SEM studies and discovery of an intriguing new Rhamphomyia (Pararhamphomyia) (Diptera, Empididae, Empidinae) species from the Kashmir Himalayas

Abstract Here we present an intriguing new species of the Empididae genus Rhamphomyia (Pararhamphomyia) from the Kashmir Himalayas. The new species, Rhamphomyia (Pararhamphomyia) aquila, has a distinctive appearance due to its highly deformed male hind legs with extremely shortened hind tibia, a feature very peculiar in the genus. The new species is described along with elements of mating behaviour, the mechanism of male hind leg articulation, and its possible role during mating. Also, scanning electron microscopy analysis is used to elucidate the general morphology and sensilla in both sexes of the new species. Details of the male and female internal reproductive system are described and illustrated. A preliminary checklist of 55 species of the Empididae of India is provided. http://www.zoobank.org/urn:lsid:zoobank.org:pub:A28B850F-6A2D-4FB2-9AC3-E89015C1C5A7 http://www.zoobank.org/urn:lsid:zoobank.org:act:C0249B1D-D0C8-4998-96A2-C25D3DB89810


Introduction
The genus Rhamphomyia Meigen, 1822 (Diptera: Empididae) is widespread across multiple zoogeographical regions (Saigusa 2012), and currently about 610 species are known worldwide (Rhodén & Wahlberg 2020). These are important pollinators in high mountains and in boreal environments (Barták & Kubík 2009); they are also important biocontrol agents and important bioindicators (Grootaert et al. 2000). So far most of the species have been reported from the Holarctic Region, with some unpublished estimates suggesting the richest diversity to be present in the Nearctic (Barták & Kubík 2009;Sinclair et al. 2019). Globally it is estimated that there may be as many as 1500 species of Rhamphomyia (Sinclair et al. 2019). The genus consists of several subgenera which helps provides a better overview of its diversity (Rhodén & Wahlberg 2020). The first attempt at subgeneric grouping was carried out by Frey (1909Frey ( , 1922, and recent papers by Barták (1981Barták ( , 1982, Chvála and Wagner (1989) and Saigusa (2012) remain very relevant in this regard. Currently, the genus consists of about 18 subgenera (Yang et al. 2007; Barták & Kubík 2009;Saigusa 2012), almost exclusively relevant for the Palaearctic fauna; some of those that

Materials, light and scanning electron microscopy
Specimens were collected by net sweeping and hand picking from the fruit orchards of the Central Institute of Temperate Horticulture (CITH) during the years 2015-2021. CITH is situated at 33. 59°N, 74.50°E, at an altitude of 1640 m a.s.l. (Figure 1). The field photographs were obtained using a Canon 80D DSLR fitted with a 100 mm macro lens. Taxonomic analyses were conducted using an Olympus SZX16 stereo zoom microscope. For digital images, ProgRes0 Capture Pro v. 2.8.0. evolution digital camera was used on the same microscope with Combine ZP-Montage software. Later, images were cleaned up with Adobe Photoshop CS6. Genitalia together with 2-3 pregenital segments were removed and macerated in potassium hydroxide solution (10%) in small vials, for 1-2 h, treated with 8% acetic acid and dissected in glycerine. For bright-field microscopy, prepared slides were placed on a stage prepared from cardboard and provided with an LED lamp (3 W) to produce the transmission light beam. For dark-field microscopy a dark-field illumination filter was used to illuminate unstained specimens. Microphotographs were captured with a digital mirrorless camera (Nikon Z50) with attached infinity-corrected microscope objectives (Lomo 3.7× and Nikon CFI Plan Achromat 10X). The details and description of the type locality and the illustration procedure are similar to those of Barták et al. (2021).
The morphological terms used here follow Sinclair and Cumming (2006). All the specimens are deposited in the Central Institute of Temperate Horticulture Insect Collection, Srinagar (CITH). Two paratypes will be deposited in the Natural History Museum, London, UK (NHMUK), four at the Czech University of Life Sciences Prague (CULSP) and two at the University of Silesia, Katowice (DZUS). For studies on leg articulation, the dissection procedure described by Földvári et al. (2019) was followed. Ethanol-stored specimens were bleached and rehydrated in 35% H 2 O 2 for 24 h and then transferred to anhydrous glycerol. Specimens were dissected in glycerol with fine forceps, insect pins and razor blades on concave slides. The straightening and bending movements of the leg were observed to understand 1326 S. A. Akbar et al. interaction between the proximal tibial flexor tendon and the ventral femoral wall. For this the bleached legs were moved with insect pins; these pins were also used to dislodge the locking sclerites, similarly to Földvári et al. (2019). The term "sclerite" was used for less flexible areas of the exoskeleton that are connected to each other by more flexible conjunctiva, as detailed in Klass and Matushkina (2012) and Ronquist and Nordlander (1989). All the procedural videos were taken with the use of a camera attached to the microscope and are provided as supplementary files. Specimens for SEM analyses were preserved in 70% ethanol for several days, processed as described by Barták et al. (2021)  Distribution. India (Jammu and Kashmir).
Legs. Black to dark brown, microtrichose, with black setae (Figures 2b-c, 3c-e). Fore femur with sparse rows of very short anteroventrals and somewhat longer posteroventrals (about half as long as femur depth), dorsal setae shorter. Fore tibia with very short setae, posteroventrals fine and about as long as tibia depth. Mid femur with anteroventral row of setae slightly shorter than femur depth and similar row of posteroventrals, shorter on proximal part, on distal part with several slightly stronger setae, dorsal setae short. Mid tibia with short and fine dorsal setae, only 1-2 antero-or posterodorsals slightly longer than tibia depth, ventral setae stronger, almost spine-like, arranged in two rows, subequal in length to tibia depth. Hind femur with row of strong preapical setae; strongly modified (Figures 2 b-c, 3c-d): swollen, with long ventral protuberance on basal third to half and strong ventral incision on apical third, on apical third with long ventral setae and comb of posteroventrals, dorsally with short setae. Hind tibia extremely shortened, roundly expanded at base, with dorsal knob, with several strong and long setae. Hind basitarsus extremely elongate, strongly constricted on basal third, much longer than tibia, covered with dense short slightly widened setae on both sides. Tarsi of both fore and mid legs narrow with short setae, with apical circlets of setae and long claws. Posteroapical comb of hind tibia with 1 long seta.
Female ( Figure 4). Similar to male, but body setae much shorter and lighter; legs simple and unremarkable, without deformed hind femorotibial joint. Eyes dichoptic with all facets almost equal in size (Figure 4a). Frons approximately 0.14-0.18 mm, broad with 4-5 black, rather long setae on sides. Labrum black, labella with rather long setae. Thoracic chaetotaxy: acrostichals and dorsocentrals black, dorsocentrals distinctly longer. Both fore and mid femora with short setae, longest (distal) posteroventrals shorter than femur depth. Both fore and mid tibiae very short, without prominent setae. Hind femur short, anteroventral setae and prominent posteroventrals, shorter than femur depth. Antero-and posterodorsal setae of hind tibia as long as tibia diameter (Figure 4b-c). Length: body 3-3.3 mm; wing 3.1-3.4 mm. Female and male internal genital tract ( Figure 5). Internal reproductive tract of female comprises paired ovaries, lateral oviducts, common oviduct, long tubular vagina, spermatheca, spermathecal duct and two accessory glands (Figure 5a-c). Vagina appears as elongate muscular tube lined internally with thin cuticle; anteriorly fused with common oviduct. Spermathecal duct and ducts of accessory glands open anterolaterally and posterolaterally, respectively, into vagina. Ovaries broad leaf-like with four follicular cell layers, with youngest oocytes near apical germarium and most mature ones near basal region. Eggs empty into lateral oviducts, unite into common oviduct. Spermatheca with broad basal stalk, extending into elongate duct and terminating as round reservoir for sperm mass collection and storage (Figure 5d). Accessory gland appears as small bisected delicate sacs, with thicker basal sac compared with thinner apical sac.
Internal reproductive tract of male comprises paired testes, vas deferens, pair of accessory glands and common ejaculatory duct (Figure 5e-f). Each testis bulb-shaped, comprising testicular follicles enclosed in common outer sheath. Vas deferens appears as long tube-like structure, without enlarged and differentiated region distinguished as seminal vesicle. Tubular accessory gland ducts along with vas deferens open into anterior portion of ejaculatory duct. Ejaculatory duct short without distinct folds.
Etymology. The species epithet is Latin for eagle and appropriately symbolises the aerial grabbling skills   The male is generally similar to the female, except for the normal sexual dimorphism. Variations in general morphology, eyes and terminalia can be observed (see Figure 6).

Head, antennae and mouthparts.
In females, the ommatidia are tightly and regularly deployed (20-25 μm), uniform without dimorphism between the upper and the lower halves ( Figure 7a). The palpi are long, cylindrical, rarely strongly compressed laterally (Figure 7b), bearing 3-4 long setae, in addition to fine microtrichia throughout ( Figure 7c). Labium with dense cover of microtrichia and several thick, long chaetic sensilla along the dorsal side of the labellum (Figure 7d). Labrum is long. The stylet is covered by a wax layer, with pointed epipharyngeal blades (Figure 7d, e). The antennae are rather short. Scape, pedicel, postpedicel and stylus are covered by numerous fine sclerotic microtrichia and several types of sensilla especially on the ventral side of the postpedicel (Figure 7f). Most of the basiconic sensilla (most probably type I) are located along the ventral side of the postpedicel, placed regularly, lengthwise (Figure 7g, h). They are 5-9 μm long, sometimes covered by a wax layer. Some sensilla are characterised by a multiporous surface (Figure 7i, j), whereas others have rather grooved surfaces (Figure 7k, l). In males, the ommatidia of the compound eye exhibits distinct dimorphism with a larger upper half and smaller lower half, tightly and regular deployed (Figure 8a, b). The distinct pseudotracheae channels and ground surface of the labellum provide capillary action (Figure 8c, d), whereas the proximal part of the labellum is characterised by a very granular surface (Figure 8e). The labrum has an array of rasps and teeth on the apical part of the epipharyngeal blades, used for piercing (Figure 8f). The antennae are similar to those of the female, and most of the sensilla are located on the ventral side of the postpedicel, a larger segment having an expanded and convex ventral basal part (Figure 8g). The postpedicel is densely covered by prominent microtrichia and numerous, regularly arranged basiconic sensilla (Figure 8h). The basiconic sensilla are located in depressions and characterised by a wide and rounded basal part, and they are mostly without a porous surface (Figure 8i, k). On the cuticle of the postpedicel some small pores of sensilla ampullacea are also apparent (Figure 8i,  j). The stylus has a dense covering of microtrichia with the most apical part bare (Figure 8l). The sensillum surface of the apical part of the stylus seems to be smooth but at higher magnification it is clear that the cuticle is covered by numerous longitudinal pores (Figure 8m-o). The basal portion of the male postpedicel latero-ventrally consists of numerous regularly placed sensilla coeloconica, located in deep depressions and surrounded by a large ring-like cuticular collar (Figure 9a-c). The sensilla are visibly tapered with a wider basal part and a more pointed distal part (Figure 9d, e). On the other hand, the lateral side of the postpedicel bears more sensilla coeloconica characterised as a raised peg with terminal bulb-like tip and 6-7 well-visible projections (Figure 9f-i).

Wings.
Wings in males and females are similar, with a distinct wax layer and a microtrichose wing membrane, regularly deployed, fine, slightly pointed and slightly ribbed. The wing edges and wing articulation bear chaetic sensilla, long and ribbed with basal groove raised ( Figure 10). The halter is covered by numerous microtrichia with robust scabellum, with well-visible sensilla plates, pedicellus and oval capitellum (Figures 11a, b). Sensilla on the dorsal scabellum and dorsal pedicellus distinct (Figure 11c), scabellus with densely  Figure 11f). The capitellum is characterised by a single trichoid and campaniform sensillum along the ventral side and single prominent trichoid sensillum along the dorsal in addition to the general dense microtrichia (Figure 11g, k).

Legs.
Legs in males and females are densely covered by numerous short, fine, pointed microtrichia and mechanoreceptors with longer trichoid sensilla mainly on the coxae. In males, the hind femur is strongly modified and swollen and the hind tibia is extremely reduced (Figure 12a, b). The hind femur is characterised also by a prominent ventral protuberance on the basal third with a ventral incision on the apical third having long and stout trichoid and chaetic sensilla in the form of a comb, posteroventrals pointed and grooved (Figure 12c-g). On the basal inner side of the femora, a single campaniform sensillum of about 5.5-6.5 μm in diameter can be found (Figure 12h). The sensillum is characterised by a protuberant inner part with a small but visible pore (Figure 12i, j). The hind basitarsus is extremely elongate, having larger numbers of grooved chaetic sensilla (Figure 12k, l).The tarsomeres have large chaetic sensilla, while distal parts bear more trichoid sensilla (Figure 12m, n). The claws are long, curved, with proximal halves covered by pointed microtrichia and distal halves curved and ribbed, ventral side of the pulvilli with regularly arranged tenent setae with flat capitate terminal plates (Figure 12o-r). cerci (Figure 13f). The terminal part of the male abdomen is covered by dense, very long, rigid and pointed chaetic sensilla (Figure 13g-k). The cercus and epandrium have numerous microtrichia, and trichoid and long chaetic sensilla (Figure 13i, k). The basal part of the phallus is smooth, broad, enlarged, and covered by waxy secretions, and the apex is swollen, bulb-shaped (Figure 13j-l).

Remarks
Among the known Indian species belonging to the genus Rhamphomyia, the new species appears distinct with its highly modified male hind legs. In addition to the distinct morphological differences between the new species and another recently discovered Indian species (Rhamphomyia bhagati Barták, Akbar, Kanturski, Wachkoo & Maqbool, 2021), there is also a difference in the male internal reproductive tract. The testis is more or less rounded with a basal neck leading into the vas deferens in R. bhagati, compared with an oval testis leading into the vas deferens without having a distinct neck in the new species. Although the male hind leg does appear to be modified very similarly to the R. basalis group, the male terminalia, especially the cercus shape and absence of a subepandrial lobe, do not match the basalis group. The cercus is more similar to those of the pusilla group (sensu Saigusa; see also Sinclair et al. (2019)). Moreover, the females of the new species are without pennate setae on the legs and without infuscated wings, both characteristic features of the basalis group (Sinclair & Saigusa 2018). From the members of the pusilla group, the new species differs in not having darkened halteres, midlegs without modified tibiae and tarsomere and phallus not long and very fine. Rhamphomyia merzi Bartak, 2000 from Kazakhstan, a species similar to the basalis group on the basis of male terminalia, also has the hind femora deformed, but asymmetrical; only the right femur is strongly swollen while the left hind femur is only slightly swollen (Barták 2000), and also not to the extent reported here. Male hind leg articulation mechanism and involved components (Figure 14). We present here perhaps the first finding of extremely modified male hind legs in Empidinae; the only remotely similar finding comes from Daugeron et al. (2010) regarding the occurrence of highly modified hind legs in Empis subgenus Enoplempis (see Sinclair et al. 2013).
Modified hind femora occur in males of some species, e.g. R. merzi. The modified basal part of the tibia is a common feature among the basalis group and many others. A highly thickened or elongated basitarsus of the hind leg is also often reported in the genus. However, a distinctly modified and shortened hind tibia, as reported here, has never been reported previously. The hind basitarsus which is greatly elongated in fact takes over a function of the normal tibia, which is of peculiar occurrence. The enlarged tibial flexor apodeme, presence of robust teeth on the ventral surface, and thickened femur having an enlarged tibial flexor muscle mass, indicates its usage in grasping/holding; this is confirmed by our behavioural observations in the field. The dark-field microphotographs of the femorotibial joint in the male hind leg also reveal a unique locking mechanism. It allows the leg to remain in a flexed position for an extended period of time; this is perhaps an efficient energy-saving feature useful during mating and for prolonged holding of the female. The occurrence of similar structural features and their diverse implicational usage in Figure 12. Scanning electron microscopy of Rhamphomyia aquila sp. nov. legs. (a-b) Swollen male hind femur and extremely reduced hind tibia; (c-g) long and stout trichoid and chaetic sensilla on apical third of femur, posteroventrals; (h-j) single campaniform sensillum on the basal inner side of femora (arrow) with small pore near the centre (star); (k-l) elongate basitarsus with large grooved chaetic sensilla; (m-n) trichoid and chaetic sensilla on third tarsomere; (o-q) curved claws and pulvilli (r) fine structure of tenent setae and terminal plates.
insect legs are detailed by Földvári et al. (2019). Of the three stated universal locking mechanisms in insect legs, the flexor sclerite lump-engaging mechanism (Type 1) explains best the femorotibial joint articulation of the new species. The locking apparatus consists of an enlarged tibial flexor muscle, relatively reduced tibial extensor muscles, tibial flexor sclerite (TFS), genuflexor apodeme (GFS) and Heitler's lump (HL) (Figure 14a-f). The lock is present between the tibial flexor sclerite and Heitler's lump. In the locked state (Figure 14g), the convex transparent ventral surface of the tibial flexor sclerite remains in physical contact with Heitler's lump ( Figure 14i); thereafter the leg can only be opened after forcing the tibial flexor sclerite over and in front of Heitler's lump (Figure 14h-j).
Our laboratory observations and the field observations lead us to postulate that the apparent male hind leg deformation is a secondary sexual character employed to exhibit territoriality and for attracting females. We have also observed the presence of a single campaniform sensillum on the proximal part of the hind leg femur, the presence of which is also supportive of enhanced muscle synergies in substrate grip, similar to that discussed in other studies (Zill et al. 2017).  (Figure 15)

Courtship rituals
The courtship behaviour of the new species illustrated here in brief is somewhat similar to that discussed for a recently described sympatric species in Barták et al. (2021). However, capturing photographic evidence of dance fly behaviour is challenging, as these are fast-moving insects and difficult to focus on, and the interpretation of intriguing aerial images is at times quite difficult. As in the new species of Barták et al. (2021), and some of the other previously reported species (Downes 1970), males raid and prey on small insects and form aerial swarms to display their catch to nearby females in "aerial holding flights". Such mating swarms often use visual environmental markers and always appear at those certain stationary positions. Once the female accepts the gift and commits to mating, the tangled couple moves at a slower pace and drops down towards the nearby vegetation, where they remain until copulation is completed. Descriptive analysis of observed courtship characters (such as lek height, copulation duration, etc.) mainly depend on the environment and are more or less the same as stated in Barták et al. (2021). Some significant differences do occur among the two species, however. The new species exhibits prey polymorphism, preying on a number of insect species such as psyllids, small flies, etc. Prey polymorphism was not reported in R. bhagati, which preyed only on a specific chironomid species. In both species chironomids are mostly preferred, but the new species preys on relatively larger chironomid species compared with R. bhagati which preyed on a smaller and different chironomid species. The new species is more frequent in occurrence compared with the other species, exhibits strong territoriality and often displays offensive aerial postures (Figure 15a-c). The modified hind legs are displayed and used as specialised structures for holding females in male bias mating swarms (Figure 15d-e). At places with similar lekking sites, the males of the two species are observed to encounter each other. The males of the new species chase off males of the other species during such encounters and retain the area (Figures 15f-h). The distinct trait morphology of the new species is certainly associated with mating success and prolonged copula duration (Figure 15im). We are currently quantifying more precisely the role of legs as specialised structures in aerial display signalling, and their selective advantage. The deformed legs have given a selective advantage to the species and have favoured their predominance in the region.

Discussion
Males have a much larger potential benefit from engaging in copulation (Parker 1979). Selection pressure leads males towards mating, while females shy away. This pressure on occasion leads to the development of male-specific morphological characters for coercing unwilling females to mate (Bergsten 2005). We observed that the deformed male hind legs are used to take hold of the female abdomen for a proper genital union and to detain an initially wilting female until the ejaculate is transferred (Figure 15i-m). In future research we will generate predictions based on these functions and test whether these predictions are consistent with observations. Among possible male phenotypes the degree of leg deformation should covary with the duration of copulation and vice versa. Our discovery adds to the list of already apparently deceptive traits in empidid flies and suggests them as a potential model to evaluate inconclusive empirical evidence about sexual selection and secondary sexual traits as drivers for speciation.
The external as well as the internal genital tract in Diptera is generally poorly known, with only a few detailed comparative studies available (Sinclair et al. 2007;Puniamoorthy et al. 2010). The same is true in the family Empididae, where apart from Smith (1969) nothing much can be cited. In addition to the external male and female genital tract we herewith have detailed the internal genital system, which perhaps represents the first such detail for the genus as well. Previously works of Loew (1841), Dufour (1851), Keuchenius (1913) and Smith (1969) are rather brief without labels or proper descriptions. The spermatheca is here illustrated in greater detail for the family Empididae for the first time, although it was previously reported and illustrated for other insect orders and various families within the order Diptera (Pascini & Martins 2017). Whether the structure in this species aids post-copulatory sperm competition remains a fascinating topic to be explored. Not only do the details of the internal reproductive tract have taxonomic significance, but its functional advantages vary among species. It is postulated that the length of the sperm duct between the sperm pump and opening roughly corresponds to the length of the male phallus (Barták, unpublished data). By clasping copulating pairs quickly by hand, and subsequently dissecting them, it can be observed that the whole length of the phallus, even if it is very long (as in Rhamphomyia (Holoclera) nigripennis group), is inserted into the female.

Checklist of Indian Empididae
An updated checklist of the family Empididae (Diptera) of India is presented. It includes 55 species belonging to 13 genera. The checklist is primarily based on the available literature with all the names of described species being presented in accordance with the most recent classification. Notes about type localities, depositories, and relevant references to each species record are given. The list provides a synthesis of the regional taxonomical work carried out to date and will serve as a baseline for future studies. Within the Indian Empididae, the genus Chelifera is the most species rich, represented by 10 species, followed by Clinocera (nine), Empis (eight), Hemerodromia (six) and Rhamphomyia (four). The genera Heleodromia, Hilara, and Roederiodes are each represented by three species, while Trichoclinocera and Chelipoda are each represented by two species and the genera Trichopeza and Wiedemannia are each represented by a single species. For the genus Rhamphomyia we had previously stated the presence of four species -R. bhagati Barták, Akbar, Kanturski, Wachkoo and Maqbool, 2021, R. griseonigra Brunetti, 1913, R. himalayana Brunetti, 1913and R. unifasciata Brunetti, 1913 -from the region (Barták et al. 2021). However, R. unifasciata is now treated as Ocydromia unifasciata (Brunetti, 1913), a member of Hybotidae, and is therefore excluded from the list. As such, the genus in India is represented by three previously described species only. The new species described here represents the fourth species belonging to the genus Rhamphomyia to be reported from India. The classification of Empidoidea suggested by Wahlberg and Johanson (2018) is followed here. According to their study, the family Empididae globally comprises 3200 known species, and more than 80 genera, in three subfamilies (Brachystomatinae, Clinocerinae and Empidinae).