Revision and morphological analysis of the Ragadidae ( Insecta , Diptera )

Several taxonomic groups within Empidoidea Latreille, 1809 have been subject to unclear phylogenetic assignments along with multiple parallel hypotheses causing difficulties in classification and morphological identification. This study reviews the internal classification of the Ragadidae and includes a diagnosis and description of all included subfamilies and genera based on the results of an analysis of morphological characters using maximum parsimony. Illustration of important characters and a key to all genera in the family is given. The genus Hormopeza Zetterstedt, 1838 is found to be most closely related to Anthepiscopus Becker, 1891 and Iteaphila Zetterstedt, 1838, and the subfamily Iteaphilinae Wahlberg & Johanson, 2018 is therefore expanded to also include that genus. Hormopeza is consequently excluded from Ragadinae Sinclair, 2016. This study provides diagnoses, descriptions and keys in a contribution to a thorough classification of the empidoid groups and increased ease in morphological recognition.

In a recent analysis (Wahlberg & Johanson 2018), the Ragadinae and the 'Iteaphila-group' were brought together within the family Ragadidae, and the 'Iteaphila-group' was included in the newly erected subfamily Iteaphilinae Wahlberg & Johanson, 2018.Supported by DNA sequence data, the Ragadidae were hypothesized to form the sister group to the Empididae.This recent change in taxonomic status and generic composition, however, left internal relationships unresolved.The objectives of this paper are to provide the results from an analysis of the internal relationships within the family, updated diagnostic characters to reflect the new classification, and determination keys to the included genera applicable to both males and females.

Material and methods
This study comprises all genera included in the Ragadidae and is based on material borrowed from the institutions listed under each taxon in the results.Each genus is represented by the type species, in most cases also the type material.For photographic purposes and detailed studies, non-types were applied to reduce the damage risk.Photographic equipment for dry material was a Nikon DS-Ri2 mounted on a motorized Nikon SMZ microscope.Automatic focus stacking was performed in Nikon NIS-Elements 5.10 connected to the camera.Dissected material was mounted in Euparal or glycerol and photographed using a Nikon D7100 mounted on a Leitz Orthoplan large field microscope, with manual focusing and subsequent stacking in Helicon Focus 6.8.0.Photos were edited and finalized in Adobe Photoshop CC 20.0.1.Pencil drawings were edited in Adobe Illustrator CC 23.0.1.A phylogenetic analysis based on 28 morphological characters, applied by Sinclair (1999Sinclair ( , 2016)), Sinclair & Cumming (2006) and Wahlberg & Johanson (2018) with the addition of new characters (Table 1), was executed in PAUP* ver.4.0a164 (Swofford 2003).The most parsimonious trees were found using the heuristic search option with random stepwise addition with 1000 replicates and TBR branch swapping, MulTrees enabled, and 1000 bootstrap replications.All characters were unordered and unweighted, and the character matrix is presented in Table 2.The species of the Dolichopodidae Latreille, 1809 Dolichopus ungulatus Linnaeus, 1758, and two species of the Empididae, Trichopeza longicornis Meigen, 1822 and Clinocera nigra Meigen, 1804, were selected as outgroups to root the character transformations.Characters were mapped and visualized on the resulting tree in MacClade 4 (Maddison & Maddison 2001), and the tree was annotated in Adobe Illustrator CC 23.0.1.The terminology applied for morphological details follows Cumming et al. (1995) for the male terminalia, McAlpine (1981) for the morphology of adult and wing, and Stuckenberg (1999) for the antennae.In the case of the second anterior branch of the cubitus in the wing (traditionally referred to as CuA2), the more recent interpretation is followed (Cumming & Wood 2017).In this case the CuA2 is now referred to as CuA.The dorsal and ventral projections of the epandrium is interpreted as dorsal and ventral surstyli, respectively.

Institutional abbreviations
The following depository institutions are mentioned in the text below, following Evenhuis (2019).

Phylogeny
The strict consensus tree based on a total of 14 trees (Fig. 1) from the maximum parsimony analysis (tree length = 54, consistency index = 0.611, retention index = 0.632, and rescaled consistency index = 0.386) showed that the Ragadidae form a monophyletic unit in relation to the outgroup taxa.Anthepiscopus and Iteaphila together form a monophyletic sister group to Hormopeza.The close relationship between Hormopeza and the Iteaphilinae is supported by unique characters in the male terminalia; tergite 8 is broad and rectangular (character 23:2) and has lost the differentiated ventral surstylus (character 25:1).The phylogenetic relationships between the remaining genera are presently unresolved.

Diagnosis
The Ragadidae are separated from the Dolichopodidae and the Hybotidae Meigen, 1820 by the presence of unrotated and symmetrical male genitalia, and with a point of origin of R S at a distance from crossvein h being equal to, or longer than, crossvein h.The family is distinguished from the Atelestidae Hennig, 1970 in having a circumambient costa.It is separated from the Empididae by a prosternum that is separated from the proepisternum (except in species of Hydropeza).The representatives of Hydropeza are distinguished by the presence of a recurved labrum, which is straight in the Empididae.

Comments
The diagnostic characters showed below are based on the results of Wahlberg & Johanson (2018), reflecting the expanded concept of including the Iteaphilinae in the family.The Ragadidae are recorded from all the biogeographic regions except Antarctica, and fossil records in Baltic amber suggest this group to be at least 40 million years old (Sinclair 1999).Subfamily Ragadinae Sinclair, 2016 Figs 2A-C, 3, 4A, 9A-D

Diagnosis
The species of the subfamily Ragadidae are distinguished from those of the Iteaphilinae by the length of the postpedicel and the shape of tergite 8 in males (see the description below and the key above for details).The stout and spine-like setae on fore coxa are synapomorphies for this group.Its included species can be further distinguished by the presence of a recurved labrum in both males and females (in the Iteaphilinae, the labrum is recurved only in the females of Hormopeza).

Comments
The first formal description of this group included the genera Dipsomyia, Hormopeza, Hydropeza, Ragas, and Zanclotus (Sinclair 2016).In the analysis herein, it is found that this subfamily, excluding Hormopeza, can be distinguished only by a few diagnostic characters.

Diagnosis
The species in the genus Ragas are recognized by having spine like setae on the postgena and on the inner ventral margin of the fore trochanter, and lack costal bristles.These character stages are shared with species in the genus Zanclotus, from which they can be distinguished by having bare instead of pubescent eyes, and straight instead of recurved CuA.

Distribution
Holarctic, with records from North America through Europe and Russia to East Asia (Sinclair & Saigusa 2001).

Diagnosis
Species in the genus Dipsomyia are distinguished from those in Ragas and Zanclotus by the lack of stout setae on postgena and the fore trochanter.The lack of pubescence on the eyes, the setose laterotergite and the isolated prosternum separates the species of Dipsomyia from Hydropeza.

Distribution
The only known species in this genus is endemic to Chile.

Diagnosis
The species in the genus Hydropeza resemble those of Dipsomyia by lacking the stout setae on the postgena, and the fore trochanter that is present in species in the genera Ragas and Zanclotus.Species in Hydropeza are distinguished from those in Dipsomyia by having pubescent eyes, a bare laterotergite, and a prosternum that is fused with the proepisternum and forming a precoxal bridge.

Distribution
This genus is known from Chile, west Australia and New Zealand (Sinclair 2016).

Diagnosis
The species in the genus Zanclotus are most similar to those in the genus Ragas, particularly by the presence of spine-like setae on postgena, and the inner margin of the fore trochanter.The species are distinguished from the species of Ragas by the pubescent eyes and recurved CuA in the wings.A unique character of species of Zanclotus is the presence of a tubercle on the coxa of each fore leg.

Distribution
Only two species are known, both from Washington State, USA.

Diagnosis
The species in this subfamily are recognized by the broad and rectangular tergite 8 in the males.All species lack spine like setae on fore coxa, in contrast to species in the subfamily Ragadinae, and the labrum is straight, except for females in the genus Hormopeza.

Diagnosis
The synapomorphies of this genus and Anthepiscopus are the loss of epipharyngal blades and the tergite 10 fused with the cercus in females.Both males and females possess a straight labrum, in contrast to Hormopeza spp., where only males possess a straight labrum.Further similarities with species in Anthepiscopus are the setose scape, the loss of costal bristle and the recurved CuA in the wings (except for 'Iteaphila setosa-group' for the latter two characters).The species in the genus are distinguished from those of Anthepiscopus by having a branched R 4+5 .

Distribution
The genus Iteaphila is the most widely spread group within the Ragadidae, with representatives in the Nearctic, Palaearctic and Oriental regions (Shamshev & Sinclair 2009;Sinclair & Shamshev 2012).Sinclair & Saigusa (2001) transferred Ragas setosa (Bezzi 1924) to the genus Iteaphila based on the presence of a straight labrum, the absence of spine like setae on fore coxa and postgena, the palpus projection and the shape of the male terminalia.Shamshev & Sinclair (2009) assigned this species together with several other from the southern Europe, the northern Africa and the Middle East to the 'Iteaphila setosa-group'.This group, however, possesses species with numerous peculiar characters such as both ventral and dorsal surstyli, costal bristle, antennal shape and modification in male hind femur, and its affinity to other species in Iteaphila is unclear.For the analysis herein, the characters present in the type species were used and the above listed characters treated as uniquely or possibly secondarily derived in the group.Further examination of morphological characters such as mouthparts, as well as DNA data is needed to clarify the phylogenetic position of this group.

Diagnosis
The species of this genus share synapomorphies and similarities with the species of the genus Iteaphila, which also separate these two genera from Hormopeza.These characteristics include the lack of epipharyngal blades, the straight labrum in both sexes, the setose scape, the loss of costal bristle in the wings, the recurved CuA, and the tergite 10 fused to the cercus in females.The species of this genus are distinguished from those of Iteaphila by the presence of an unbranched R 4+5 .

Description
Eyes bare; males holoptic.Scape with setae; postpedicel tapering, at least three times as long as wide; stylus cylindrical.Labrum straight in both sexes; epipharyngal blades and ventroapical comb absent.Postgena bare or with setae, not spine like.Prosternum separated from proepisternum forming an isolated sclerite; laterotergite bare.Stout spine like setae on anterior face of fore coxa and on inner ventral margin of fore trochanter absent; tubercle on fore coxa absent; male fore tarsal claws present.In wings, costal bristle absent; subcosta reaching costa; R 4+5 unbranched; CuA recurved (Fig. 9F).Female: tergite 10 fused to cercus.Male: postgonites as fused processes from hypandrium; apex of phallus simple.

Distribution
This genus is represented in the Nearctic and west Palearctic.In addition, one species is recorded from Australia.

Diagnosis
The species of the genus Hormopeza are separated from the species of Iteaphila and Anthepiscopus by the presence of epipharyngal blades and costal bristle, the scape is bare and CuA straight.Males lack claws on the fore legs, and females have a recurved labrum a tergite 10 separate from to cercus.

Discussion
When Chvála described the subfamily Oreogetoninae (Chvála 1976), he expressed a probable close relationship between the genera Anthepiscopus and Iteaphila and the genera Hormopeza and Ragas.However, more recent authors considered the groups of Iteaphila and Ragas only as distant relatives with the 'Iteaphila-group' as incertae sedis within the Empidoidea, and the 'Ragas-group' as incertae sedis within the Empididae (Sinclair & Cumming 2006), or later as a subfamily within the Empididae (Sinclair 2016).In Wahlberg & Johanson (2018), the close relationship between Anthepiscopus, Iteaphila and Ragas was re-established, and the family Ragadidae was established based on the molecular distances separating this group and the Empididae.The present study further explores this concept, and the analysis of morphological data indicates a distinct clade including Iteaphila, Anthepiscopus and Hormopeza separate from the remaining Ragadidae.The synapomorphy of this group including Hormopeza in the 'Ragas-group', according to Sinclair (1999) and Sinclair & Cumming (2006), is a membranous distiphallus.However, this character may vary in different degree of sclerotization and form, even within empidoid genera (e.g., in Hesperemis Melander, 1906, see Cumming et al. 2013).The recurved labrum is found to be a homoplastic character in the phylogenetic analysis in the present study.The genus Hormopeza is therefore transferred from this subfamily.The diagnosis and description of the Iteaphilinae and the Ragadinae are updated to reflect the results herein, based on labrum shape, chaetotaxy of fore coxa, and characters in male genitalia.The monophyly of the genus Iteaphila in relation to Anthepiscopus is dubious (Sinclair & Shamshev 2012), which was also indicated in Walberg & Johanson (2018).Further molecular studies including a broader taxon sampling are required to resolve this relationship, as well as the monophyly in regard to the 'Iteaphila setosagroup'.The morphological similarities reviewed herein between Ragas and Zanclotus is coherent with the results in Sinclair (1999).The spine like setae on fore coxa and postgena, and the fore coxal tubercle in Zanclotus, are hypothesized to be morphological adaptations to catch and handle prey.The presence of epipharyngal blades strengthens the notion of a predatory lifestyle as these may be used in cutting prey tissue (Bletchly 1954;McAlpine 1981).Species in the genus Hormopeza lack the spine-like setae on for coxa present in species of the Ragadinae, and species in Anthepiscopus and Iteaphila also lack epipharyngal blades.Iteaphila has been observed feeding on flowers (Shamshev & Sinclair 2009), especially in early spring and may be an early pollinator.Many of the species of the Ragadidae are rarely collected, and there have been few observations of feeding and mating behaviour.A few genera remain unplaced, e.g., Oreogeton within the Empidoidea.Further studies, preferably combinations of molecular and morphological methods, are warranted to properly assign Oreogeton and the other unplaced genera to formal taxonomic groups.Steps towards accessible and thorough classification, not only in this group, but also within other groups within the Empidoidea, together with up-to-date determination keys may, hopefully, lead to more attention to these flies and their roles in ecological systems.
) branch of the radial vein R 4+5 = branch 4 plus 5 of the radial vein h = humeral crossvein Museum Wien, Vienna, Austria MZLU = Lund University, Lund, Sweden NZAC = New Zealand Arthropod Collection, Landcare Research, Auckland, New Zealand USNM = National Museum of Natural History, Smithsonian Institution, Washington DC., USA

Fig. 1 .Fig. 2 .
Fig. 1.The strict consensus tree from maximum parsimony analysis with characters mapped.Numbers to the right of character symbols and character numbers show bootstrap support values.

Table 1 .
Characters used in the analysis.

Table 2 .
Matrix of scored morphological characters used in analysis.