The genus Dasydorylas Skevington in Iran, with the description of two new species (Diptera: Pipunculidae)

The genus Dasydorylas Skevington, 2001 is recorded from two provinces in Iran (Sistan-o Baluchestan and Kermanshah Provinces). Dasydorylas derafshani sp. nov. and D. zardouei sp. nov. are illustrated and characterized morphologically and by DNA barcoding of the mitochondrial COI gene. Eudorylas antennalis Kapoor, Grewal & Sharma, 1987 is transferred to Dasydorlyas (comb. nov.). An existing identification key to the males of the western Palaearctic species of Dasydorylas is complemented to include the newly described species.


Introduction
To date, more than 1400 species of Pipunculidae or big-headed flies are recognized, placed into four subfamilies and 20 genera (Rafael & Skevington 2010;Kehlmaier et al. 2014).However, the diversity is still considered to be poorly known and it is estimated that well over 2000 species exist on a world scale (Skevington & Yeates 2001).Similar to Syrphidae, adults are frequently seen hovering among vegetation.Male and female pipunculids primarily feed on honeydew, a sugar-rich secretion produced by many Hemiptera, but most of the energy necessary for reproduction is accumulated during the larval stage of the fly (Kehlmaier 2015).Pipunculid larvae develop as endoparasitoids in Tipulidae (Nephrocerus Zetterstedt, 1838) and in various families of Auchenorrhyncha (all other Pipunculidae), but information on host specificity is limited (Skevington & Marshall 1997;Koenig & Young 2007;Kehlmaier & Floren 2010).Females deposit a single egg into the host during flight (Williams 1918;Huq 1985;Rafael & Skevington 2010).Together with Dryinidae (Hymenoptera) and Strepsiptera, Pipunculidae are considered the most important parasites of Auchenorrhyncha (Freytag 1985).As many species of Auchenorrhyncha are known to transmit plant diseases, Pipunculidae may have the potential to become biological control agents of economically important pest species of leafhoppers.
The genus Dasydoryas Skevington, 2001 was first described in a comprehensive phylogenetic study of world Eudorylini by Skevington & Yeates (2001).Currently, the genus includes 30 species, occurring in the Afrotropical (nine species), Palaearctic (seven species), Australasian and/or Oriental (11 species) and in the Nearctic and/or Neotropic region (three species) (Skevington & Yeates 2001;Kehlmaier 2005aKehlmaier , 2005b;;Földvári 2013).Being placed within the diverse Eudorylini, the genus can best be separated from other genera by a combination of characters such as the funnel-shaped ejaculatory apodeme, a rather strongly tapering flagellum and a well-developed apical hair fringe on the scutellum (Skevington & Yeates 2001).Kehlmaier (2005aKehlmaier ( , 2005b) ) revised part of the Palaearctic and Oriental faunas, whereas Földvári (2013) revised the Afrotropical members of the genus.
The Iranian fauna of pipunculid flies is poorly known.In previous studies, only 19 species were recorded.Becker (1913) described two new species from Sistan-o Baluchestan Province; Gharali et al. (2008) cited two species from Ilam Province; Kehlmaier & Majnon Jahromi (2015) listed 15 species from Alborz Province; Motamedinia et al. (2017) described two new species from Sistan-o Baluchestan and Southern Khorasan Provinces.Due to a wide range of habitat diversity, favouring the existence of more than 390 leafhopper species in Iran (Mozaffarian & Wilson 2016), it is estimated that well over 100 species of pipunculid flies should be present in the country.Collecting material of Dasydorylas from the Eastern (Sistan-o Baluchestan) and Western (Kermanshah) Provinces of Iran revealed two new species of this genus that are described below.

Material and methods
Specimens were collected by Malaise traps and by hand netting in the eastern and western provinces of Iran (Sistan-o Baluchestan and Kermanshah).The collecting sites are characterized by a warm and dry (Sistan-o Baluchestan Province) or a humid and temperate mountain climate (Kermanshah Province).The traps were situated among herbaceous plants between rows of a Tamarix plantation (Tamarix aphylla (L.) Karst.)(Sistan-o Baluchestan) and between oak trees (Quercus brantii Lindl.and Q. infectoria Oliv.) (Kermanshah) (Fig. 1A-B).Malaise traps were emptied every 14 days.Pipunculids collected by hand net were captured using an aspirator and dropped into 75% ethanol.After sorting, a series of voucher specimens was preserved in 100% ethanol and kept in a refrigerator for DNA extraction.Other specimens were dried using the AXA-method according to van Achterberg (2009).The examined material is deposited in the Hayk Mirzayans Insect Museum, Insect Taxonomy Research Department, Iranian Research Institute of Plant Protection, Tehran, Iran (HMIM) and the Senckenberg Natural History Collections Dresden, Museum of Zoology, Germany (SMTD).Male genitalia was separated from the abdomen, heated in lactic acid (85%) for 30 minutes and then placed in a drop of glycerin on a microscopic slide.Illustrations were prepared using an Olympus TM AX70 microscope and a Motic TM SMZ-168 stereo microscope equipped with a Moticam TM 480 digital imaging system.A series of ten images were merged using the image-stacking software ZereneStacker v. 1.04.Line drawings of genitalia were traced using the software Inkscape ® , based on digital photographs, and subsequently mounted in Adobe Photoshop CS3 ® .
The morphological terminology follows Skevington (2002) and Kehlmaier (2005a), with the following abbreviations being used throughout the paper:

LF:WF
= ratio of length of flagellum to its width LW:MWW = ratio of length of wing to maximum width of wing LS:LTC = ratio of length of pterostigma to length of third costal segment LTC:LFC = ratio of length of third costal segment to length of fourth costal segment LT35:W5 = ratio of length of tergites 3-5 to maximum width of tergite 5 WT5:LT5 = ratio of width of tergite 5 to its length T5R:T5L = ratio of length of right margin of tergite 5 to length of its left margin LT35:WS8 = ratio of length of tergites 3-5 to width of syntergosternite 8 LS8:HS8 = ratio of length syntergosternite 8 to its height MLE:MWE = ratio of maximum length of epandrium to its maximum width (viewed dorsally) LP:LB = ratio of length of piercer to length of base (viewed laterally) LDP:LPP = ratio of length of distal part of piercer to length of its proximal part (viewed laterally) For DNA barcoding, a 658 bp fragment of the 5' end of the mitochondrial coding gene cytochrome oxidase subunit I (COI) was sequenced, using the primer pair LCO1490 and HCO2198 (Folmer et al. 1994).Laboratory procedures are outlined in Kehlmaier et al. (2012).Sequence accession numbers issued by the European Nucleotide Archive (ENA) are provided for each species.Specimens sampled for molecular analysis received an additional label stating an individual voucher number "DNA voucher CKxxx".Uncorrected pairwise genetic distances (p-distance) were computed with MEGA7 (Kumar et al. 2016) using all sites, transitions and transversions with uniform rates and pairwise deletion for missing data.

Differential diagnosis
Dasydorylas derafshani sp.nov. is closely related to D. setosus (Becker, 1908), redescribed by Kehlmaier (2005a) and known from the Canary Islands, Morocco and Spain, and to D. gradus Kehlmaier, 2005, described from Israel by Kehlmaier (2005b).The males of all three species have long spines at the apex of the phallic guide that differ in number and direction between species, with D. derafshani sp.nov.having eight downward directed spines on either side.Additional diagnostic characters are the differently shaped surstyli and, compared to D. setosus, the shorter and more pilose scutellar hair fringe.

Etymology
This species is named in honour of Hossein Ali Derafshan, who collected the type series material.The surname is to be used as a noun in genitive case.head.Face dark, silver-gray pollinose.Scape dark, pedicel brown with a pair of short upper bristles and one short lower bristle; flagellum yellow, short tapering and gray pollinose (LF:WF = 2-2.2);arista dark, flattened, with thickened base.Eyes converging but not meeting and separated by less than diameter of frontal facets (Fig. 2B).Frons dark, silver-gray pollinose; vertex dark, lacking pollinosity; occiput dark, gray pollinose.thorax.Pleura, prescutum, scutum and scutellum dark but prescutum light yellow at lateral margin.Pleura gray pollinose.Postpronotal lobe pale, gray pollinose and with 3-5 postpronotal setae along upper margin.Prescutum and scutum gray pollinose, with two uniseriate dorsocentral rows of conspicuous setae and supra-alar setae.Scutellum gray pollinose, with an apical fringe of up to ten pale setae (up to 0.1 mm).Subscutellum gray pollinose.
halter.Length: 0.4 mm.Base dark, stem narrowly white and knob paler than base, somewhat gray pollinose.
legs.Coxae dark but yellow on apical margin, gray pollinose.Mid coxa with three brown anterior bristles.Trochanters light brown, partly gray pollinose.Femora dark, distinctly light brown at apex, gray pollinose.All femora bearing two rows of dark, smaller, peg-like anteroventral spines on apical one third.Tibiae light brown, distinctly dark on apical half, gray pollinose with three rows of brown setae on anterior and posterior sides, without apical spines.Hind tibia with a wrinkled indentation midanteriorly.Tarsi light brown and paler than apical half of tibiae, brown pollinose, with some black setae dorsally.Distitarsi brown.Pulvilli shorter than distitarsi.
genitalia.Genital capsule in dorsal view: epandrium dark but brownish near surstyli and tergite 5, gray pollinose and longer than wide (MLE:MWE = 1.5).Surstyli paler than epandrium, gray pollinose and symmetrical (Fig. 3E).Both with a broad and rectangular base and a pair of finger-like projections at apices which are bent towards each other by 45° (Fig. 3E).Genital capsule in ventral view: gonopods rather large and almost symmetrical (Fig. 3A); phallus trifid, narrow, with long and straight ejaculatory ducts (Fig. 3B); phallic guide of medium length, rather broad, on either side with eight downwards directed long spines at its apex (Fig. 3A).Genital capsule in lateral view: epandrium without projecting lobe on either side.Both surstyli distinctly convex and broad in basal two thirds, distally narrowed to form a pair of finger-like processes which are bent towards each other (Fig. 3F-G).Phallic guide broad, gently bent towards surstyli (Fig. 3C).Ejaculatory apodeme funnel-shaped (Fig. 3D).

Female
Unknown.

Distribution
Iran.

Differential diagnosis
Dasydorylas zardouei sp.nov. is closely related to the western Palaearctic D. holosericeus (Becker, 1897) and D. roseri (Becker, 1897), both redescribed by Kehlmaier (2005a), to the Afrotropical D. evanidus (Hardy, 1949), redescribed by Földvári (2013), to the Oriental D. orientalis (Koizumi, 1959), redescribed by Kapoor et al. (1987), and, judging from the original figures, also to D. antennalis (Kapoor et al., 1987) comb.nov.from southern India.Being morphologically hardly distinguishable from each other, the D. holosericeus species group is a good example of how challenging pipunculid taxonomy can be.The males of D. zardouei sp.nov.differ from those of the other species by a different sclerotization pattern of the gonopods and by the length of the phallus.The females can be separated by the shorter length of tergite 9 (piercer of ovipositor).

Etymology
The species is named in honour of Maryam Zardouei who collected the type series material.The surname is to be used as a noun in apposition.head.Face dark, silver-gray pollinose.Scape dark, pedicel brown with three short upper bristles and one short lower bristle; flagellum brown, short tapering and gray pollinose (LF:WF = 2.1-2.2);arista dark, with thickened base.Eyes meeting for twelve facets (Fig. 4B).Frons dark, silver-gray pollinose; vertex dark, lacking pollinosity, shining black; occiput dark, gray pollinose.thorax.Pleura, prescutum, scutum and scutellum dark, but prescutum light yellow at lateral margin.Pleura gray pollinose.Postpronotal lobe pale, gray pollinose and with 2-3 postpronotal setae along upper margin.Prescutum and scutum gray pollinose, with two uniseriate dorsocentral rows of setae and patches of supra-alar setae.Scutellum gray pollinose, with a fringe of up to six dark setae (up to 0.1 mm).Subscutellum gray pollinose, only in dorsocentral area with some brown pollinosity.
halter.Length: 0.5 mm.Base dark, stem narrowly white and knob yellow.Base and stem somewhat gray pollinose.legs.Coxae dark, gray pollinose.Front and mid coxae with two dark anterior bristles.Trochanters dark, partly gray pollinose.Femora dark, distinctly yellow at apex, gray pollinose.All femora bearing two rows of dark, smaller, peg-like anteroventral spines on apical one third.Tibiae dark, with basal third and sometimes also apices pale, gray pollinose, with three rows of setae on anterior and posterior side, without apical spines.Hind tibia with some weak wrinkles midanteriorly, bearing one or two stronger bristles.Tarsi brown and paler than tibiae, gray pollinose, with some brown setae dorsally.Distitarsi dark.Pulvilli longer than distitarsi.distally by 90°; base of right surstylus slightly wider than left surstylus (Fig. 5G).Genital capsule in ventral view: gonopods minute and symmetrical, with elongated regions of distinctly stronger sclerotization (Fig. 5A); phallus trifid, straight and short (almost reaches apex of surstyli), with a membranous nose at base (Fig. 5B); phallic guide narrow and straight, with two dorsolateral spines at the end of basal half on either side (Fig. 5C).Genital capsule in lateral view: epandrium without projecting lobe on either side.Both surstyli in basal half broad, in apical half narrowed to form a finger-like process which is bent towards the sternites by 90° (Fig. 5F-G).Phallic guide bow-like bent towards surstyli (Fig. 5C).Ejaculatory apodeme funnel-shaped (Fig. 5D).4C-D, 5H-I) Body length.3.2 mm (excluding antennae).

Female (Figs
head.Scape dark, with two upper bristles.Pedicel with three to four short upper bristles and a pair of short lower bristles.Flagellum paler than pedicel and long, tapering.LF:WF = 3.1.Eyes separated (Fig. 4D).Front facets enlarged (0.05 mm).Frons dark, lower half silver-gray pollinose, otherwise shining.Frons with a weak median keel, ending in a tubercle shortly before antenna.Occiput gray pollinose.
legs.Hind coxa paler and larger than fore and mid coxae, with 4-6 black or brownish bristles.Mid femur with two small ventral rows of dark peg-like spines on apical half.Tibiae light brown, distinctly darkened on apical half, without apical spines.Pulvilli longer than distitarsi.

Distribution
Iran.

DNA barcoding
Uncorrected pairwise genetic distances (p-distance) were calculated from partial mitochondrial COI based on a dataset comprising five out of six European species of Dasydorylas -D.filiformis Kehlmaier, 2005 (n = 1), D. holosericeus (n = 1), D. horridus (Becker, 1897) (n = 4), D. roseri (n = 4) and D. setosus (n = 1) -and will be published in a forthcoming larger project (Kehlmaier et al. in prep.).Based on this, Dasydorylas derafshani sp.nov.stands closest to D. setosus, differing by 11.2%.The third Iranian species of the genus, D. horridus, differs by 14.3% (LK391738), whereas D. zardouei sp.nov. is differentiated by 12.6%.The latter species stands closest to D. holosericeus/D.roseri, differing by a minimum interspecific p-distance of 6.8% and 11.6% respectively.From D. horridus it is distinguished by 11.7%.and other previously unnamed species (Motamedinia et al. 2017) is not very surprising, considering not only the limited amount of research carried out on big-headed flies in Iran, but also the geographic location of the Middle East in general and the large habitat diversity present in Iran in particular.Being situated at the "border triangle" of the Palaearctic, Afrotropical and Oriental zoogeographic regions, the Middle East and Iran act as a transition zone of these realms.An extensive and long-lasting survey would be essential in order to assess the true diversity of Pipunculidae and other taxa present in Iran.