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

Molecular Phylogenetics and Evolution 49 (2008) 639–652

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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 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 b

a r t i c l e

i n f o

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

a b s t r a c t 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. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction With approximately 7000 species in four families, the Muscoidea constitute approximately 5% of the described dipteran diversity. Many of the muscoids are familiar flies that we encounter on a daily basis. For example, the most speciose family, the Muscidae, includes the housefly (Musca domestica) and the stable fly (Stomoxys 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. * Corresponding author. E-mail address: [email protected] (R. Meier). 1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.08.012

The Muscoidea are one of the three superfamilies in the Calyptratae, 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 Muscoidea as ‘‘the Calyptratae less the Hippoboscoidea and Oestroidea”. However, the non-monophyly of the Muscoidea is far from universally 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

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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 relationships between these families are far from understood and additional research based on molecular, morphological, and other data is necessary before this significant portion of Diptera diversity 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 species 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 A2. Fanniid larvae are furthermore characterized by lateral fleshy projections. While the monophyly of the Fanniidae may seem strongly corroborated, the phylogenetic relationships within the family are still poorly understood. The monotypic Australofannia Pont is currently considered the sister group to the remaining members of the family (Pont, 1977) because it retains 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 biogeographical regions. The larvae are usually saprophagous while adults can be saprophagous, predacious, hematophagous, or feeders on nectar and pollen. Many muscids are vectors of disease. Presumably 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 generally 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 spiracle 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 monophyly was recently corroborated using molecular data (Schuehli et al., 2007). Currently, eight subfamilies are recognized (Achanthipterinae, Atherigoninae, Azeliinae, Cyrtoneurininae, Coenosiinae, Muscinae, Mydaeinae, and Phaoniinae), but the subfamilies and tribes in this family have undergone many classificatory changes and various hypotheses of relationships have been proposed (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 region. 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 larvae 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 conditions can be put forward to demonstrate that the family, as presently 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, hairlike setulae subapically on the underside of scutellum, a surstylus with a sclerotised connection to the cercus, a surstylus that is distally 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 further split into two or more subfamilies. Hucket (1965) considered the genera Fucellia Robineau-Desvoidy and Circia Malloch [=Alliopsis 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 Myopinini. This classification has since been completely abandoned, and the family now stands without any subfamilies (e.g. Suwa and Darvas, 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 taxon (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 unusually varied natural history ranging from saprophagy over phytophagy 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 morphological characters, but two recent molecular studies have brought considerable progress with the result that phylogenetic

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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 species (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 Scathophagidae was corroborated with strong support. The two recognized 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 Muscoidea 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 Fanniidae 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 Anthomyiidae 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 character states, that a taxon composed of Anthomyiidae, Muscidae, and Fanniidae is monophyletic, which suggested that the Scathophagidae 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 obtained conflicting results. Therefore, it appears timely to use a different source of data; i.e. DNA sequences. A small-scale phylogenetic study using the genes Cytochrome oxidase subunit I and II was carried out by Bernasconi et al. (2000b), but the authors had to conclude 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 position 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 sequence 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 Muscoidea is our third contribution to a better understanding of calyptrate 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

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remaining two calyptrate superfamilies, we included two species from the Hippoboscoidea (Glossinidae and Hippoboscidae, respectively), while the Oestroidea are represented by ten species from the four major families (Calliphoridae, Rhinophoridae, Sarcophagidae, 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 protocol as described in Kutty et al. (2007). DNA extractions for some species were also carried out according to manufacturer’s instructions using the QIAamp tissue kit (QIAGEN, Santa Clara, CA). 2.2. DNA amplification Standard PCR amplifications were carried out using either Takara Ex-Taq or Bioline Taq on 1–5 ll 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 ribosomal RNA, 28S ribosomal RNA, Elongation factor 1-a, and a fragment 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 conservative nature of the 18S sequences, we only amplified this gene for 20 species representing the major taxa in the analysis (see Table 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 approximately 5 min was also added. The amplified gene products were purified using Bioline Sure-Clean solution following the manufacturer’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 alignments 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-repeatable 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 inspected 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

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Table 1 List of taxa used in study with GenBank Accession Numbers Taxa

Voucher number RMBR #

Author name

Carnidae Hemeromyia anthracina

102770

Collin (1949)

Celyphidae Celyphidae sp.

102769

Coelopidae Lopa convexa Gluma nitida

102737 102710

Tephritidae Ceratitis capitata Bactrocera dorsalis Bactrocera oleae

GenBank Accession Numbers 12S

16S

28S

COI

CYTB

EF1a

CAD4

18S

FJ025402

FJ025464

FJ025553

FJ025644

N/A

N/A

N/A

N/A

FJ025401

FJ025463

FJ025552

FJ025643

N/A

N/A

FJ025568

N/A

McAlpine (1991) McAlpine (1991)

N/A N/A

AF403450 AF403468

FJ025535 FJ025517

EU435768 EU435770

EU435900 EU435902

AY048515 AY048533

N/A N/A

EU435620 EU435622

102676 102670 102669

Wiedemann (1824) Hendel (1912) Rossi (1790)

AJ242872 DQ845759 AY210702

AJ242872 DQ845759 AY210702

N/A N/A N/A

AJ242872 DQ845759 AY210702

AJ242874 DQ845759 AY210702

N/A N/A N/A

N/A N/A N/A

DQ490237 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 Fannia manicata

102705 102706

Linnaeus (1761) Meigen (1826)

DQ656884 DQ656885

DQ648647 N/A

DQ656961 DQ656962

DQ657037 DQ657038

DQ657051 DQ657052

N/A N/A

EF531184 N/A

FJ025489 FJ025490

Muscidae Musca domestica Mesembrina mystacea Stomoxys calcitrans Coenosia testacea Coenosia tigrina Drymeia alpicola Drymeia hamata Eudasyphora cyanella Haematobosca stimulans Phaonia pallida Haematobia irritans Hebecnema fumosa Hebecnema umbratica Helina celsa Helina evecta Helina impuncta Helina lasiophthalma Hydrotaea cyrtoneurina Hydrotaea dentipes Hydrotaea irritans Limnophora exuta Limnophora maculosa Limnophora olympiae Limnophora riparia Lispe tentaculata Mesembrina meridiana Morellia aenescens Morellia hortorum Morellia simplex Musca autumnalis Muscina levida Muscina stabulans Mydaea ancilla Mydaea rufinervis Mydaea urbana Myospila meditabunda Phaonia subventa Polietes lardarius Potamia littoralis Spilogona caliginosa Spilogona dispar Thricops aculeipes Thricops cunctans Thricops genarum Thricops nigritellus Villeneuvia aestuum

102741 102745 102784 102690 102691 102697 102698 102701 102722 102779 102727 102717 102723 102713 102726 102718 102721 102715 102716 102719 102730 102732 102733 102734 102736 102744 102738 102742 102748 102740 102743 102749 102739 102747 102752 102751 102781 102776 102777 102785 102787 102805 102806 102809 102810 102811

Linnaeus (1758) Linnaeus (1758) Linnaeus (1758) Robineau-Desvoidy (1830) Fabricius (1775) Rondani (1871) Fallén (1823) Meigen (1826) Meigen (1824) Fabricius (1787) Linnaeus (1758) Meigen (1826) Meigen (1826) Harris (1780) Harris (1780) Fallén (1825) Macquart (1835) Zetterstedt (1845) Fabricius (1805) Fallén (1823) Kowarz (1893) Meigen (1826) Lyneborg (1965) Fallén (1824) De Geer (1776) Linnaeus (1758) Robineau-Desvoidy (1830) Fallén (1817) Loew (1857) De Geer (1776) Harris (1780) Fallén (1817) Meigen (1826) Pokorny (1889) Meigen (1826) Fabricius (1781) Harris (1780) Fabricius (1781) Robineau-Desvoidy (1830) Stein (1916)) Fallén (1823) Zetterstedt (1838) Meigen (1826) Zetterstedt (1838) Zetterstedt (1838) Villeneuve (1902)

DQ656896 DQ656895 DQ656886 FJ025367 FJ025368 FJ025370 FJ025371 FJ025373 FJ025375 FJ025409 NC007102 N/A N/A FJ025376 FJ025377 FJ025378 FJ025379 FJ025380 FJ025381 N/A FJ025384 FJ025385 FJ025386 FJ025387 FJ025388 FJ025390 FJ025391 FJ025392 FJ025393 FJ025394 FJ025395 FJ025396 FJ025398 N/A FJ025399 FJ025400 FJ025410 FJ025411 FJ025412 FJ025414 FJ025415 FJ025417 FJ025418 FJ025419 FJ025420 FJ025421

DQ648650 FJ025453 EF531122 FJ025426 FJ025427 FJ025430 FJ025431 FJ025433 FJ025437 FJ025469 FJ025436 N/A N/A FJ025438 FJ025439 FJ025440 N/A FJ025441 FJ025442 FJ025443 FJ025446 FJ025447 FJ025448 FJ025449 FJ025450 FJ025452 FJ025454 FJ025455 FJ025456 FJ025457 FJ025458 EF531117 FJ025460 FJ025461 N/A FJ025462 FJ025470 FJ025471 FJ025472 FJ025474 FJ025475 FJ025477 FJ025478 FJ025479 FJ025480 FJ025481

DQ656974 DQ656973 DQ656963 N/A FJ025503 FJ025508 FJ025509 FJ025511 FJ025518 FJ025555 N/A FJ025519 FJ025520 FJ025521 FJ025522 FJ025523 FJ025524 FJ025526 FJ025527 N/A FJ025530 FJ025531 FJ025532 FJ025533 FJ025534 FJ025537 FJ025539 FJ025540 FJ025541 FJ025542 FJ025544 EF531145 FJ025547 N/A FJ025548 FJ025549 N/A FJ025557 FJ025558 N/A FJ025560 N/A FJ025564 N/A N/A N/A

AF104622 DQ657046 DQ657039 FJ025605 FJ025606 FJ025608 FJ025609 FJ025611 FJ025615 FJ025651 DQ029097 FJ025616 FJ025617 FJ025618 FJ025619 FJ025620 FJ025621 FJ025622 FJ025623 FJ025624 FJ025626 FJ025627 FJ025628 FJ025629 FJ025630 FJ025633 FJ025634 FJ025635 FJ025636 FJ025637 FJ025638 EF531210 FJ025639 FJ025640 FJ025641 FJ025642 FJ025652 FJ025653 FJ025654 FJ025657 FJ025658 FJ025660 FJ025661 FJ025662 FJ025663 FJ025664

DQ657064 DQ657063 DQ657053 FJ025707 FJ025708 FJ025710 FJ025711 N/A FJ025716 FJ025747 FJ025715 N/A FJ025717 FJ025718 FJ025719 N/A FJ025720 FJ025721 FJ025722 FJ025723 FJ025725 FJ025726 FJ025727 FJ025728 FJ025729 N/A FJ025731 FJ025732 FJ025733 FJ025734 FJ025735 FJ025736 FJ025737 FJ025738 FJ025739 N/A FJ025748 N/A N/A FJ025750 FJ025751 FJ025752 FJ025753 FJ025754 FJ025755 FJ025756

DQ657113 N/A FJ025698 N/A N/A FJ025669 FJ025670 FJ025671 FJ025673 N/A N/A N/A N/A FJ025674 FJ025675 FJ025676 N/A FJ025678 FJ025679 FJ025680 FJ025684 FJ025685 FJ025686 N/A FJ025687 N/A N/A N/A N/A N/A FJ025688 FJ025689 FJ025690 N/A FJ025691 FJ025692 N/A FJ025695 N/A N/A N/A FJ025699 FJ025700 FJ025701 FJ025702 FJ025703

FJ025591 N/A EF531173 FJ025569 FJ025570 FJ025572 FJ025573 FJ025574 FJ025576 FJ025596 N/A N/A N/A N/A N/A N/A FJ025577 FJ025578 FJ025579 FJ025580 FJ025581 FJ025582 FJ025583 FJ025584 FJ025585 FJ025586 FJ025587 FJ025588 FJ025589 FJ025590 N/A EF531167 FJ025592 N/A FJ025593 FJ025594 N/A FJ025597 FJ025598 N/A FJ025599 N/A FJ025600 N/A N/A N/A

N/A FJ025493 FJ025499 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A

Anthomyiidae Botanophila fugax Botanophila rubrifrons Delia platura Emmesomyia grisea Eutrichota frigida Hydrophoria lancifer Hylemya vagans Hylemya variata Lasiomma latipenne Lasiomma seminitidum Mycophaga testacea Paregle coerulescens Pegomya winthemi Pegoplata aestiva Pegoplata infirma

102671 102673 102700 102702 102704 102720 102724 102725 102731 102735 102750 102774 102782 102771 102775

Meigen (1826) Ringdahl (1933) Meigen (1826) Robineau-Desvoidy (1830) Zetterstedt (1845) Harris (1780) Panzer (1798) Fallén (1823) Zetterstedt (1838) Zetterstedt (1845) Gimmerthal (1834) Strobl (1893) Meigen (1826) Meigen (1826) Meigen (1826)

DQ656890 FJ025364 DQ656894 FJ025372 N/A DQ656891 FJ025382 FJ025383 DQ656892 DQ656893 DQ656890 FJ025403 FJ025406 FJ025407 FJ025408

N/A FJ025423 N/A N/A N/A N/A N/A FJ025444 FJ025445 DQ648649 N/A N/A N/A N/A N/A

DQ656967 N/A DQ656972 FJ025510 FJ025513 DQ656968 N/A N/A DQ656970 DQ656971 DQ656967 N/A N/A N/A FJ025554

DQ657042 FJ025602 DQ657045 FJ025610 FJ025613 DQ657043 FJ025625 N/A DQ657044 AF104624 DQ657042 FJ025645 FJ025648 FJ025649 FJ025650

DQ657057 N/A DQ657062 FJ025712 FJ025714 DQ657058 N/A FJ025724 DQ657060 DQ657061 DQ657057 FJ025741 FJ025744 FJ025745 FJ025746

FJ025665 N/A N/A N/A N/A N/A N/A N/A FJ025683 DQ657112 N/A FJ025693 N/A N/A N/A

N/A N/A N/A N/A N/A EF531164 N/A N/A N/A N/A N/A N/A N/A N/A N/A

N/A N/A FJ025486 FJ025487 N/A N/A N/A N/A FJ025491 N/A N/A N/A N/A N/A FJ025497

Scathophagidae Acanthocnema glaucescens Acerocnema macrocera

102667 102668

Loew (1864) Meigen (1826)

DQ656897 DQ656898

DQ648651 DQ648652

DQ656975 DQ656976

AF181023 AF181025

DQ657065 DQ657066

DQ657114 N/A

N/A N/A

N/A N/A

643

S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652 Table 1 (continued) Taxa

Voucher number RMBR #

Author name

12S

16S

28S

COI

CYTB

EF1a

CAD4

18S

102666 102684 102685 102689 102694 102696 102693 102677 102678 102683 102688 102692 102674 102679 102686 102675 102681 102687 102699 102707 102709 102708 102711 102714 102728 102746 102754 102755 102756 102757 102758 102759 102753 102765 102760 102761 102762 102763 102764 102766 102767 102772 102780 102773 102790 102791 102792 102793 102794 102795 102796 102797 102798 102799 102800 102801 102802 102803 102804 102786 102788 102808

Loew (1863) Curtis (1832) Coquillett Meigen (1826) Meigen (1826) Meigen (1826) Walker (1849) Walker (1849) Meigen (1826) Curran (1929) Meigen (1826) Loew (1873) Fallén (1819) Cresson (1918) Loew (1863) Zetterstedt (1846) Loew (1869) Linnaeus (1758) Fallén (1819) Coquillett (1908) Zetterstedt (1838) Zetterstedt (1846) Fallén (1819) Loew (1863) Fabricius (1794) Zetterstedt (1838) Becker (1894) Johnson (1927) Meigen (1826) Fallén (1819) Becker (1894) Zetterstedt (1838) Malloch (1924) Fabricius (1794) Collin (1958) Wiedemann (1826) Šifner (1974) Fallén (1819) Meigen (1826) Zetterstedt (1838) Loew (1863) Fabricius (1805) Becker (1900) Zetterstedt (1838) Meigen (1826) Curtis (1832) Meigen (1826) Say (1823) Becker (1900) Meigen (1826) Fallén (1819) Fabricius (1794) Fallén (1819) Oldenberg (1923) Linnaeus (1758) Fabricius (1794) Rondani (1866) Becker (1894) Malloch (1931) Loew (1863) Fallén (1819) Meigen (1826)

DQ656899 DQ656914 DQ656915 DQ656916 DQ656900 DQ656901 DQ656913 DQ656904 DQ656905 DQ656908 DQ656911 DQ656912 DQ656902 DQ656906 DQ656909 DQ656903 DQ656907 DQ656910 DQ656889 DQ656917 DQ656919 DQ656918 DQ656920 DQ656921 DQ656922 DQ656923 DQ656924 DQ656925 DQ656926 DQ656927 DQ656928 DQ656929 DQ656930 DQ656936 DQ656931 DQ656932 DQ656933 DQ656934 DQ656935 DQ656937 DQ656938 DQ656939 DQ656941 DQ656940 DQ656942 DQ656943 DQ656944 DQ656945 DQ656946 DQ656947 DQ656948 DQ656949 DQ656950 DQ656951 DQ656952 DQ656953 DQ656954 DQ656955 DQ656956 DQ656957 DQ656958 DQ656959

DQ648653 DQ648668 DQ648669 DQ648670 DQ648654 DQ648655 DQ648667 DQ648658 DQ648659 DQ648662 DQ648665 DQ648666 DQ648656 DQ648660 DQ648663 DQ648657 DQ648661 DQ648664 DQ648648 DQ648671 DQ648673 DQ648672 DQ648674 DQ648675 DQ648676 DQ648677 DQ648678 DQ648679 DQ648680 DQ648681 DQ648682 DQ648683 DQ648684 DQ648690 DQ648685 DQ648686 DQ648687 DQ648688 DQ648689 DQ648691 DQ648692 DQ648693 DQ648695 DQ648694 DQ648696 DQ648697 DQ648698 DQ648699 DQ648700 DQ648701 DQ648702 DQ648703 DQ648704 DQ648705 DQ648706 DQ648707 DQ648708 DQ648709 DQ648710 DQ648711 DQ648712 DQ648713

DQ656977 DQ656992 DQ656993 DQ656994 DQ656978 DQ656979 DQ656991 DQ656982 DQ656983 DQ656986 DQ656989 DQ656990 DQ656980 DQ656984 DQ656987 DQ656981 DQ656985 DQ656988 DQ656966 DQ656995 DQ656997 DQ656996 DQ656998 N/A DQ656999 DQ657000 DQ657001 DQ657002 DQ657003 DQ657004 DQ657005 DQ657006 DQ657007 DQ657013 DQ657008 DQ657009 DQ657010 DQ657011 DQ657012 DQ657014 DQ657015 DQ657016 DQ657018 DQ657017 DQ657019 DQ657020 DQ657021 DQ657022 DQ657023 DQ657024 DQ657025 DQ657026 DQ657027 DQ657028 DQ657029 DQ657030 DQ657031 DQ657032 DQ657033 DQ657034 DQ657035 DQ657036

AF181030 AF180792 AF181017 AF181016 AF181031 AF181024 AF180996 AF180988 AF180989 AF180992 AF180991 AF180990 AF180995 AF180993 AF180994 DQ657047 AF180997 AF180987 AF181029 AF181009 AF181008 AF181011 AF181010 AF181015 AF181014 AF181021 DQ657048 AF181006 AF181003 AF181005 AF181004 AF181007 AF181027 AF180998 DQ657049 AF181001 AF181002 AF180999 AF181000 AF181020 AF181026 AF181028 AF181018 AF181019 AF180783 AF181003 AF180784 AF180777 AF180786 AF180781 AF180789 AF180779 AF180790 AF180785 AF180759 AF180773 AF180768 AF180079 AF180788 AF181012 AF181013 AF181022

DQ657067 AF180986 DQ657082 DQ657083 DQ657068 DQ657069 DQ657081 DQ657072 DQ657073 DQ657076 DQ657079 DQ657080 DQ657070 DQ657074 DQ657077 DQ657071 DQ657075 DQ657078 DQ657056 DQ657084 DQ657086 DQ657085 DQ657087 DQ657088 DQ657089 DQ657090 DQ657091 DQ657092 DQ657093 DQ657094 DQ657095 DQ657096 DQ657097 DQ657103 DQ657098 DQ657099 DQ657100 DQ657101 DQ657102 DQ657104 DQ657105 DQ657106 DQ657108 DQ657107 AF180977 AF180981 AF180978 AF180974 AF180980 AF180976 AF180983 AF180975 AF180984 AF180979 AF180971 AF180973 AF180972 AF180985 AF180982 DQ657109 DQ657110 DQ657111

N/A DQ657123 DQ657124 N/A N/A DQ657115 FJ025668 DQ657117 N/A DQ657120 N/A N/A N/A DQ657118 DQ657121 DQ657116 DQ657119 DQ657122 N/A DQ657125 N/A N/A N/A DQ657126 N/A N/A N/A DQ657127 N/A N/A N/A DQ657128 DQ657129 DQ657133 DQ657130 DQ657131 DQ657132 N/A N/A N/A DQ657134 N/A N/A N/A DQ657135 DQ657136 DQ657137 DQ657138 N/A N/A DQ657139 DQ657140 N/A N/A DQ657141 DQ657142 N/A FJ025697 DQ657143 N/A N/A N/A

N/A N/A N/A N/A N/A N/A N/A N/A EF531159 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A

N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FJ025485 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A

Sarcophagidae Peckia gulo Sarcophaga arizonica

102783 102789

Fabricius (1805) Townsend (1919)

FJ025405 FJ025413

FJ025467 FJ025473

N/A FJ025559

FJ025647 FJ025655

FJ025743 FJ025749

N/A FJ025696

N/A N/A

N/A FJ025498

Tachinidae Tachina ferox

102807

Panzer (1809)

FJ025416

FJ025476

FJ025562

FJ025659

N/A

N/A

N/A

FJ025500

Calliphoridae Bengalia peuhi Calliphora vomitoria Compsomyiops fulvicrura Chrysomya megacephala Eurychaeta palpalis Lucilia caesar

102672 102695 102680 102682 102703 102729

Villeneuve (1914 Linnaeus (1758) Robineau-Desvoidy (1830) Fabricius (1794) Robineau-Desvoidy (1830) Linnaeus (1758)

FJ025363 FJ025365 FJ025369 FJ025366 FJ025374 FJ025389

FJ025422 FJ025424 FJ025428 FJ025425 FJ025434 FJ025451

FJ025501 FJ025502 FJ025504 N/A FJ025512 N/A

FJ025601 FJ025603 FJ025607 FJ025604 FJ025612 FJ025632

FJ025704 FJ025705 FJ025709 FJ025706 FJ025713 FJ025730

N/A FJ025666 FJ025667 N/A FJ025672 N/A

FJ025566 FJ025567 FJ025571 N/A FJ025575 N/A

N/A FJ025482 FJ025484 FJ025483 N/A FJ025492

Rhinophoridae Paykullia maculata

102778

Fallén (1815)

FJ025404

FJ025466

N/A

FJ025646

FJ025742

FJ025694

FJ025595

FJ025496

Americina adusta Ceratinostoma ostiorum Chaetosa (Opsiomyia) palpalis Chaetosa punctipes Chylizosoma vittatum Cleigastra apicalis Cordilura (Achaetella) varipes Cordilura (Cordilura) carbonaria Cordilura (Cordilura) ciliata Cordilura (Cordilura) ontario Cordilura (Cordilura) pudica Cordilura (Cordilura) umbrosa Cordilura (Cordilurina) albipes Cordilura (Parallelomma) dimidiata Cordilura (Parallelomma) pleuritica Cordilura atrata Cordilura latifrons Cordilura pubera Delina nigrita Gimnomera cerea Gimnomera dorsata Gymnomera cuneiventris Gymnomera tarsea Hydromyza confluens Hydromyza livens Microprosopa pallidicauda Nanna articulata Nanna brunneicosta Nanna fasciata Nanna flavipes Nanna inermis Nanna tibiella Neorthacheta dissimilis Norellia (Norellia) tipularia Norellia (Norellisoma) flavicorne Norellia (Norellisoma) liturata Norellia (Norellisoma) mirusae Norellia (Norellisoma) spinimana Norellia (Norellisoma) striolata Okeniella caudata Orthacheta cornuta Phrosia albilabris Pogonota (Lasioscelus) sahlbergi Pogonota (Pogonota) barbata Scathophaga analis Scathophaga calida Scathophaga cineraria Scathophaga furcata Scathophaga incola Scathophaga inquinata Scathophaga litorea Scathophaga lutaria Scathophaga obscura Scathophaga pictipennis Scathophaga stercoraria Scathophaga suilla Scathophaga taeniopa Scathophaga tinctinervis Scathophaga tropicalis Spaziphora cincta Spaziphora hydromyzina Trichopalpus fraterna

GenBank Accession Numbers

alignment with ClustalX 2.0 (Thompson et al., 1997) for the ribosomal 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 optimality criterion, the alignment that yielded the shortest tree was used

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in all subsequent analyses, but we also confirmed that the trees based on the remaining alignments were very similar (see Supplementary materials). 2.4. Tree search strategies The aligned Muscoidea dataset had 146 taxa and 7202 characters. It was analyzed using both maximum parsimony (MP) and maximum likelihood (ML). The tree was rooted using the acalyptrate Hemeromyia anthracina (Carnidae), although any other acalyptrate 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 parsimony analyses were carried out in TNT v2.0 (Goloboff et al., 2000: new technology search at level 50, initial addseqs = 9, find minimum 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 options as above) obtained at 36% deletion as recommended by Farris et al. (1996). For the likelihood analyses, we used MrModeltest version 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.

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 indels coded as a fifth character state yielded three most parsimoni-

645

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 sharing approximately 85% of the nodes, which suggests that indel coding 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 congruent 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)

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S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652

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 Hippoboscoidea, which are polyphyletic on the ML tree where the Glossinidae emerge as the sister group of a clade consisting of the nonfanniid Muscoidea plus the Oestroidea. There are also other topological differences within the muscoid and oestroid families (compare 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 superfamily 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 composed 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 N and majority rule consensus of all ten guide trees indicated by r.

S.N. Kutty et al. / Molecular Phylogenetics and Evolution 49 (2008) 639–652

647

Fig. 2 (continued)

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 Lasiomma is paraphyletic. The two subfamilies Anthomyiinae and Pegomyinae are para- or poly-phyletic on the MP tree while the subfamily Anthomyiinae is monophyletic on the ML tree. The Myopininae 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 Paykullia maculata (Rhinophoridae) being the sister group to Eurychaeta palpalis.

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4. Discussion

4.2. Calyptrate monophyly

The Muscoidea are of economic importance, because many of its species are pests on both agricultural crops and livestock, while others are of medical importance with some species being vectors of disease. 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 remained poorly understood, and past analyses yielded very conflicting hypotheses. Even the taxonomic composition of the Muscoidea within 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 Muscidae in the superfamily Muscoidea. In the absence of convincing evidence to the contrary, Hennig (1973) used the monophyly of Muscoidea as a working hypothesis, but kept the interfamilial relationships unresolved and considered the relationships to the other calyptrate superfamilies (Oestroidea and Hippoboscoidea) unknown. Michelsen (1991), however, explicitly acknowledged the lack of support 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.

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 paraphyletic (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 inclusion of acalyptrate outgroups from four different superfamilies. All remaining molecular studies only included few outgroup taxa.

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 relationships are uncontroversial regardless of which indel treatment or the analysis strategy is used. The calyptrate monophyly is supported on all trees. Well supported is the position of the monophyletic Oestroidea, which are always placed as the sister group to the clade Anthomyiidae + Scathophagidae. The interfamilial relationship (Fanniidae + (Muscidae + (Anthomyiidae + Scathophagidae) + Oestroidea))) is also recovered irrespective of the analytical method. Of the approximately 80% nodes shared between the MP and ML trees, many relationships at the subfamily 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. Tephritoidea: 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 topologies obtained using different analysis methods such as parsimony 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 monophyletic on the MPTs, which is in agreement with the currently accepted 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.

4.3. 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 overlap 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 relationship between Hippoboscoidea and the remaining calyptrate flies (McAlpine, 1989), whereas in Petersen et al. (2007) the Hippoboscoidea were deeply nested within the Calyptratae, although outgroup sampling was sparse and the support for this hypothesis 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 synapomorphies relative to the (hypothetical) groundplan of the Schizophora; 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 suggest 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) proposed 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 Diptera 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 superfamily-level classification that would either require that the Oestroidea 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 superfamilies, and at least two would contain a single family only and thereby be redundant.

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4.4. Interfamilial relationships within the muscoid grade The Fanniidae are placed as the sister group to a clade consisting of the Oestroidea plus the remaining families of the muscoid 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 Anthomyiidae + Muscidae (McAlpine, 1989). On the likelihood tree, the Fanniidae are in a similar position, but surprisingly Glossina pallidipes is the sister group of Muscoidea + Oestroidea. Given the strong morphological support for a monophyletic Hippoboscoidea, we believe that the overall evidence supports the most parsimonious topology with Fanniidae being sister group to Muscoidea + 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 molecular 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 + Anthomyiidae). We also consistently find that Anthomyiidae + Scathophagidae 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 A1 + CuA2 reaching the wing margin, which is probably a symplesiomorphy, and on the larval morphology 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 morphological support for Anthomyiidae + Scathophagidae, but it is consistently 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 subfamilies 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 Phaoniinae are not monophyletic in the MP tree, although the monophyly 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

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the Mydaeinae + Phaoniinae + Coesnosiinae clade has moderate support. However, it is puzzling that the azeliine Muscina is sister group to Phaoniinae + Mydaeinae + Coenosiinae with moderate 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 Michelsen (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 sampled 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 Phaonantho should be included. The Scathophagidae and most of its genera are monophyletic. However, the relationships between some of the genera and species 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 Scathophaginae 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 transitions 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 prohibitive. Most scathophagid genera are monophyletic as proposed by Kutty et al. (2007), but some relationships between the genera differ 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 evidence 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 Eurychaeta 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 nonmonophyly as argued by Rognes (1997) and Pape and Arnaud (2001) is caused by the Rhiniinae falling outside and the Oestridae 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 constituent families are currently under study based on a much larger set of exemplar species and will be the focus of a future publication.

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4.6. Alignments of the ribosomal genes The use of ribosomal genes for the reconstruction of phylogenetic relationships has its advantages and disadvantages (Hillis and Dixon, 1991; Simon et al., 1994; Caterino et al., 2000). Ribosomal genes vary in length across species of different ages and the genes can thus provide valuable information for many phylogenetic 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 techniques 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 Clustal (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 (Morrison 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-alignments 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 technique 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 regions 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 regions) while Clustal’s guide trees are calculated from a distance matrix based on dissimilarity scores between sequence pairs. However, such dissimilarity scores are unlikely to reflect true evolutionary 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 category (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 ribosomal genes in our dataset. The guide tree used in our alignments was not based on subjective opinions about calyptrate relationships. Instead, we use the phylogenetic signal from those sequences 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 computationally tractable. It also does not require the deletion of sequence 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 phylogenetic 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 informative. 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 Muscidae, 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 higher-level hypotheses for calyptrates. It thus appears that in this case the phylogenetic signal from the hypervariable regions of the ribosomal 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 phylogenetic 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 secondary 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 particular for the paraphyly of, and phylogenetic relationships within, what we suggest to call the muscoid grade, i.e., the Fanniidae, Muscidae, Anthomyiidae, and Scathophagidae. The position of this grade within the Calyptratae and the relationships of its constituent families to the remaining calyptrate superfamilies, viz. Hippoboscoidea and Oestroidea, are resolved. The relationships between the four muscoid families are established and the monophyly of the Fanniidae, Muscidae, and Scathophagidae is further corroborated while no molecular support was obtained for anthomyiid monophyly.

Acknowledgments This research was funded by the Academic Research Fund Grants R-154-000-207-112 from the Ministry of Education in Singapore 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-

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ural History Museum of Denmark; Dr. D.M. Ackland, Oxford University Museum of Natural History; Dr J. Ziegler, Zoologisches Museum 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. References Benavides, E., Baum, R., McClellan, D., Jack, W.J., 2007. Molecular phylogenetics of the lizard genus Microlophus (Squamata: Tropiduridae): aligning and retrieving indel signal from nuclear introns. Syst. Biol. 56, 776–797. Bernasconi, M.V., Pawlowski, J., Valsangiacomo, C., Piffaretti, J.C., Ward, P.I., 2000a. Phylogeny of the Scathophagidae (Diptera, Calyptratae) based on mitochondrial DNA sequences. Mol. Phylogenet. Evol. 16, 308–315. Bernasconi, M.V., Valsangiacomo, C., Piffaretti, J.C., Ward, P.I., 2000b. Phylogenetic relationships among Muscoidea (Diptera: Calyptratae) based on mitochondrial DNA sequences. Insect Mol. Biol. 9, 67–74. Carvalho, C.J.B., 1989. Classificaca~o de Muscidae (Diptera): uma pro-posta atrave´s da ana´lise cladi´stica. Rev. Bras. Zoolog. 6, 627–648. Carvalho, C.J.B., Couri, M.S., 2002. Cladistic and biogeographic analyses of Apsil Malloch and Reynoldsia Malloch (Diptera: Muscidae) of southern South America. Proc. Entomol. Soc. Wash. 104, 309–317. Carvalho, C.J.B., de Couri, M.S., Pont, A.C., Pamplona, D., Lopes, S.M., 2005. A catalogue of the Muscidae (Diptera) of the Neotropical Region. Zootaxa 860, 1– 282. Caterino, M.S., Cho, S., Sperling, F.A.H., 2000. The current state of insect molecular systematics: A thriving Tower of Babel. Ann. Rev. Entomol. 45, 1–54. Coquillett, D.W., 1901. A systematic arrangement of the families of the Diptera. Proc. US Natl. Mus. 23, 653–658. Couri, M.S., Carvalho, C.J.B., 2003. Systematic relations among Philornis Meinert, Passeromyia Rodhain & Villeneuve and allied genera (Diptera, Muscidae). Braz. J. Biol. 63, 223–232. Couri, M.S., Pont, A.C., 2000. Cladistic analysis of Coenosiini (Diptera: Muscidae: Coenosiinae). Syst. Entomol. 25, 373–392. Danforth, B.N., Lin, C.P., Fang, J., 2005. How do insect nuclear ribosomal genes compare to protein-coding genes in phylogenetic utility and nucleotide substitution patterns? Syst. Entomol. 30, 549–562. De Rijk, P., Robbrecht, E., de Hoog, S., Caers, A., Van de Peer, Y., De Wachter, R., 1999. Database on the structure of large subunit ribosomal RNA. Nucleic Acids Res. 27, 174–178. Dittmar, K., Porter, M.L., Murray, S., Whiting, M.F., 2006. Molecular phylogenetic analysis of nycteribiid and streblid bat flies (Diptera: Brachycera, Calyptratae): Implications for host associations and phylogeographic origins. Mol. Phylogenet. Evol. 38, 155–170. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Evenhuis, N.L., Pape, T., Pont, A.C., Thompson F.C. (Eds.), 2008. Biosystematic Database of World Diptera, Version 10.5. Available from: , accessed on 15 June 2008. Farris, J.S., Albert, V.A., Källersjö, M., Lipscomb, D., Kluge, A.G., 1996. Parsimony jackknifing outperforms neighbor-joining. Cladistics 12, 99–124. Giribet, G., Wheeler, W.C., 1999. On gaps. Mol. Phylogenet. Evol. 13, 132–143. Goloboff, P., Farris, S., Nixon, K., 2000. TNT (tree analysis using new technology) (BETA) version 2.0. Published by the authors, Tucumán, Argentina. Griffiths, G.C.D., 1972. The phylogenetic classification of Diptera Cyclorrhapha, with special reference to the male postabdomen. Series Entomologica 8, 1– 340. Gutell, R.R., 1994. Collection of small-subunit (16s- and 16s-like) ribosomal-RNA structures. Nucleic Acids Res. 22, 3502–3507. Han, H.Y., Ro, K.E., 2005. Molecular phylogeny of the superfamily Tephritoidea (Insecta: Diptera): new evidence from the mitochondrial 12S 16S, and COII genes. Mol. Phylogenet. Evol. 34, 416–430. Hennig, W. 1955–1964. Muscidae. In: Lindner, E. (Ed.), Die Fliegen der Palaearktischen Region, vol. 7(2), pp. 1–1110. Hennig, W., 1965. Vorarbeiten zu einem phylogenetischen System der Muscidae (Diptera: Cyclorrhapha). Stuttg. Beitr. Natkd. 141, 1–100. Hennig, W. 1973. Diptera (Zweiflügler). In: Helmcke, J.G., Starck, D., Wermuth, H. (Eds.), Handbuch der Zoologie, vol. 4(31) pp. 1–337. Hennig, W. 1976. 63a. Anthomyiidae [part]. In: Lindner, E. (ed.), Die Fliegen der Palaearktischen Region 7(1), i–xxvii. Hickson, R.E., Simon, C., Perrey, S.W., 2000. The performance of several multiplesequence alignment programs in relation to secondary-structure features for an rRNA sequence. Mol. Biol. Evol. 17, 530–539.

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