees (Bombus spp.) are important pollinators attacked by different pests. Present studies were conducted to know the genetic relatedness among bumble bee species Bombus haemorrhoidalis; Bombus rufofaciatus and their important pests i.e. conopid fly, (Physocephala tibialis) and bee moth (Aphomia sociella). DNA was isolated from all the insects under study and quantified. PCR was carried out using 14 random OPA primers. In all the primers bands were observed ranging from 300-700 bp in B. haemorrhoidalis whereas in B. rufofaciatus they were recorded between 300-900 bp regions. In case of Physocephalatibialis amplified DNA bands were observed between 200-700 bp region whereas in A. sociella bands were obtained between 300-1000 bp. Based on these studies 62% genome similarity was found between B. haemorrhoidalis and B. rufofaciatus whereas very less similarity (51%) was recorded between A. sociella, P. tibialis, B. haemorrhoidalis and B. rufofaciatus. In future such studies can be used for finding the relation between different insect species which are parasitically or socially related to each other.
... Species identification and species delimitation are more accurate when based not only on morphological but also on molecular-genetic characters. Unfortunately, there are very few papers devoted to the DNA barcoding of the Muscidae and most of them have used the sequence data to perform phylogenetic analyses (Savage et al. 2004;Schuehli et al. 2004Schuehli et al. , 2007Kutty et al. 2008Kutty et al. , 2014Haseyama et al. 2015). Nevertheless, there is one paper in which the relevance of DNA barcoding as a taxonomic tool in muscid fly identification has been established (Renaud et al. 2012). ...
... The recent molecular data confirmed the monophyly of Coenosiinae, whereas Limnophorini was found to be a paraphyletic group (Kutty et al., 2014(Kutty et al., , 2019. Besides, the genus Spilogona, which in most morphological classifications belongs to the tribe Limnophorini, was placed in the tribe Coenosiini together with Xenomyia and Villeneuvia according to quite reliable results of molecular analysis (Kutty et al., 2008(Kutty et al., , 2014. In the opinion of the cited authors, these data are inconsistent with morphological results, especially as concerns the autapomorphies of Coenosiini, and remain to be confirmed by including other genera in analysis. ...
Article
Full-text available
The abdominal and pregenital segments and genitalia were studied in males of Spilogona Schnabl, 1911: S. tundrae (Schnabl, 1915), S. sanctipauli (Malloch, 1921), and S. zaitzevi (Schnabl, 1915) (Muscidae, Coenosiinae). The examined species are very similar in the structure of the sclerites and muscles of their terminal segments, and more strongly resemble the previously studied members of Mydaeinae (especially the genus Graphomya) than Muscinae. The well-developed pregenital sclerites and muscles in Spilogona indicate the basal position of this genus within Muscidae relative to Muscinae and probably Mydaeinae.
... We had into account geographical information (e. g, www.gbif.org) and possible synonyms [13,70,72,73,[117][118][119][120][121][122][123][124]. All these discordant BINs were due to correctable errors: synonyms/erroneous information in BOLD, morphological misidentification due to the preservation conditions of samples or BINs shared by species with too low molecular distance. ...
Article
Species identification with DNA barcodes has been proven to be effective on different organisms and, particularly, has become a routinely used and quite accurate tool in forensic entomology to study necrophagous Diptera species. In this study, we analysed 215 specimens belonging to 42 species of 17 genera, from 9 different Diptera families. Flies were collected in 39 Spanish localities of the Iberian Peninsula sampled across three years in the four seasons. Intraspecific variation ranged from 0 to 2.46% whereas interspecific variation fluctuated from 3.07 to 14.59%, measuring 651 pb of the cytochrome oxidase subunit one (COI) gene. Neighbour-Joining analysis was carried out to investigate the molecular identification capabilities of the barcoding region, recovering almost all species as distinct monophyletic groups. The species groupings were generally consistent with morphological and molecular identifications. This work, which is the first with this intensive and extensive sampling in this area, shows that the COI barcode is an appropriate marker for unambiguous identification of forensically important Diptera in Spain.
... Despite the high number of described species, we still lack comprehensive phylogenies for the family (the Darwinian shortfall; Diniz-Filho, Loyola, Raia, Mooers, & Bini, 2013). Schuehli, de Carvalho, and Wiegmann (2007) provided the first molecular phylogeny for Muscidae with 24 terminals, and Kutty, Pape, Pont, Wiegmann, and Meier (2008) reconstructed the relationship between inner clades of Calyptratae, which included 46 terminals of the family. More recently, a molecular phylogeny for the family that included 84 terminals was provided by Kutty, Pont, Meier, and Pape (2014) and the time-calibrated phylogeny, provided by Haseyama et al. (2015) and adopted in this study, included 141 terminals of all biogeographic regions. ...
Article
Aim The housefly family Muscidae originated during the Paleocene–Eocene and is currently distributed worldwide. We investigated how muscid genera have assembled in the continents by historical dispersal events. Location Global (except Antarctica). Taxon Muscidae, Diptera, Insecta. Methods We compiled a dataset of 181 species, which provided geographic information for 67 genera as terminals. Phylogenetic and geographic data were fitted by five dispersal–extinction–cladogenesis models based on dispersal constraints. Based on the event inferences of the best‐fit model, we quantified the direction and frequency of dispersals through time in absolute numbers and in relation to the number of lineages. Results The best‐fit model was ‘unrestricted routes’, which inferred 48 events of dispersals: 66% were trans‐climatic events and 81% were transoceanic events. The direction of the dispersals was strongly asymmetric: the Neotropical (NEO) region was the primary source of lineages for all other regions and the Palearctic (PAL) was the secondary source. The relative frequency of dispersal events through time plot was marked by three phases: the early peak of the frequency (40–35 million years ago [Ma]), a valley of low frequencies (30–20 Ma), and the recent peak (15–5 Ma). Trans‐climatic dispersals from the NEO to the PAL region occurred in all phases. Lineages of the Cyrtoneurininae showed a low frequency of dispersal events and most of the terminals (11 of 15) were endemic to the NEO region. Conclusion The Muscidae family originated in the Neotropical region and more recent dispersal events to the Palearctic region established the scenario for lineages to diversify and spread worldwide. Trans‐climatic dispersals were more frequent than cis‐events and tropical niche conservatism was not the underlying mechanism for the whole clade. We showed that dispersal events between biogeographic regions of the same and different climatic zones were required to achieve the current distribution of extant genera of muscids across the globe.
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The genus Haematobosca Bezzi, 1907 (Diptera: Muscidae) contains haematophagous flies of veterinary importance. A new fly species of this genus was recognised from northern Thailand based on morphological characters and described as Haematobosca aberrans Pont, Duvallet & Changbunjong, 2020. In the present study, the mitochondrial cytochrome c oxidase I (COI) gene was used to confirm the morphological identification of H. aberrans. In addition, landmark-based geometric morphometrics was used to determine sexual dimorphism. The molecular analysis was conducted with 10 COI sequences. The results showed that all sequences were 100% identical. The sequence was not highly similar to reference sequences from GenBank and did not match any identified species from Barcode of Life Data Systems (BOLD). Phylogenetic analysis clearly differentiated this species from other species within the subfamily Stomoxyinae. For geometric morphometric analysis, a total of 16 wing pictures were analysed using the landmark-based approach. The results showed significant differences in wing shape between males and females, with a cross-validated classification score of 100%. The allometric analysis showed that wing shape has no correlation with size. Therefore, the COI gene is effective in species identification of H. aberrans, and geometric morphometrics is also effective in determining sexual dimorphism.
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The Diptera, or true flies (mosquitoes, gnats, and house flies) comprise 12-15% of animal species, and are the most ecologically diverse order of insects, spanning ecological roles from detritivory to vertebrate blood feeding and leaf mining. The earliest known fossil Diptera are from the early Triassic 240 mya, and the order probably arose in the late Permian. The earliest brachyceran fossils are found in the late Triassic and earliest Jurassic, but the diversification of the extremely diverse Calyptrata (ca. 30% of described species) began in the late Creataceous. The monophyly of the order is supported by numerous morphological and biological characters and molecular data sets. The major lineages within the order are well established, and we summarize major recent phylogenetic analyses in a supertree for the Diptera. Most studies concur that the traditional subordinal group Nematocera is paraphyletic, but relationships between the major lineages of these flies are not recovered consistently. There is particular instability around the placement of the tipulids and their relatives and the families of the Psychodomorpha as traditionally defined. The other major suborder, Brachycera, is clearly monophyletic, and the relationships between major brachyceran lineages have become clearer in recent decades. The Eremoneura, Cyclorrhapha, Schizophora and Calyptrata are monophyletic, however the “Orthorrhapha” and “Aschiza” are paraphyletic, and it is likely that the “Acalyptrata” are also. Ongoing phylogenetic analyses that span the diversity of the order shall establish a robust phylogeny of the group with increased quantitative rigor. This will enable a more precise understanding of the evolution of the morphology, biogeography, biology, and physiology of flies.
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Abstract- Because they are designed to produced just one tree, neighbor-joining programs can obscure ambiguities in data. Ambiguities can be uncovered by resampling, but existing neighbor-joining programs may give misleading bootstrap frequencies because they do not suppress zero-length branches and/or are sensitive to the order of terminals in the data. A new procedure, parsimony jackknifing, overcomes these problems while running hundreds of times faster than existing programs for neighbor-joining bootstrapping. For analysis of large matrices, parsimony jackknifing is hundreds of thousands of times faster than extensive branch-swapping, yet is better able to screen out poorly-supported groups.