Diversity, distribution and biology of Romanian flat-footed flies (Diptera, Opetiidae and Platypezidae) with taxonomic notes on Callomyia saibhira Chandler

Abstract Altogether 18 species of the families Opetiidae and Platypezidae are reported from Romania, based on newly studied material and previously published records. The following three species are recorded from Romania for the first time: Agathomyia vernalis Shatalkin, 1981, Callomyia saibhira Chandler, 1976, and Lindneromyia hungarica Chandler, 2001. The presented differential diagnosis and a detailed redescription of body and genitalia of the male of Callomyia saibhira are based on one specimen which is the first male found in Europe. Information about distribution and biology of all 18 Romanian species is provided as well as photographs of selected important species. Finally, a new checklist of all Romanian species is given.


Introduction
The Opetiidae and Platypezidae are basal cyclorrhaphous families of Diptera, belonging to the superfamily Platypezoidea. The European species are small brachycerous flies, ranging from 1.4 to 6.0 mm in wing length. Their coloration is often black (males) or composed of black, orange and grey (females), and some species have silvery grey reflective patterns. The males have larger heads with holoptic eyes, while the female eyes are dichoptic. The larvae may be flat or cylindrical. All known larvae are mycophagous and feed by burrowing in the tissue of fungus fruiting bodies, at the surface of the gills of gill fungi, or on fungal mycelia under bark of dead trees; one species, Agathomyia wankowiczii (Schnabl, 1884), is gall-forming on sporocarps of a polypore. Adults of European species may be observed running rapidly on broad leaves in forested habitats; females may be observed ovipositing on host fungi.
These two families of flat-footed flies include 44 species in 13 genera in Europe (Chandler 2001(Chandler , 2004. Current literature (Chandler 2001(Chandler , 2004 lists only six species from Romania. However, during the preparation of this paper we have noticed that some localities of Platypezidae given by Thalhammer (1899) and Szilády (1941) are in fact in the present territory of Romania. Unfortunately, the material of Thalhammer (1899) and Szilády (1941) was destroyed by fire in 1956 (Papp 2001), but we consider their determinations to be correct and thus their records are included in the present paper. The monograph of the European species by Chandler (2001) summarizes all known (up to year 2000) data on adult and larval morphology, biology, distribution, systematics, including keys to the species. The nomenclature and classification used here therefore follow Chandler (2001).

Material and methods
Specimens were examined with an Olympus SZX10 binocular microscope. Photographs were taken by Canon 600D and/or 60D with MPE-65 macro lens and in some cases combined from multiple layers using Helicon Focus Pro 5.2. Drawings and photographs were edited in CorelDRAW 12 and Corel PHOTO-PAINT 12 graphic software. Morphological terminology follows Cumming and Wood (2009) and Chandler (2001), terminology of male genitalia follows Chandler (2001), Chandler and Shatalkin (1998), and is supplemented in parentheses by terminology adopted from Cumming and Wood (2009). The material examined is now deposited in the Silesian Museum, Opava, Czech Republic (SMOC, all specimens collected by J. Roháček) and the National Museum, Praha, Czech Republic (NMPC, remaining specimens).
The following abbreviations are used in the text: I-XII -January to December, BMNH -The Natural History Museum, London, United Kingdom, JR -Jindřich Roháček, ER -European Russia, FE -Far East of Russia, MT -Michal Tkoč, NMPC -National Museum, Praha, Czech Republic, SMNS -Staatliches Museum für Naturkunde, Stuttgart, Germany, SMOC -Silesian Museum, Opava, Czech Republic. The species with asterisk (*) in front of their names represent new records for Romania. The translations of original localities from Romanian are in square brackets [ ], together with names of the respective county and historical region.
Biology. The adults run on broad leaves in wooded biotopes, where they sometimes form swarms. Its larvae are unknown. The adults were reared from very rotten beech wood and leaf litter (Speight et al. 1990, Chandler 2001) but the exact development substrate of the larvae remains unknown and thus these records need confirmation. The males can sometimes be caught by light trap (Chandler 2001), while the females can be collected by pitfall traps (Vaňhara 1986). The adults (mostly males) can be collected by sweeping undergrowth of various forests, sweeping on Atropa belladonna leaves proved to be particularly productive (pers. obs.). The species is bivoltine, adult flight period in central Europe is V-VI and VIII-X.
Biology. Most common species of the genus in the Palaearctic Region. The adults of both sexes are often found running on Petasites sp. leaves (pers. obs.). Larvae develop in Bjerkandera adusta (Ševčík 2010). Adult flight period is in IV-IX.
Biology. Very rare species with early flight period. The individuals are collected only in IV and V and were almost exclusively females. Larval biology and host fungus are unknown. Tkoč and Barták (2013) recorded this species in numbers from a pyramidal (emergence) trap baited with dead wood.  Distribution. Palaearctic species. Recorded in the Czech Republic, Denmark, Finland, France, Germany, Great Britain, Hungary, Ireland, Lithuania, Montenegro, the Netherlands, Norway, Poland, Romania, Slovakia, Sweden, Switzerland and Russia (ER, FE).

Agathomyia viduella (Zetterstedt, 1838)
Biology. Uncommon species with adults occurring in undergrowth and along brooks in humid deciduous and mixed forests (Roháček and Ševčík 2009) and are often observed running on Petasites sp. leaves together with A. antennata (pers. obs.). The main flight period of adults ranges from V to VII. Host fungus is unknown.   Distribution. Palaearctic species. Recorded in Austria, Belgium, the Czech Republic, Denmark, Finland, France, Germany, Great Britain, Hungary, Ireland, Italy, Lithuania, the Netherlands, Norway, Poland, Slovakia, Spain, Sweden, Switzerland, Romania and Russia (ER).

Callomyia amoena Meigen, 1824
Biology. Common species, the larvae live on mycelia under bark of fallen trunks of various trees. A record from mycelia on bark on the underside of aspen trunks lying on the ground was mentioned by Krivosheina (2008). Flight period of adults ranges from V to X.    (Figure 17), 300 m a.s.l., 44°42'12"N, 21°43'52"E, sweeping vegetation along brook, JR leg. Differential diagnosis. Male of C. saibhira differs from C. amoena and C. elegans by having darker halteres that are not orange (brown with knob black). C. speciosa have longer arista and shorter first flagellomere and upper part of pleura is not so silvery grey dusted as in C. saibhira. From its most similar species, C. dives Zetterstedt, 1838, it differs by a clear wing membrane, brown palpus and different genitalia, basal lobe of gonopod is shorter (Figure 10). Females have unique abdominal coloration: tergites 1-4 (T1-4) are orange yellow with narrow brown hind margins on T2-4, only T5 is black, T6 and terminal segments are silvery grey dusted. Head black with silvery grey dusting. Antenna dark brown, scapus with dorsal seta reaching to tip of pedicel, pedicel with one strong dorsal seta reaching to the middle of first flagellomere, both sides of pedicel with 3 short setae, 1 short seta on ventral position. First flagellomere conical, twice as long as pedicel. Second and third flagellomere long. Arista half of antennal length. Two pairs of small frontal setae. Ocellar tubercle dull brown, with one pair of ocellar setae and one pair of small postocellar setae. Postocular setae long, their apices visible in anterior view. Face and parafacial bare, silvery grey dusted. Gena, occiput and postgena with long black setae. Occiput black, silvery grey dusted. Palpi brown with short black setae, proboscis brown with pale pubescence.
Wing hyaline with brown to dark brown veins. Subcostal cell (sc) yellow tinted and with microtrichia. Wing surface not uniformly covered with microtrichia, microtrichia present on anal lobe, posterior and distal part of wing. First longitudinal vein (R 1 ) bearing 9-10 spines. Anterior (r-m) and posterior (dm-cu) crossveins present. Costal cell (c) equal to sc in length. Posterior crossvein (dm-cu) twice as long as distal part of the fifth longitudinal vein (CuA 1 ). Anal cell (cup) elongated, its length about three times portion of anal vein (A 1 +CuA 2 ) beyond it.
Legs slender, brown, slightly silvery shiny. All coxae silvery dusted with black setae, yellow distally. Fore femur with longer fine ventral setae distally. Fore femur with 1 oxhorn seta. Apices of femora and basal parts of tibiae (="knees") yellow. Fore tibia with 1 anteroventral spur. Fore tarsomeres I−II yellow. Mid tibia bearing short dorsal seta above middle (anterodorsal seta absent) and two long ventral apical spurs. Hind femur of the same width as hind tibia. Hind tarsomere I with ventral seta above middle.
Abdomen black with silver-grey coloured markings. Setae on abdomen fine and black. Tergites 1 and 2 (T1+2) more setulose than the others, T3+T4 sparsely setulose. T1 black, its anterior half shiny silvery grey in lateral view. T2 black with silvery grey marking on posteroventral area. In lateral view this marking occupies posterior third of T2. T3 black, with similar (but smaller) marking, mainly on ventral part. T4 black, with silvery grey marking on posteroventral area occupying posterior two thirds in lateral view. T5 black. T6 black with posterior border grey. T7 small, entirely grey. Sternite 8 also grey, without setae. Genitalia ( Figure 10) with epandrium grey, cercus brownish, surstylus and hypandrium shiny amber-brown. Paramere (postgonite) slightly curved dorsally with base narrower, its broader apical part slightly tapered towards the rounded apex. Aedeagus (phallus) broad in lateral view, its dorsal apex with sharp tooth anteriorly, bluntly rounded posteriorly. Hypandrium with small inner hypandrial lobe (ihl) sub-basal to gonopod. Gonopod (hypandrial lobe) trifid, with shorter basal gonopodal lobe (bgl) and longer terminal part deeply bifid forming two slender apical lobes. Surstylus with basal part narrower than its apical rounded part, the latter with a digitiform dorsal process. Terminal lobe of epandrium gradually tapered and slightly curved, covered by setulae and with 2 longer setae. Ventral part of epandrium with 5 prominent setae two smaller setae and two additional smaller setae positioned more ventrally. Cercus covered by microtrichia and with short curly setulae on apex.
Female. Not studied, for description see Chandler (1976Chandler ( , 2001. Distribution. Palaearctic species. Hitherto recorded only from Bulgaria (Chandler 1976) and the Far East of Russia (Shatalkin 1985). New record for Romania.
Biology. Unknown. The larvae of other European Callomyia species develop on mycelia under bark of various trees (see above under C. amoena). The adult male examined was swept from vegetation close to a cave along a small brook (Figure 17). The known flight period in Europe is in VI.
Comments. This is the second specimen and first male of the species from Europe. Other known material (♂♂ and ♀♀) was collected in the Far East of Russia, Amur region (Shatalkin 1985). It is similar to Callomyia dives in the morphology of males, but the silver coloration on the thorax and abdomen is less developed. Also, the basal lobe of the gonopod (bgl) is shorter (Figure 10). This species seems to have an inland distribution, whereas C. dives is found mostly on islands or close to coastal zones of Europe. Chandler (2001) figured genitalia of an Amur specimen collected by Shatalkin. However, the genitalia of our specimen do not entirely fit into the description and figure of Chandler (2001). The main differences appear to be as follows (see Figure 10): presence of inner hypandrial lobe (ihl) positioned sub-basally to gonopod (this character is present in more Callomyia species, but usually omitted in the descriptions and figures in the literature); paramere (postgonite) is wider in lateral view, curved dorsally, with narrow basal part; morphology and position of basal gonopodal lobe (bgl) is very similar to that of Chandler (2001), but the bifurcation of terminal part of gonopod is more profound; the apical part of surstylus is less rounded and its basal part is only slightly narrower than apical part; cerci have shorter curly setulae.  Distribution. Palaearctic species. Recorded in Andorra, Austria, Belgium, the Czech Republic, Denmark, Finland, France, Germany, Great Britain, Greece, Hungary, Italy, Israel, Lithuania, the Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey, Russia (ER) and Caucasus.

Callomyia speciosa Meigen, 1824
Biology. Less common than C. amoena but widely distributed throughout Europe. The larvae also live on mycelia under bark of various trees (reared from mycelium on surface of a fallen hazel trunk by Krivosheina (2008)). Adults fly in V-IX.
Biology. Rare species associated with Pluteus sp. The adults were reared from Pluteus cervinus several times by the first author (not published). Adult flight period ranges from VIII to X.   Distribution. Palaearctic species. Recorded in Andorra, Austria, Belgium, Cyprus, the Czech Republic, Denmark, France, Germany, Great Britain, Greece, Hungary, Italy, Israel, Morocco, the Netherlands, Poland, Portugal, Romania, Slovakia, Spain, Switzerland, Turkey and Russia (ER).

*Lindneromyia hungarica Chandler, 2001
Distribution. Palaearctic species. Recorded in Austria, the Czech Republic, France, Germany, Great Britain, Hungary, Portugal, Slovakia, Spain and Switzerland. New record for Romania.
Biology. The species was only recently separated from L. dorsalis and their larvae can develop together with those of L. dorsalis in the same fruiting body of an Agaricus sp. (Chandler 2001, Tkoč andVaňhara 2008). Occasionally, the species can be caught on sporocarps of other fungi, e.g. Roháček and Ševčík (2013) collected one female on Meripilus giganteus in a park of Opava city (Czech Republic). Adults can be found in V-X.

Family Opetiidae
Opetia nigra Meigen, 1830 Agathomyia setipes Oldenberg, 1916 Agathomyia vernalis Shatalkin, 1981 Agathomyia viduella (Zetterstedt, 1838) Callomyia amoena Meigen, 1824 Callomyia elegans Meigen, 1804 Callomyia saibhira Chandler, 1976 Callomyia speciosa Meigen, 1824 Subfamily Platypezinae Seri obscuripennis (Oldenberg, 1916) Bolopus furcatus (Fallén, 1826) Polyporivora ornata (Meigen, 1838) Paraplatypeza atra (Meigen, 1804) Paraplatypeza bicincta (Szilády, 1941) Lindneromyia dorsalis (Meigen, 1804) Lindneromyia hungarica Chandler, 2001 Discussion Altogether 18 species of the families Opetiidae and Platypezidae are reported from Romania, representing 40.9 % of all flat-footed flies known from Europe. This number is far from the total number of species in this country and more research on flatfooted flies is needed to understand their distribution in Romania and Europe as a whole. Comparing the species number of the family Opetiidae and Platypezidae from Romania with the countries related to the Carphathian-Pannonian region it is an average number of species: Austria has 17 species (Chandler 2004), the Czech Republic 34 (Vaňhara 2009), Hungary 27 (Papp 2001, Weele 2001, Chandler 2004), Poland 25 (Chandler 2004), Slovakia 34 (Vaňhara 2009); the flat-footed fly fauna of Croatia, Serbia, Slovenia and Ukraine has not been systematically studied, thus the numbers are very low and not useful for comparison. The discovery of the first male of Callomyia saibhira in Romania is the most important result of this study. The genitalia figured herein do not entirely agree with the description and figure of Chandler (2001). The differences in male genitalia between the Romanian and Amur specimen highlighted above may be the result of simple variation, or could be caused by comparing our specimen (Figure 10) with the somewhat simplified illustration of Chandler (2001) or may point to a more complicated taxonomic problem. To resolve this question, additional fresh material from both Europe and the Far East of Russia is needed in order to study variability in the morphology of the male genitalia and to test for differences using molecular methods. It also needs to be determined, if the males assigned to this species are correctly associated with the females, which appears to be a problem in the Nearctic species of Callomyia (Chandler 2001;Tkoč 2012).