Diptera: Sarcophagidae)

Anchored hybrid enrichment challenges the traditional classification of flesh flies (Diptera: Sarcophagidae)

Buenaventura, Eliana; Szpila, Krzysztof; Cassel, Brian K.; Wiegmann, Brian M.; Pape, Thomas

Published in: Systematic Entomology

DOI: 10.1111/syen.12395

Publication date: 2020

Document version Publisher's PDF, also known as Version of record

Document license: CC BY-NC-ND

Citation for published version (APA): Buenaventura, E., Szpila, K., Cassel, B. K., Wiegmann, B. M., & Pape, T. (2020). Anchored hybrid enrichment challenges the traditional classification of flesh flies (Diptera: Sarcophagidae). Systematic Entomology, 45(2), 281-301. https://doi.org/10.1111/syen.12395

Download date: 01. okt.. 2021 Systematic Entomology (2020), 45, 281–301 DOI: 10.1111/syen.12395

Anchored hybrid enrichment challenges the traditional classification of flesh flies (Diptera: Sarcophagidae)

ELIANA BUENAVENTURA1,2 , KRZYSZTOF SZPILA3 , BRIAN K. CASSEL4, BRIAN M. WIEGMANN4 and THOMAS PAPE5

1Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Berlin, Germany, 2National Museum of Natural History, Smithsonian Institution, Washington, DC, U.S.A., 3Department of Ecology and Biogeography, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, Torun,´ Poland, 4Department of Entomology & Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A. and 5Natural History Museum of Denmark, Universitetsparken 15, Copenhagen, Denmark

Abstract. Sarcophagidae is one of the most species-rich families within the superfamily Oestroidea. This diversity is usually represented by three lineages: Miltogram- minae, Paramacronychiinae and Sarcophaginae. Historically, the phylogenetic relationships among these lineages have been elusive, due to poorly supported hypotheses or small taxon sets, or both. This study provides a dramatic increase in molecular data, more balanced sampling of all three lineages from all biogeographical regions and a reassessment of morphological characters using scanning electron microscopy in the most comprehensive assessment of subfamily-level phylogeny in Sarcophagidae to date. This analysis of the largest molecular dataset ever produced for a phylogenetic analysis of a fly lineage, with 950 loci from anchored hybrid enrichment comprising 435930bp from 101 species, revealed Paramacronychiinae as sister to Miltogramminae, not to Sar- cophaginae, as suggested by adult morphology. Maximum likelihood analysis produced a well-supported topology, with 91% of the nodes receiving strong bootstrap proportions (> 97%). In contrast to the molecular data, three out of nine morphological characters studied point to a sister-group relationship of (Sarcophaginae + Paramacronychiinae) and the remaining six characters are either silent on subfamily relationships or in need of further study. Re-examination of morphological structures provides new insights into the evolution of male genitalic traits within Sarcophagidae and highlights their convergence producing conflicting phylogenetic signal. Our phylogeny reconciles older and widely used systems of classification with tree-based thinking and sets up a classification of flesh flies that is more aligned with their evolutionary history.

Introduction with > 600 species in 37 genera (Piwczynski´ et al., 2017), have larvae that are mostly known as kleptoparasites in the Sarcophagidae are known as flesh flies as they are usually con- nests of solitary bees and aculeate wasps (Pape, 1996), while sidered to be sarcosaprophagous scavengers (i.e. feeding upon others are parasitoids, predators, saprophages and scavengers dead and decaying flesh). However, the increasing number of (Lopes, 1982; Mullen et al., 1984; Ferrar, 1987; Verves & known breeding habits reveals much more diversity and special- Khrokalo, 2006; Szpila, 2010). Species of the other two sub- izations in the life-history strategies. This diversity is usually families – Paramacronychiinae, with 97 species in 23 genera organized into three main lineages of flesh flies: Miltogrammi- (Zhang et al., 2016a), and the largest and most diverse subfamily nae, Paramacronychiinae and Sarcophaginae. Miltogramminae, Sarcophaginae, with about 2300 species in 46 genera (Bue- naventura & Pape, 2018) – are mostly known as necrophagous in invertebrate and vertebrate carrion (Bänziger & Pape, 2004; Correspondence: Eliana Buenaventura, Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Berlin, Ramírez-Mora et al., 2012). Still, their ecological diversity also Germany. E-mail: [email protected] includes facultative and/or obligate parasitoids of invertebrates

© 2019 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society. 281 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 282 E. Buenaventura et al.

(McKillup et al., 2000), saprophages (Méndez, 2012), agents Some sarcophagid classifications have subsequently been of myiasis (Mello-Patiu & Luna-Dias, 2010; Hall et al., 2016) tested in published phylogenetic studies. For example, the and scavengers in pitcher plants (Beaver & Shinonaga, 1979; hypothesis of subfamilial relationships by Pape (1986a, 1987) Dahlem & Naczi, 2006). Flesh flies perform ecological ser- was consistent with a parsimony molecular-based phylogenetic vices such as carrion decomposition and pollination and act as analysis by Kutty et al. (2010) using eight molecular markers, potential biological control agents (Pape & Bänziger, 2000; Der but rejected by the maximum likelihood (ML) analysis of the Niet et al., 2011), which are essential for the sustainability and same study as well as by Piwczynski´ et al. (2014), who used well-being of urban, rural and wild ecosystems. available GenBank sequences and recovered Sarcophaginae as Sarcophagidae include about 3000 named species, being sister to (Miltogramminae + Paramacronychiinae). The clade the second most species-rich family within the superfamily (Miltogramminae + Paramacronychiinae) was also recovered Oestroidea, after the bristled flies (Tachinidae; ca. 10.000 by Zhang et al. (2016a) based on mitogenomes, as well as in species) (Pape et al., 2011). One of the earliest classifica- a recent multilocus analysis using three mitochondrial genes tions of the Oestroidea considered Miltogramminae as part of (COI, cytB, ND4) and one nuclear gene (Ef-1𝛼), although Tachinidae (Williston, 1908), but later this taxon was trans- with larger taxon sampling of Miltogramminae and Para- ferred to Sarcophagidae (Rohdendorf, 1930; Curran, 1934). macronychiinae, by Piwczynski´ et al. (2017). The most recent North American dipterists divided the Sarcophagidae into the morphology-based phylogenies focusing on Sarcophaginae subfamilies Miltogramminae and Sarcophaginae, but differed did not include a sufficient number of representatives of Mil- in considering taxa of the tribe Agriini (= Paramacronychiinae) togramminae and Paramacronychiinae to thoroughly examine as part of either Miltogramminae (Downes, 1955, 1965) or subfamilial phylogenetic relationships (Giroux et al., 2010; Sarcophaginae (Roback, 1951, 1954). The system proposed Buenaventura & Pape, 2018). However, both phylogenies coded by Rohdendorf (1937, 1965, 1967) increased the subdivi- the juxta (of the phallus; see later) as ‘present’ and the position sions within Sarcophagidae by considering five subfamilies: of the acrophallus as ‘ventral’ for both Paramacronychiinae Chrysogrammatinae, Macronychiinae, Miltogrammatinae, and Sarcophaginae, which would support the hypothesis of Sarcophaginae and Sarcotachininae. Rohdendorf’s classifi- Pape (1986a, 1987) and Verves (1998) of a clade composed cation found partial support in the dipterological community of (Paramacronychiinae + Sarcophaginae). In summary, the in subsequent systems that considered four subfamilies: monophyly of the subfamilies of Sarcophagidae seems well sub- Macronychiinae, Miltogrammatinae, Paramacronychiinae and stantiated, but there is no phylogenetic consensus with regard to Sarcophaginae (Lopes, 1969, 1983, 1989; Lopes et al., 1977; subfamilial relationships using molecular data, and topological Dear, 1980; Rognes, 1986; Verves, 1989a). conflict remains between trees inferred by molecules versus These classifications were proposed without an explicit set morphology. of arguments, and with no cladistic framework even when Here, we investigate subfamilial and generic phylogenetic based on a series of characters considered of evolutionary rele- relationships of Sarcophagidae using 101 flesh fly species and vance. Thus, providing an explicit synapomorphy scheme, Pape molecular data from 950 loci captured using a genomic approach (1986a, 1987) instead proposed a simplified system of three of anchored hybrid enrichment (AHE; including 435 930 sites). subfamilies with Miltogramminae as sister to (Paramacrony- The comparison of our AHE-based phylogeny with recently chiinae + Sarcophaginae). Alternative morphology-based published adult morphology-based phylogenies of Sarcophagi- classifications considering additional subfamilies such as dae (Giroux et al., 2010; Whitmore et al., 2013; Buenaventura Eumacronychiinae and Macronychiinae also concurred in plac- & Pape, 2015, 2018) provides new insights into the evolu- ing Paramacronychiinae as sister to Sarcophaginae (Rognes, tion of male genitalic traits within the family and highlights 1986; Verves, 1998). Thus, the two most recent classifica- convergent characters generating a conflicting phylogenetic tions of Sarcophagidae (Pape, 1992, 1996; Verves, 1998) are signal. in agreement in recognizing a clade (Paramacronychiinae + Sarcophaginae), even if they disagree on the genera Gonio- phyto Townsend and Sarcotachina Portschinsky; Pape (1992, Materials and methods 1996) included them in Paramacronychiinae, whereas both were included in Eumacronychiinae by Verves (1998). Another Taxon sampling difference between these two systems is in the criteria for taxonomic ranking, as the subfamily Macronychiinae of Verves Our AHE dataset included an ingroup of 101 species rep- (1989a) is equivalent to the genus Macronychia Rondani of Pape resenting all three subfamilies of Sarcophagidae and all (1996). However, taxonomic conventions for what constitutes biogeographical regions. We further included 12 Anthomyiidae sufficient distinction for a particular rank are not formalized and outgroup taxa. Morphological identifications of specimens were appear to differ among organismal groups (Avise & Liu, 2011), provided by V. Michelsen (Anthomyiidae), KS (Miltogrammi- or even within the same group, as in the case for the different nae and Paramacronychiinae) and EB + TP (Sarcophaginae). subfamily concepts for Sarcophagidae. A modern approach Voucher specimens have been deposited at the Museum für towards organizing the group requires that we reconcile older Naturkunde (ZMHB), Germany, the Natural History Museum and widely used systems of classification with evidence-based of Denmark (NHMD), the University of Copenhagen, Den- groupings that reflect their evolutionary history. mark, and the National Museum of Natural History (NMNH),

Table 1. Taxa included in the morphological study

Family/Subfamily Genus Subgenus Specific epithet Author

Miltogramminae Eumacronychia – persolla Reinhard (1965) – sternalis Allen (1926) Macronychia – aurata Coquillett (1902) Opsidia – intonsa Aldrich (1928) Sphecapatoclea –sp. Paramacronyichiinae Angiometopa – falleni Pape (1986) Brachicoma – devia Fallén (1820) Dexagria – ushinskyi Rohdendorf (1978) Oophagomyia – plotnikovi Rohdendorf (1978) Paramacronychia – flavipalpis Girschner (1881) Sarcophila –sp. Sarcotachina – subcylindrica Portschinsky (1881) Sarcophaginae Blaesoxipha Gigantotheca plinthopyga Wiedemann (1830) Oxysarcodexia trivialis Wulp (1895) Peckiamyia abnormalis Hall (1937) Ravinia columbiana Lopes (1962) Sarcophaga Sarcophaga carnaria Linnaeus (1758) Tricharaea simplex Aldrich (1916) Tripanurga albicans Wiedemann (1830)

Smithsonian Institution, U.S.A. (see Table S1 for specimen Morphological study, nomenclature and terminology identifiers). Morphological studies included five species of Miltogrammi- Classification, names and authorship for species and gen- nae, seven of Paramacronychiinae and seven of Sarcophaginae era/subgenera follow Pape (1996). The terminology of (Table 1). These species are representatives of genera also structures, except the male terminalia, follow McAlpine (1981). included in the AHE phylogenetic analysis, except for species For male terminalia structures, we follow Buenaventura & Pape of Eumacronychia Townsend and Tripanurga Brauer & Bergen- (2018). Several morphological characters were studied, mainly stamm, for which no material was available for the molecular from the male genitalia, which, according to recent phylogenies study. (Giroux et al., 2010; Buenaventura & Pape, 2018) and previous taxonomic studies (Downes, 1955), provide phylogenetic signal on subfamilial relationships. The following abbreviations were used for morphological structures in figures: ac, acrophallus; Specimen preparation and documentation bp, basiphallus; dp, distiphallus; ep, epiphallus; hi, hillae; j, juxta; ls, lateral stylus; ms, median stylus. The preparation of the terminalia of male specimens for pro- duction of scanning electronic microscopy (SEM) images and morphological study followed Buenaventura & Pape (2018). Anchored hybrid enrichment laboratory protocols Soft tissues of dissected male terminalia were digested with hot 85% lactic acid for about 5 min, after which terminalia were We extracted whole-genomic DNA using nondestructive transferred to a smaller dish to separate the phallus, pregonites (specimen retained after extraction) methods from thoracic and postgonites from the remaining terminalia and rinsed twice and leg muscular tissue of individual specimens by proteinase in distilled water and once in 96% ethanol. The phallus, prego- K digestion using the DNeasy extraction protocol (Qiagen, nites and postgonites were examined using a Carl Zeiss Stemi Hilden, Germany). We quantified DNA for each sample using 305 stereomicroscope with front optics 5 Apo 2.0x (Wash- a Qubit fluorometer (High Sensitivity Kit; Life Technologies, ington, D.C., U.S.A.). Subsequently, phallus, pregonites and Inc.), and sheared 7.9–110 ng/𝜇L (47 ng/𝜇L mean) of each postgonites were air-dried, mounted on adhesive electrical tape DNA sample to a target size of c. 300 bp by sonication with attached to aluminium stubs, coated with platinum/palladium a Covaris E220 Focused-ultrasonicator with a Covaris micro- and studied in a JEOL JSM-6335 F SEM housed in the NHMD TUBE (Covaris). We used this sheared DNA as input for a or an EVO MA-15 SEM housed in the NMNH. Part of the modified version of the genomic DNA library preparation SEM images were kindly provided by M. Giroux as indicated and indexing protocol of Meyer & Kircher (2010), in which in the relevant captions, and produced as noted by Giroux et al. we included a size-selection step after blunt-end repair using (2010). All SEM images were edited using Adobe photoshop SPRIselect beads (0.9 × ratio of bead to sample volume; Beck- cs6 and assembled in plates with indesign cs6 and Adobe man Coulter, Inc.). Indexes were added to samples, which were illustrator cs6 (Adobe, San Jose, CA, U.S.A.). subsequently assembled into two pools (48 samples per pool).

We enriched each 48-sample pool using the Diptera AHE kit Drosophila melanogaster (Diptera: Drosophilidae) and (Young et al., 2016), an Agilent Custom SureSelect kit (Agilent Glossina morsitans Westwood (Diptera: Glossinidae). In our Technologies) containing 57 681 tiled, custom-designed probes orthograph implementation, we used default settings except (design described later) targeting 559 loci. We sequenced the that, for ‘substitute-u-with X’ (activated) and ‘extend-orf’, pooled libraries using two lanes of an Illumina HiSeq 2500 we replaced putative internal stop codons and the amino run (single read, 100 bp). All AHE laboratory procedures and acid Selenocysteine U with ‘X’ (amino acid level) or ‘NNN’ sequencing were conducted in laboratory facilities of the North (nucleotide level) with a custom Perl script of Misof et al. (2014) Carolina State University, Department of Entomology & Plant Pathology (Wiegmann Lab). Alignment and trimming

Probe design FASTA files of sequences were created for each orthologous gene set and aligned using mafft (v.7.273) with the L-INS-i The AHE Diptera Probe kit design we used is described in algorithm (Katoh & Standley, 2013). To assess the quality of the Young et al. (2016). In short, 559 target loci were first selected amino acid multiple sequence alignments (MSAs), we followed through comparison of protein-coding genes from available the procedure of Misof et al. (2014) with the addfragments genome data and from available official gene sets for 13 insect algorithm implemented in mafft to refine each alignment species (Niehuis et al., 2012). These potential target loci were through identification of outlier sequences, and remove out- selected based on the criteria that each are at least 150 bp in liers from both amino acid MSAs and nucleotide sequences length, contain no exon boundaries and no indels, and represent (Evangelista et al., 2019). We also removed the orthologue set differing levels of pairwise sequence variability (45–84%). reference species from all MSAs, as well as all empty or X-only These genes were then mapped to single-copy orthologues in data columns in each MSA. To obtain nucleotides, we used a Drosophila melanogaster Meigen and then compared across modified version of pal2nal v.14 (Suyama et al., 2006; Misof seven dipteran genomes and 14 transcriptomes provided by the et al., 2014) to generate the corresponding nucleotide MSAs 1KITE consortium (Young et al., 2016; Table S2) to select loci with the amino acid MSAs as a guide. We checked the amino that strongly match in the majority of compared fly taxa and acid MSAs of each orthologous gene set for ambiguously contain low numbers of gaps in multiple sequence alignments. A aligned regions with the software aliscore v.1.2 (Misof & total of 559 loci were selected for probe construction, with each Misof, 2009; Kück et al., 2010; Misof et al., 2014). We used covering 150 bp with 1.72 × tiling density for a total of 57 681 the -e option for gappy datasets and forced a comparison of all probes (Lemmon et al., 2012; Young et al., 2016). sequence pairs. Except as detailed earlier, default parameters were used in the settings for these procedures. Ambiguously aligned regions were identified at the amino acid level and Processing and AHE bioinformatic data analysis subsequently removed from the nucleotide MSAs. To do this, erroneously aligned sections identified by aliscore v.2.2 were Raw reads were demultiplexed using cassava 1.8.2 at the removed from the amino acid alignment, and corresponding NCSU Genomic Sciences Laboratory and trimmed of adapters nucleotide codons were removed using alicut and custom and low-quality sequences using trimmomatic v.0.36 (Bolger Perl scripts (Misof & Misof, 2009; Kück et al., 2010). These 2014). A cleaning step consisted of an exhaustive locus-by-locus refined nucleotide sequences were realigned using the amino check and removal of nonfly and low-quality reads based on acid alignment as blueprint in Pal2Nal (Suyama et al., 2006), expected values (E-value) reported by blast (National Center as modified in Misof et al. (2014). for Biotechnology Information, Bethesda, MD, U.S.A.). We computed summary statistics on the data and assembled the cleaned reads using trinity v.2.4 (Grabherr et al., 2011). We Phylogenetic analysis selected the files containing very low numbers of reads and rechecked for contamination (five species were excluded due to We performed ML phylogenetic analyses of our AHE dataset high incidence of microbial contamination). To ensure that loci with the MSAs at codon (nucleotide) level using raxml v.8.0.22 captured with the probe set and used in the phylogenetic anal- (Stamatakis, 2014), under default parameters, 50 replicates and ysis are clusters of orthologous sequences of selected reference random starting trees, and with 500 nonparametric, slow boot- species [single-copy orthologues, also called orthologue groups strap replicates, and GTR + G as substitution model partitioned (OGs)], an additional orthology assessment was carried out by locus (codon level including all three codon positions). We using the program orthograph v.0.5.14 (Petersen et al., 2017) used Anthomyia monilis (Meigen) to root the tree. Analyses were with the orthologue set ‘Mecopterida’, which includes a set of carried out on the High-Performance Computing Cluster of the 3145 single-copy nuclear genes. The ‘Mecopterida’ orthologue North Carolina State University Bioinformatics Research Center set is available on Mendeley (Pauli et al., 2018) and includes (NCSU BRC). five reference species: Bombyx mori Linnaeus (Lepidoptera: Our AHE phylogenetic dataset contains 435 930 nucleotides Bombycidae), Danaus plexippus (Linnaeus) (Lepidoptera: from 950 loci concatenated in fasconcat-g (Kück & Longo, Nymphalidae), Aedes aegypti (Linnaeus) (Diptera: Culicidae), 2014), with leading and trailing gaps in each MSA recoded as

‘N’ using custom Perl scripts of Misof et al. (2014) available to nonspecific binding of probes to regions contained in the from the 1KITE project (http://software.zfmk.de/The-1KITE- ‘Mecopterida’ orthologue reference set. Overall, we recovered project_evolution-of-insects.zip.) We performed ML phyloge- an average of 20 AHE loci per taxon and a mean locus length netic analyses of our AHE dataset with the MSAs at codon of 562 bp. The concatenated dataset contained 80.2% of miss- (nucleotide) level using raxml v. 8.0.22 (Stamatakis, 2014), ing data, corresponding to the result that 678 of 950 loci were under default parameters, 50 replicates and random starting represented in 10 or fewer taxa and only 97 loci were recovered trees, and with 500 nonparametric, slow bootstrap replicates, in 90 or more sampled taxa. The species Sarcophaga (Hadrox- and GTR + G as substitution model partitioned by locus (includ- ena) karakoncolos (Whitmore et al., 2018), Sarcophaga (Nes- ing all three codon positions). The GTR + G model with bittia) munronis Pape and Mesomelena mesomelaena (Loew) locus-specific rates was applied separately in each locus to allow were removed as outliers based on low yield of assignable fly for among-site rate heterogeneity but fixed for each partition orthologues in recovered sequences for these taxa. to balance the effects of model complexity and computational load (Abadi et al., 2019). Analyses were carried out on the High-Performance Computing Cluster of the NCSU BRC. Phylogeny Branch support is presented as strong for bootstrap proportions (BP) > 97, moderate for BP = 80–97, and weak for Maximum likelihood analysis produced a completely resolved BP = 50–79. Clades with BP < 50 are considered unsupported topology of 99 nodes, of which 90 (91%) received strong BP > and are not reported on the tree. ( 97) (Fig. 1). Of the remaining nine nodes, five received moderate BP support (80–97, including two outgroup nodes), two had weak BP support (50–79), and two had BP support < 50. Of the latter two nodes, one corresponds to a clade containing the Coalescent species tree estimation genera Apodacra Macquart, Miltogramma Meigen and Pterella Robineau-Desvoidy of Miltogramminae and the other is within We estimated species trees using gene tree summation meth- the genus Sarcophaga Meigen of Sarcophaginae. ods with the astral 4.9.7 algorithm (Mirarab & Warnow, 2015) The species tree estimated by astral was very similar to the from individual gene trees estimated with raxml v.8.022, again tree estimated in the partitioned ML analysis, with most nodes using the by-codon partitioned nucleotides and 100 rapid boot- showing 100% BP (Fig. 2). The interspecific nodes with low strap replicates. In general, tree summation methods have been support in the partitioned ML analysis also received lowered shown to be statistically consistent under the multispecies coa- support in the species tree analysis, although BP values were lescent model (Rannala & Yang, 2003), and in particular, astral slightly improved (Fig. 2). Our phylogenetic trees had a basal has been shown to outperform concatenation or other summary split into two well-defined and strongly supported clades with methods under differing levels of incomplete lineage sorting Sarcophaginae (BP = 100) as sister to (Miltogramminae + (Mirarab & Warnow, 2015). astral handles very large numbers Paramacronychiinae) (BP = 100). of taxa and/or loci using a heuristic search to find the species tree that agrees with the largest number of quartet trees in the set of input gene trees, i.e. individual gene trees estimated for Miltogramminae each locus with raxml v.8.2.00. Miltogramminae, with the inclusion of Sarcotachina subcylindrica, formed a strongly supported clade (BP = 100). The Data availability first split within this subfamily was between a smaller cladeof ‘lower miltogrammines’ (Dolichotachina Villeneuve, Phyllote- Anchored hybrid enrichment raw data from this study les Loew, Sarcotachina and Sphecapatoclea Villeneuve) and a are available from the NCBI SRA database (Bioproject no. larger clade of ‘higher miltogrammines’ (cf. Piwczynski´ et al., PRJNA548116). The phylogenetic dataset and individual loci 2017). Two out of six genera represented by more than one used in this study can be obtained from the alignment files avail- species were not reconstructed as monophyletic: (i) Pterella was able in the Mendeley Database (doi: 10.17632/j3mfhwmfz8.1). resolved as paraphyletic with regard to the genera Apodacra and Miltogramma, and (ii) Senotainia Macquart was part of a cluster including the genera Amobia Robineau-Desvoidy and Results Oebalia Robineau-Desvoidy.

AHE capture Paramacronychiinae Trimmed alignments contained 101 taxa (including out- groups) and 435 930 sites from 950 AHE loci, of which 50 916 Species of Paramacronychiinae, excluding Sarcotachina (11.68%) sites are parsimony-informative. It is notable that we subcylindrica, were clustered in one strongly supported clade recovered and confirmed orthology for roughly twice the num- (BP = 100). The potential monophyly of the genera of the ber of loci that were targeted by the AHE probe set. This is Paramacronychiinae could not be tested, as only one species of due to recovery of regional fragments of targeted orthologs and each genus was included.

Fig. 1. Maximum likelihood tree of our analysis using our anchored hybrid enrichment dataset and raxml. Values on branches are bootstrap proportions. Coloured branches assigned to subfamilies are as follows: blue, Miltogramminae; purple, Paramacronychi- inae; turquoise, Sarcophaginae. Species and/or genera included in the morphological study are in bold. Insets on the left show habitus images of representative flies: (A) Miltogramminae (Protomiltogramma sp., South Africa); (B) Paramacronychiinae (Wohlfahrtia sp., United States); (C) Sarcophaginae (Microcerella sp., Chile). Habitus photos are courtesy of Steve Marshall. [Colour figure can be viewed at wileyonlinelibrary.com].

Fig. 2. Bootstrapped maximum likelihood tree estimated with astral. Support values in red (only reported for bootstrap proportions < 50) indicate bootstrap proportions as estimated in the partitioned maximum likelihood analysis (see Fig. 1). Coloured branches assigned to subfamilies are as follows: blue, Miltogramminae; purple, Paramacronychiinae; turquoise, Sarcophaginae. Species and/or genera included in the morphological study are in bold. [Colour figure can be viewed at wileyonlinelibrary.com].

Sarcophaginae which may be the clade (Oestridae + Mystacinobiidae) (Kutty et al., 2019). Both Mystacinobia zelandica Holloway and the Monophyly was strongly supported (BP = 100), and the majority of bot flies appear to have the pregonite rigidly con- first split recovered the cladeOxysarcodexia ( Townsend + nected to the hypandrium (Patton, 1935, 1937; Grunin, 1965, Ravinia Robineau-Desvoidy) as sister to a large clade with 1966; Holloway, 1976: figs 40, 41). This would indicate that the the genera laddered as follows: (Blaesoxipha Loew + (Sar- condition in Miltogramminae and Paramacronychiinae is best cophaga + (Peckia Robineau-Desvoidy + (Engelimyia Lopes + considered as plesiomorphic. Lipoptilocnema Townsend)))). Five out of seven genera were recovered as monophyletic. The monophyly of the remaining Shape of phallotrema. The phallotrema is an external opening two genera, Engelimyia and Ravinia, could not be tested, as only or the secondary gonopore at the apex of the phallus, as opposed one species of each genus was included. Our taxon sampling to the internal opening or primary gonopore at the end of the of species was strongly biased towards the genus Sarcophaga, ejaculatory duct coming from the sperm pump (Hennig, 1973; which was represented by 27 subgenera, of which 14 were rep- Ulrich, 1974; Sinclair, 2000; Cumming & Wood, 2017). In resented by more than one species. Of these, nine were resolved Calyptratae, the parameral sheath encloses the aedeagus, and its as monophyletic. distal opening constitutes the phallotrema (Sinclair, 2000; Cum- ming & Wood, 2017). Thus, identifying features like the external Morphology opening and the small denticles of the distiphallus can help in recognizing the phallotrema across Sarcophagidae (Giroux The study of a broad sample of species from across the three et al., 2010; Buenaventura & Pape, 2018). These features are subfamilies allowed us to re-examine the morphological char- also observed in the phallus of Miltogrammninae and Para- acters that have been considered as supporting subfamilial rela- macronychiinae. All miltogrammine and paramacronychiine tionships in previous studies, and to reassess their homology in (Figs 4,5) taxa studied bear a phallotrema in its plesiomorphic the context of the terminological framework recently proposed form, as a single external opening clothed by the denticulated for male genitalia structures (Giroux et al., 2010; Buenaventura acrophallic rim (Figs 4B, G, 5D). An apomorphic form of the & Pape, 2018). We re-examined five characters in particular, as phallotrema consisting of three openings has evolved indepen- follows: (i) connection between pregonite and hypandrium; (ii) dently several times across Diptera, e.g. in Cylindrotomidae, shape of phallotrema; (iii) position of acrophallus; (iv) juxta; Blephariceridae, Tanyderidae, Asilinae, some Rhinophoridae, and (v) epiphallus (Fig. 3). Additional characters are discussed some Tachinidae (Andersen, 1988; Wood, 1991; Dikow, 2009; based on data from the literature, including (vi) labrum of first Cerretti et al., 2014a) and twice within Sarcophagidae: once in instar larva, (vii) shape of testes, (viii) shape of female accessory the ancestor of the subfamily Sarcophaginae (Figs 5,6) and once glands and (ix) shape of male accessory glands. in the miltogrammine species Senotainia trifida Pape (Pape, 1989, 1992; Buenaventura & Pape, 2018). In Sarcophaginae, the apomorphic ground plan of the phallotrema is the result Connection between pregonite and hypandrium. The pre- of a folding of the acrophallic rim creating three more or less and postgonites are structures of uncertain homology found separated outlets or openings (Figs 5J, 6C, G) (Buenaventura throughout Cyclorrhapha (Cumming et al., 1995; Sinclair & & Pape, 2015), giving two lateral and one median stylus. The Cumming, 2006). The pregonites may be derived from the sarcophagine acrophallus also has a paired capitis flanking the gonocoxite or the hypandrium, or both (Merz & Haenni, 2000), median stylus and a pair of hillae flanking the lateral part of and the Eremoneuran ground plan is considered to display a each lateral stylus (see Buenaventura & Pape, 2018, for a more fusion or nonarticulated connection between hypandrium and detailed definition). Most Sarcophaginae have three openings, pregonites (Cumming et al., 1995; Sinclair & Cumming, 2006). but some have only two or even a single opening (Fig. 5H), Our studies across the Calyptratae have revealed the widespread which is here interpreted as a secondary loss (Buenaventura & presence of a demarcation clearly, even if narrowly, separating Pape, 2018). the pregonite from the hypandrium, which is also exemplified by the traditional way of depicting the pregonite as a well-defined sclerite separate from the hypandrium (e.g. Michelsen, 2009). Position of acrophallus. The acrophallus in Miltogramminae The demarcation is, however, not always present, as described and Paramacronychiinae consists of a single tube formed by the for Tachinidae by Tschorsnig (1985). Downes (1965) diagnosed unfolded and often denticulated acrophallic rim (Figs 4A–C, E, his Miltogramminae, i.e. (Miltogramminae + Paramacronychi- G, I, 5C–G), which gives a single opening, the phallotrema. inae), as displaying a complete fusion between the pregonite In Miltogramminae, the acrophallus is the apicalmost part of (as ‘anterior clasper’) and the hypandrium. Our studies indi- the distiphallus (Figs 3B, 4B, C), which takes up most of the cate that the pregonites of both the Miltogramminae and the distiphallic length. It is not surrounded by accessory distiphallic Paramacronychiinae are connected to the hypandrium, but structures but always fully exposed and visible (Figs 4A–C). always with a narrow line of demarcation. The pregonites of In Paramacronychiinae, the acrophallus is placed ventrally on Sarcophaginae are more loosely connected to the hypandrium the distiphallus and partly covered by the juxta (Figs 3C, 4D–I, and probably have some mobility. An assessment of the polarity 5A–C), and is only partially visible in lateral and ventral views would require comparisons to the most probable sister taxon, of the phallus (Figs 3C, 4E, I, 5A). The acrophallus is much

Eumacronychia persolla Outgroup Eumacronychia sternalis Macronychia aurata MILTOGRAMMINAE Opsidia intonsa Sarcotachina subcylindrica Sphecapatoclea sp. Angiometopa falleni Brachicoma devia Dexagria ushinskyi PARAMACRONYCHIINAE Oophagomyia plotnikovi Paramacronychia flavipalpis Sarcophila sp. Blaesoxipha plinthopyga Oxysarcodexia trivialis Peckiamyia abnormalis SARCOPHAGINAE Ravinia columbiana Sarcophaga carnaria Tricharaea simplex A Tripanurga albicans

B ep C D bp ep

dp bp dp ac

dp ac j ac j

Fig. 3. (A) Subfamilial phylogenetic relationships of Sarcophagidae lineages based on our anchored hybrid enrichment tree. Five morphological characters were traced across the subfamilies, which are indicated by numbers and squares explained in the legend. Colours assigned to subfamilies are as follows: blue, Miltogramminae; purple, Paramacronychiinae; turquoise, Sarcophaginae. (B–D) Phallus in left lateral view: (B) Eumacronychia persolla;(C)Paramacronychia flavipalpis;and(D)Tripanurga albicans. ac, acrophallus; bp, basiphallus; dp, distiphallus; ep, epiphallus; j, juxta. [Colour figure can be viewed at wileyonlinelibrary.com].

B C A ep bp

dp dp dp ac ac ac

D E F

j ac j j

G H I

dp ac

j j j

Fig. 4. Miltogramminae and Paramacronychiinae male genitalia. (A) Distiphallus, left lateral view: Eumacronychia sternalis. (B) Phallus, left ventrolateral view: Opsidia intonsa. (C) Phallus, left lateral view: Sphecapatoclea sp. (D) Distiphallus, left lateral view: Angiometopa falleni.(E) Distiphallus, latero-apical view: Angiometopa falleni. (F) Distiphallus, left lateral view: Dexagria ushinskyi. (G) Distiphallus, ventral view: Dexagria ushinskyi. (H) Distiphallus, left lateral view: Oophagomyia plotnikovi. (I) Distiphallus, ventral view: Oophagomyia plotnikovi. ac, acrophallus; bp, basiphallus; dp, distiphallus; ep, epiphallus; j, juxta.

ACB ep

ac dp ac

j j j

D E F G

ac ac ac

j j

HIJ

ls ls dp

j j ms

Fig. 5. Paramacronychiinae and Sarcophaginae male genitalia. (A) Distiphallus, left lateral view: Paramacronychia flavipalpis. (B) Distiphallus, dorsolateral view: Paramacronychia flavipalpis. (C) Distiphallus, ventral view: Paramacronychia flavipalpis. (D) Acrophallus, ventral view: Paramacronychia flavipalpis. (E) Distiphallus, ventral view: Sarcophila sp. (F) Distiphallus, left lateral view: Sarcophila sp. (G) Acrophallus, ventral view: Sarcophila sp. (H) Distiphallus, ventral view: Blaesoxipha plinthopyga. (I) Distiphallus, left lateral view: Oxysarcodexia trivialis. (J) Acrophallus, apical view: Oxysarcodexia trivialis. ac, acrophallus; dp, distiphallus; ep, epiphallus; j, juxta; ls, lateral stylus; ms, median stylus.

A B C ms D ls ms ls bp

dp j

j j

E F G ep dp bp hi dp ls

ms dp j

Fig. 6. Sarcophaginae male genitalia. (A) Distiphallus, ventral view: Peckiamyia abnormalis. (B) Distiphallus, dorsolateral view: Peckiamyia abnormalis. (C) Acrophallus, ventral view: Tricharaea simplex. (D) Distiphallus, left lateral view: Tricharaea simplex. (E) Distiphallus, left lateral view: Ravinia columbiana. (F) Distiphallus, left lateral view: Tripanurga albicans. (G) Distiphallus, latero-apical view: Tripanurga albicans. bp, basiphallus; dp, distiphallus; ep, epiphallus; hi, hillae; j, juxta; ls, lateral stylus; ms, median stylus. more complex in Sarcophaginae than in the other flesh fly the median stylus as a landmark to point out the origin of the lineages, being composed of three styli, paired capitis and a juxta. As this stylus is absent in many Sarcophaginae, and as pair of hillae (Figs 6A, C, E) (all of which can be variously the shape and position of the juxta vary considerably, assess- reduced; see Buenaventura & Pape, 2018), but it is always ing the presence and point of origin of the juxta can be difficult placed ventrally on the distiphallus (Figs 3D, 6A, G), as in (Giroux et al., 2010; Whitmore et al., 2013; Buenaventura & Paramacronychiinae. Pape, 2015). These difficulties were addressed by Buenaven- tura & Pape (2015), who updated the definition of a juxta to Juxta. The term juxta was proposed by Zumpt & Heinz any apical extension of the posterior (= dorsal) side of the para- (1950) in a study where previous terminology (i.e. Séguy, 1928; phallus, proximal to the base of the median stylus and/or lateral Patton & Cushing, 1934; Hennig, 1937; Rohdendorf, 1937; styli (i.e. to the acrophallus), usually anteriorly (= ventrally) Graham-Smith, 1938) was summarized and male terminalia directed, often curved over the acrophallic structures and some- homologies were defined on a comparative basis. A definition times articulated to the paraphallus by a hinge near its base that of the juxta is not explicitly outlined in the study by Zumpt is commonly used as a landmark that separates the juxta from & Heinz (1950) but this structure is illustrated and its apical the remaining paraphallus. We consider a juxta to be absent in position on the phallus, texture and a few other features can the studied species of Miltogramminae (Fig. 3), as the dorsal be deducted from previous interpretations cited in this study surface of the paraphallus does not show any apical extension, (i.e. Séguy, 1928; Rohdendorf, 1937). A revised interpretation the acrophallus being the apicalmost structure of the distiphallus of this structure was provided by Giroux et al. (2010), who used (Figs 3B, 4B, C). In the large majority of Miltogramminae, the

© 2019 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society. 45, 281–301 AHE challenges classification of Sarcophagidae 293 acrophallus is not protected or covered by any curving structure most other calyptrate families (Ferrar, 1987), appears to provide originating from the dorsal surface of the paraphallus, and the strong evidence that the condition in Sarcophaginae is derived. few exceptions (e.g. Eumacronychia persolla; Fig. 4A) appear to be most parsimoniously considered as having evolved inde- Shape of testes and female and male accessory glands. The pendently. All the studied species of Paramacronychiinae pos- ground plan condition of the female reproductive system in sess an apical extension of the dorsal surface of the paraphallus, Diptera includes paired ovaries and a pair of female acces- proximal to the base of the acrophallus, anteriorly (= ventrally) sory glands, which are unsclerotized organs of variable shape directed and curved over the acrophallic stylus (Figs 3C, 4D–I, (pear-shaped, subspherical, long and cylindrical, or complex 5A–C, E, F). This apical extension of the paraphallus is inter- ramose), each having a duct that opens into the anterodorsal preted as a juxta (Fig. 3). In Paramacronychiinae, the juxta show part of the genital chamber, usually close behind the spermath- no hinge or other sort of discontinuity with the remaining para- ecal openings (McAlpine, 1981; Cumming & Wood, 2017). phallus (Figs 4D, F, H, 5B, F). The presence of a juxta in Para- The situation is similar for the male, where a pair of testes are macronychiinae was reported in previous studies (Giroux et al., accompanied by a pair of accessory glands, with the ducts of 2010; Buenaventura & Pape, 2018), but without any further the latter opening at the upper part of the common ejaculatory details. Thus, here we confirm the presence of a nondemarcated duct (Sinclair et al., 2007). Based on the few available illustra- juxta for this subfamily. The juxta was studied in Sarcophaginae tions and scattered information from a few authors (Rohdendorf, by Giroux et al. (2010) and its presence has been confirmed for 1937; Lopes, 1941; Downes, 1958; Hori, 1961; Pape, 1992), it all genera of Sarcophaginae by Buenaventura & Pape (2018), seems that Sarcophaginae (or at least the genus Sarcophaga) which is consistent with our results (Fig. 3). In some lineages of share the following traits, which are here considered as derived: Sarcophaginae the juxta is not clearly delimited from the remain- female accessory glands ellipsoidal, oval or subspherical (long ing paraphallus (Figs 5I, 6D, E), while a hinge or poorly sclero- and cylindrical in Miltogramminae and other oestroids); male tized strip is clearly recognizable in other lineages (Figs 6B, G) accessory glands elongated and coiled (sausage-shaped in Mil- and appears to have evolved only once in Sarcophaginae. Thus, togramminae and other oestroids); testis elongated, pear-shaped the ‘nondemarcated juxta grade’ mentioned by Buenaventura & with a more or less distinct constriction about one-third from the Pape (2018) is paraphyletic with regard to the ‘juxtate’ clade that base (sausage-shaped in Miltogramminae and Calliphoridae). contains the remaining Sarcophaginae. Unfortunately, no information is as yet available on the internal reproductive system of any member of Paramacronychiinae. Epiphallus. The epiphallus is a distinct lobe or spine-like pro- Hori (1960, 1961) provided data for Sarcophila cinerea (Fabri- cess arising from the dorsomedial surface of the basiphallus in cius) [= Sarcophaga (Leucomyia) alba (Schiner)], which he many Cyclorrhapha (Sinclair, 2000; Cumming & Wood, 2017). later gave as Leucomyia cinerea, and his listing of this species This structure was considered as a synapomorphy of Calyp- in his Agriinae (Hori, 1960, 1961) as well as in his phylogeny tratae, although it is absent in Fanniidae and Hippoboscoidea (Hori, 1967, fig. 1) led Pape (1996: 9) to suggest spherical (Griffiths, 1972), and its presence was subsequently confirmed female accessory glands as a possible synapomorphy between for a clade composed of Muscidae, Anthomyiidae, Scathophagi- Paramacronychiinae and Sarcophaginae. The internal reproduc- dae and Oestroidea (Sinclair et al., 2013). In Sarcophagidae, tive system needs further examination and documentation from the epiphallus arises from the proximal (Figs 3B, 4B, C) part a larger number of species before it can be properly evaluated of the dorsomedial surface of the basiphallus and is delimited as a source of phylogenetic information at the subfamily level. from this by a poorly sclerotized strip. Following this definition of an epiphallus, we found this structure to be present in Mil- Discussion togramminae (Figs 3B, 4B, C) except for all species of Amobia Robineau-Desvoidy and a single species of Macronychia (Kura- Studies of fly radiations at higher taxonomic levels are now hashi & Pape, 1996). An epiphallus-like structure is also found able to process and analyse large amounts of molecular data in the paramacronychiine genera Galopagomyia Bischof, Para- (Kutty et al., 2010; Wiegmann et al., 2011; Marinho et al., 2012; macronychia Brauer & Bergenstamm (Fig. 5B) and Turanomyia Young et al., 2016; Gillung et al., 2018; Kutty et al., 2018; Kutty Rohdendorf & Verves, and in the sarcophagine genus Tripa- et al., 2019), but AHE and massive parallel sequencing have nurga (Fig. 6F), but a discontinuity between this epiphallus-like not yet been fully exploited. Previous attempts at reconstructing structure and the basiphallus is found only in Galopagomyia. flesh fly phylogeny using molecular data (Kutty et al., 2010; Piwczynski´ et al., 2014, 2017) had low support at the subfamily Labrum of first-instar larva. Downes (1955) diagnosed level, possibly due to insufficient taxon sampling, uninformative his (Miltogrammini + Agriini) by the shared presence of a or conflicting data, or both. Therefore, a dramatic increase in well-developed labrum in the first-instar larva (LI) as opposed molecular data and a more balanced sampling make this the first to Sarcophaginae, where the labrum of LI is reduced to a comprehensive attempt at elucidating the early diversification of small knob between the closely adpressed mouthhooks (Lopes, flesh flies. 1983). However, the presence of a well-developed labrum in Previous phylogenetic studies of Sarcophagidae based on a LI of Mystacinobia zelandica (Holloway, 1976) and Oestridae few nuclear genes suggest that the group experienced periods (Grunin, 1965, 1966), as well as in the putative ground plan of of rapid evolutionary diversification (Piwczynski´ et al., 2014;

Buenaventura et al., 2017; Buenaventura & Pape, 2017). These Three of nine morphological characters studied appear to rapid divergence events, involving mostly lineages within the support a sister-group relationship of (Sarcophaginae + Para- subfamily Sarcophaginae, represent bursts of speciation that macronychiinae), and the remaining six characters are either may have impeded the elucidation of a fully resolved and silent on subfamily relationships or in need of further study. strongly supported phylogeny in previous studies. Rapid radia- The three morphological character states conflicting with our tions pose some of the most difficult phylogenetic problems, as phylogeny and a previous Sanger-based tree (Piwczynski´ et al., the low levels of phylogenetic information can cause low tree 2017) are: ventral position of acrophallus, possession of juxta, resolution and discordance between gene trees, and species trees and lack of epiphallus (Fig. 3). A nonarticulated connection may have relatively more impact when divergences happen at a between hypandrium and pregonite, a phallotrema as a sin- faster rate (Whitfield & Lockhart, 2007). An important approach gle external opening surrounded by the denticulated acrophallic to reconstructing rapid radiations is by analysing large numbers rim, and a well-developed labrum in the first-instar larva are of loci with coalescent-based species-tree inference methods symplesiomorphic at the level of the family, and shape of the (Crawford et al., 2012; Giarla & Esselstyn, 2015; Edwards testes as well as of the female and male accessory glands need et al., 2016). In our study, the adoption of this approach further study. revealed a rapid early radiation, where the deeper branches The male terminalia of all Miltogramminae (Fig. 4) and Para- are much shorter than the more distal branches (Whitfield & macronychiinae (Figs 4,5) studied here present a phallotrema Lockhart, 2007). Similar patterns were reported in previous in its plesiomorphic shape, as a single opening surrounded studies for Sarcophaginae (Piwczynski´ et al., 2014) and for the by a denticulate acrophallic rim. Most Sarcophaginae lineages genus Sarcophaga (Buenaventura et al., 2017; Buenaventura have three openings (Figs 5,6) and cases of absence/reduction & Pape, 2017), although none of these works showed strong of styli are interpreted as secondary losses (Buenaventura & branch support for all nodes. Weak branch supports in these Pape, 2018). A tripartite configuration of the phallotrema has previous studies probably resulted from using few molecular evolved independently more than once within Oestroidea, and markers. In contrast to these studies, our results show strong apart from Sarcophaginae, twice in the rhinophorid genera Ven- statistical support for a rapid radiation pattern observed mostly trops Crosskey and Stevenia Robineau-Desvoidy (Tschorsnig, within the genus Sarcophaga. Internal branches across other 1985; Pape, 1986a; Cerretti & Pape, 2009) and numerous times flesh fly lineages are longer than those within this genusand within Tachinidae (Andersen, 1988). Thus, a phallotrema with would not represent genuine events of rapid radiations. How- three openings is considered as an apomorphic feature for Sar- ever, our sampling of species and genera within Sarcophaginae cophaginae and therefore not informative for the reconstruction is limited to seven out of 46 currently recognized genera of subfamilial relationships within Sarcophagidae. (Buenaventura & Pape, 2018), leaving most lineages within We observed a plesiomorphic configuration of the acrophal- this subfamily in need of further studies. Our phylogenetic lus in apical position on the distiphallus in Miltogramminae, trees have three well-defined and strongly supported clades in the same position as in most of Oestroidea (Pape, 1992), corresponding to the three subfamilies, with Sarcophaginae while a derived state with the acrophallus in a ventral posi- (BP = 100) as sister to the clade (Miltogramminae + Para- tion on the distiphallus was observed for Paramacronychiinae macronychiinae) (BP = 100) (Figs 1,2). A possible explanation and Sarcophaginae, supporting the clade (Paramacronychiinae for this robust result is the combination of a drastic increase in + Sarcophaginae) and in conflict with (Miltogramminae + Para- macronychiinae) reconstructed by our AHE data. data – the high number of loci and bp – with a more balanced The shared possession of a fully developed juxta in Para- taxon sampling of the main lineages. This combination is macronychiinae and Sarcophaginae similarly counts as evidence required for rigorously testing the monophyly of each subfam- of a sister-group relationship between these subfamilies. This ily and to further resolve the phylogenetic relationships within morphology-based hypothesis conflicts with our AHE-based Sarcophagidae. phylogeny, as it supports the hypothesis of the juxta having evolved only once in the ancestor of the clade (Paramacronychi- inae + Sarcophaginae), whereas its absence in Miltogramminae Morphological insights can only be reconstructed as plesiomorphic. Downes (1955) cites the possession of a posterior ‘spine’ The SEM study of a broad spectrum of genera and the recently (epiphallus) to characterize his Miltogramminae, which updated terminological framework provided by Giroux et al. included the taxa currently included in Miltogramminae (2010) and Buenaventura & Pape (2018) allowed us to reassess and Paramacronychiinae. Pape (1996) observed the absence the homology of genitalia structures which have been widely of epiphallus in both Paramacronychiinae and Sarcophaginae used to infer and support phylogenetic relationships between and proposed this shared character state as a synapomorphy subfamilies. These structures are: (i) connection between pre- of the sister-group relationship between these two flesh fly gonite and hypandrium; (ii) shape of phallotrema; (iii) position lineages. Buenaventura & Pape (2018) included the epiphallus of acrophallus; (iv) juxta; (v) epiphallus; (vi) labrum of first in their phylogenetic analysis of Sarcophaginae and found an instar larva; (vii) shape of testes; (viii) shape of female acces- epiphallus-like structure to be present in the genus Tripanurga, sory glands; and (ix) shape of male accessory glands (Fig. 3), as but apparently as an autapomorphic condition, as this genus discussed later. emerged deeply within the subfamily Sarcophaginae. Here, we

© 2019 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society. 45, 281–301 AHE challenges classification of Sarcophagidae 295 confirm that the epiphallus being present in the large majority coxopleural streak; (ii) no subprimary notopleural setae; (iii) of Miltogramminae, and the epiphallus-like extension in the complete fusion of anterior clasper (= pregonite) with ninth sarcophagine genus Tripanurga are best considered as conver- sternite (= hypandrium); (iv) aedeagus (= phallus) consisting of gently evolved. The situation in Paramacronychiinae is open to one segment; (v) phallus with posterior ‘spine’ (= epiphallus); interpretation, awaiting a better-supported generic phylogeny. and (vi) first-instar larva with labrum well developed as alarge As coded by Pape (1998), an epiphallus-like structure is found hook-like structure. All of these character states are here con- in Galopagomyia, Paramacronychia and Turanomyia,butapart sidered symplesiomorphic. Our observations do not corroborate from Galopagomyia emerging at the base under a successive the fusion of the pregonite with the hypandrium either for weighting protocol, there is no conclusive evidence on the Miltogramminae or for Paramacronychiinae. The pregonite in paramacronychiine ground plan. It should be noted that the these groups is firmly connected to the hypandrium, but with a epiphallus in Miltogramminae as well as in Galopagomyia clear demarcation, which is here considered the plesiomorphic seems to be partly delimited from the basiphallus by an unscle- condition. Also, Downes (1955) defined his Miltogramminae rotized strip or discontinuity, while the epiphallus-like structure (= Miltogramminae and Paramacronychiinae) as having the observed in Paramacronychia, Turanomyia and Tripanurga ‘aedeagus (= phallus) consisting of one segment’ and his Sar- lacks this demarcation. Also, there are differences in the degree cophaginae by having an ‘aedeagus commonly with a basal and a of curvature or undulation of the epiphallus-like extension of distal segment’. We agree with the connection between basi- and Paramacronychia, Turanomyia and Tripanurga, which may distiphallus being continuous (nonarticulated) in Miltogrammi- suggest nonhomology with that observed in Miltogramminae. nae and Paramacronychiinae, while it has a discontinuity or even Without this discontinuity between the basiphallus and the a hinge between basi- and distiphallus in a large clade within epiphallus and the difference in shape, there is no anatomi- the Sarcophaginae (Buenaventura & Pape, 2018). However, we cal reference to assume homology between the epiphallus of disagree on the polarity and we consider the discontinuity as an Miltogramminae and the epiphallus-like structure observed in apomorphic condition for some of Sarcophaginae. A hinge-like Paramacronychia, Turanomyia and Tripanurga. Thus, with an discontinuity has also evolved at least once in Dexiinae epiphallus unequivocally identified only in the ground plan of (Tachinidae) (Cerretti et al., 2014b) and probably within other Miltogramminae, this structure would either have been lost Tachinidae (Herting, 1957, 1960; Verbeke, 1963; O’Hara, 2013), once in the hypothetical ancestor of (Paramacronychiinae + and similarly in Rhinophoridae (Cerretti & Pape, 2012; Cerretti Sarcophaginae), or lost twice, once in the ancestor of some et al., 2014a), where bends and desclerotizations are observed or all of Paramacronychiinae and again in the ancestor of between basi- and distiphallus. However, the continuous con- Sarcophaginae. nection between basi- and distiphallus is symplesiomorphic, as As stated earlier, further examination and documentation of it appears to be the ancestral condition in all other calyptrate the testes and female and male accessory glands of Sarcophagi- families (Sinclair, 2000; Sinclair et al., 2013). The posterodor- dae are needed in a large number of species to test Pape’s (1996) sal ‘spine’ (character 5) of the phallus is interpreted differently hypothesis of the shape of these organs supporting a sister-group as the epiphallus and is considered absent in Paramacrony- relationship between Paramacronychiinae and Sarcophaginae. chiinae, which would imply that its loss in the ancestors of The paucity of information would suggest that Sarcophaginae Paramacronychiinae and Sarcophaginae would have occurred has oval female accessory glands and elongated and coiled male in parallel, in accordance with our phylogeny. Finally the accessory glands, while Miltogramminae and other oestroids larval morphological feature (character 6) used by Downes have cylindrical female accessory glands and sausage-shaped (1955) to define his Miltogramminae (= Miltogramminae and male accessory glands. The elongate pear-shaped testes in Paramacronychiinae) is best considered symplesiomorphic. the Sarcophaginae are most likely apomorphic relative to the Both Miltogramminae and Paramacronychiinae, as many other sausage-shaped testes in Miltogramminae and Calliphoridae, Oestroidea, possess a well-developed labrum in the first-instar but without data from Paramacronychiinae the internal repro- larvae, while all Sarcophaginae have a first-instar larva with a ductive organs remain silent on subfamily-level phylogeny. vestigial labrum (Downes, 1955; Lopes, 1983). The sister-group relationship between Miltogramminae and Paramacronychiinae as suggested by our results is unexpected in the light of the morphological data studied here. Also, previous Morphology-based classifications and phylogeny research considered some anatomical features as support for the clade (Miltogramminae + Paramacronychiinae). For example, There are two competing subfamily-level classifications the absence of metasternal setosity shared by Paramacrony- for Sarcophagidae (Pape, 1996; Verves, 1998), one including chiinae and Miltogramminae (except for Chivamyia Pape) three subfamilies [Miltogramminae, Paramacronychiinae and mentioned by Pape (1992) may also support our phylogeny. Sarcophaginae (Pape, 1996)] and another considering two However, other morphological character states historically con- additional subfamilies (Eumacronychiinae, Macronychiinae). sidered in support of (Miltogramminae + Paramacronychiinae) Our results do not reject these classifications, as our analysis are in need of further examination to corroborate or reject did not include representatives of the Macronychiinae and them as symplesiomorphic. For example, Downes (1955) used Eumacronychiinae of Verves and we recover three main lin- the following character states to define his Miltogramminae eages, forming three monophyletic groups, corresponding to (= Miltogramminae and Paramacronychiinae): (i) presence of the subfamilies of Pape’s classification.

Miltogramminae The arrangement of clades of Miltogramminae species in our tree is fully consistent with the so-called ‘lower’ and Several classification schemes arranged Miltogramminae into ‘higher’ miltogrammines proposed by Piwczynski´ et al. (2017). different tribes and genera, but without reaching a stable clas- Despite the differences in taxon sampling, our results were sification (Rohdendorf, 1967; Verves, 1989b; Pape, 1996). Our partially consistent with Kutty et al. (2010) and Piwczynski´ phylogeny does not support the tribal classifications of Rohden- et al. (2014), as Miltogramma is closely related to Pterella,and dorf (1967) and Verves (1989b) with regard to the Miltogrammi- Sphenometopa to Taxigramma (Fig. 1). nae. At the genus level, only the genera assigned by Rohdendorf The genus Pterella is polyphyletic in our results (Figs 1,2) as (1967) to the tribe Miltogrammatini form a monophyletic group Pterella convergens (Pandellé) is sister to the species of Mil- (Figs 1,2). These genera are Apodacra, Craticulina Bezzi, Mil- togramma, while Pterella grisea (Meigen) is sister to a clade togramma and Pterella. The tribes Metopiini [Metopia Meigen, containing the taxa described earlier and the species of Apo- Metopodia Brauer & Bergenstamm, Oebalia, Opsidia Coquil- dacra. Interestingly, P. c o nv e rge n s and P. g r i s e a were origi- lett, Phrosinella Robineau-Desvoidy, Sphenometopa Townsend, nally assigned to the genus Miltogramma. Species of Pterella Taxigramma (in part)] and Phyllotelini (Dolichotachina, Phyl- may be divided into two distinct groups based on the presence loteles, Sphecapatoclea, Sphecapatodes Villeneuve) of Rohden- of modified chaetotaxy on the male foretarsus [e.g. P. conver- dorf (1967) are polyphyletic in our tree, while Senotainiini gens, P. penicillaris (Rondani), P. trichiosoma (Rohdendorf), [Amobia, Senotainia, Taxigramma (in part)] of Rohdendorf’s P. secunda (Rohdendorf), P. zaisanica Verves) or its absence classification are paraphyletic. (e.g. P. g r i s e a , P. melanura (Meigen), P. asiatica (Rohdendorf), The tribes Metopiini (Mesomelena Rondani, Metopia, P. nigrofasciata (Rohdendorf)]. Interestingly, Piwczynski´ et al. Opsidia, Phrosinella, Sphenometopa, Taxigramma Perris) (2017) found P. convergens with modified chaetotaxy on male and Phyllotelini (Dolichotachina, Metopodia, Phylloteles, foretarsus as nested inside Miltogramma, whereas two other Sphecapatoclea, Sphecapatodes) of Verves (1989b) are poly- species of Pterella with unmodified chaetotaxy on male fore- phyletic in our tree, while the tribes Amobiini (Amobia)and tarsus are sister to the genus Protomilitogramma Townsend. Oebaliini (Oebalia) are represented by a single species each Species of Miltogramma are generally identified by the absence and their monophyly could not be assessed. Amobiini and of a well-developed vibrissa, but this is not an exclusive feature Oebaliini are nested in a clade also containing species of the of Miltogramma (Pape & Szpila, 2012), as a weak vibrissa is also genus Senotainia, which, according to Verves (1989b), belongs present in some Pterella, including P. convergens. An affiliation to the tribe Miltogrammatini. Verves (1989b) followed Vil- of P. convergens within Miltogramma is also supported by larval leneuve (1909) in considering the genera Sphecapatoclea and morphology (Szpila, 2010). Piwczynski´ et al. (2017) also found Sphecapatodes to be closely related, and placed these genera in Miltogramma and Pterella as polyphyletic. Thus, the reconstruc- the tribe Phyllotelini. Our data do not support this phylogenetic tion of Pterella as a nonmonophyletic taxon is not surprising and closeness, nor was it supported by Sanger-based molecular evi- points to a poor delimitation of this genus with regard to Mil- dence (Piwczynski´ et al., 2017) or first-instar larval morphology togramma. The redefinition of Miltogramma and Pterella could (Szpila et al., 2017). reciprocally solve taxonomic problems for these and other gen- Pape (1996) agreed with earlier authors in considering the era of Miltogramminae, as both are currently defined in a very genera Metopodia, Hilarella Rondani and Taxigramma as form- broad morphological sense. ing a clade and argued for synonymizing Hilarella under Taxi- Our analysis does not recover the two representative species gramma. This hypothesis conflicted with Verves (1989b), who of Senotainia as forming a monophylum; instead these species had placed the monotypic Metopodia in Phyllotelini. We recover cluster separately with the genera Amobia and Oebalia,aresult Metopodia pilicornis Pandellé as sister to the genus Taxi- similar to that found by Piwczynski´ et al. (2017). As in the case gramma, in agreement with Pape’s (1996) hypothesis and with of Pterella, Pape (1996) highlighted the need for a more accurate Sanger-based molecular evidence (Piwczynski´ et al., 2017). morphological concept of Senotainia, which is also consistent Miltogramminae (sensu Pape, 1996) were recovered as with our tree. monophyletic with the inclusion of one species traditionally considered as part of Paramacronychiinae, the type species of the genus Sarcotachina. Interestingly, the morphology of Paramacronychiinae the male genitalia of this species is fully consistent with our observations for other miltogrammine species: phallotrema Paramacronychiinae form a strongly supported clade represented by a single opening (Figs 4A–C), acrophallus in (BP = 100), but excluding the type species of Sarcotachina apical position on the distiphallus (Figs 4B, C), juxta absent (Figs 1,2). Our phylogeny mirrors with high accuracy the phy- and epiphallus present (Figs 4B, C). We do not have data logenetic arrangement recovered by Piwczynski´ et al. (2017) for on the shape of the female accessory glands. Thus, based Paramacronychiinae. By contrast, our tree had few agreements on our phylogeny and morphological examinations, Sarco- with the morphology-based phylogenetic analysis published tachina subcylindrica is a member of Miltogramminae, as for this subfamily by Pape (1998). This contrast is possibly also argued by Piwczynski´ et al. (2017) from a Sanger-based explained by a conspicuous disparity in character sampling molecular phylogeny and supported by larval morphology and the differences in taxon sampling. Also, the low phyloge- (Szpila, 2010). netic resolution recovered by Pape (1998) allows only limited

© 2019 The Authors. Systematic Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society. 45, 281–301 AHE challenges classification of Sarcophagidae 297 comparisons with our results. The main agreement between the rise to questions regarding the homology hypotheses support- classification of Verves (1990) and our phylogeny is the close ing such estimations. Explaining the origin of convergence in relationship between Sarcophila Rondani, Wohlfahrtia Brauer the main Sarcophagidae lineages and obtaining a new genomic & Bergenstamm and Oophagomyia Rohdendorf, which form dataset with a taxon sampling adequately populated at the genus a clade (Fig. 1), thus closely matching subtribe Wohlfahrtiina level for a well-supported phylogeny are the two most prominent of Verves (1990), except that this author also included Asiosar- challenges that should be addressed in future studies. cophila Rohdendorf & Verves and Wohlfahrtiodes Villeneuve, taxa not included in our analysis. Supporting Information

Sarcophaginae Additional supporting information may be found online in the Supporting Information section at the end of the article. The species of this lineage were arranged into several Table S1. Voucherinformation on specimens used for molec- clades, which are fully consistent with the so-called ‘lower’ ular study. sarcophagines or the ‘nondemarcated juxta grade’, here represented by Oxysarcodexia and Ravinia, and the ‘higher’ sarcophagines or ‘demarcated juxta grade’ with the remaining genera (Figs 1,2), as proposed by Buenaventura & Pape (2018). Acknowledgements Disregarding the differences in taxon sampling, the generic topology recovered here closely matches the morphology-based EB wishes to thank the team of the Laboratory of Imaging of phylogeny by Buenaventura & Pape (2018) for Sarcophagi- the Smithsonian National Museum of Natural History, espe- nae. These phylogenies have the genera Oxysarcodexia and cially Mr Scott Whittaker, SEM Laboratory Manager, for support during the imaging work. We thank the 1KITE Antlio- Ravinia as part of some of the early divergences within the phora group (http://1kite.org/subprojects.html), Lars Posdiad- subfamily, while Blaesoxipha emerges as sister to a large lowski, Alexander Donath, Xin Zhou and Shanlin Liu for clade containing Engelimyia, Lipoptilocnema, Peckia and providing unpublished dipteran transcriptome data for during Sarcophaga. However, the morphological data recovered bait design. Moreover, we acknowledge Lars Posdiadlowski, a clade (Peckia + (Lipoptilocnema + Sarcophaga)) (Bue- Alexander Donath, Christoph Mayer, Malte Petersen, Daniela naventura & Pape, 2018), while we reconstructed a clade Bartel, Sabrina Simon, Alexandros Vasikopolous and Karen (Peckia + (Lipoptilocnema + Engelimyia)) as sister to Sar- Meusemann for their advice during orthology assignment and cophaga. Previous phylogenetic studies using DNA sequences data analyses, and KM and AD for reading and commenting on and including Lipoptilocnema also recovered this genus as an early version of the manuscript. We thank Keith Bayless for more closely related to Peckia than to Sarcophaga (Zhang et al., providing advice on data contamination. We thank Steve Mar- 2016a 2016b; Buenaventura & Pape, 2017). The position of shall for giving us permission to use his photographs. KS was Engelimyia has been uncertain in previous studies, with almost supported by a project of the Polish National Science Centre a different hypothetical sister group in each phylogenetic analy- (2015/17/B/NZ8/02453). BKC and BMW were supported by sis, including as sister to Boettcheria Parker (Kutty et al., 2010; the Schlinger Foundation and the US National Science Foun- Piwczynski´ et al., 2014), Villegasia Dodge (Buenaventura & dation (DEB-1754376). The authors declare that the research Pape, 2015) or Tulaeopoda Townsend (Buenaventura & Pape, was conducted in the absence of any commercial or financial 2018). As none of these possible sister taxa were included in relationships that could be construed as a potential conflict of the present analysis, the phylogenetic position of Engelimyia is interest. still obscure.

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