A review of the genus Lordiphosa Basden in India, with descriptions of four new species from the Himalayan region (Diptera, Drosophilidae)

Abstract All Indian species of the genus Lordiphosa Basden are reviewed, with descriptions of four new species, L. curva Fartyal & Toda, sp. n. of the denticeps species group and L. ayarpathaensis Kandpal & Singh, sp. n., L. makaibarensis Pradhan & Chatterjee, sp. n. and L. srinagarensis Sati & Fartyal, sp. n. of the nigricolor species group. Two of the new species, L. ayarpathaensis and L. makaibarensis, were found visiting flowers of Hedychium spicatum and Datura suaveolens, respectively. This is the first record of flower visitation in Lordiphosa flies. In addition, L. parantillaria (Kumar & Gupta, 1990), syn. n. is synonymized with L. antillaria (Okada, 1984). Supplementary and revised descriptions for L. antillaria and L. neokurokawai (Singh & Gupta, 1981) and a key to all Indian species of Lordiphosa are provided.


Introduction
The genus Lordiphosa Basden is a moderately-sized genus of the family Drosophilidae, currently comprising 57 species (Brake and Bächli 2008). The taxonomy of this genus had once been confused by assignment of some species to the subgenera Sophophora Sturtevant (Kikkawa and Peng 1938, Okada 1956, 1966, 1974, 1977, Lee 1959, Takada and Okada 1960, Bock and Wheeler 1972, Hirtodrosophila Duda (Okada and Sasakawa 1956, Okada 1966, 1967, 1971, 1988, Singh and Gupta 1981, or Drosophila Fallén (Duda 1935) of the genus Drosophila. However, some revisional works (Laštovka and Máca 1978, Okada 1984 reclassified those species into the subgenus Lordiphosa of the genus Drosophila. Grimaldi (1990) elevated Lordiphosa to the generic rank according to morphological characters. Furthermore, Hu and Toda (2001) showed, by a cladistic analysis focusing on Lordiphosa, that the tenuicauda species group, initially included in Lordiphosa (Toda 1983, Hu et al. 1999, is remotely related to the Lordiphosa proper, and Hu and Toda (2002) transferred all species of the tenuicauda group to the revised genus Dichaetophora Duda. A molecular phylogenetic study by Gao et al. (2011) has revealed that Lordiphosa is the sister group to the Neotropical Sophophora consisting of the Drosophila saltans and D. willistoni species groups.
The genus Lordiphosa is distributed from the tropics of Oriental Region (Okada 1984, 1988 to the subarctics of Palearctic Region (Toda et al. 1996, Bächli et al. 2004, with the highest species richness in the subtropics of Oriental Region (Okada 1966, 1984, Zhang 1993a, b, Zhang and Liang 1992, 1994, Quan and Zhang 2001, 2003. However, this genus has been poorly represented in India: only seven species have been recorded (Dwivedi and Gupta 1980, Singh and Gupta 1981, Kumar and Gupta 1990, Gupta and Gupta 1991, De and Gupta 1996, Gupta 2005, Upadhyay and Singh 2007. Four new species of Lordiphosa have been discovered from India. Two of them were found visiting flowers of Hedychium spicatum Smith (Zingiberaceae) in Kumaon, Uttarakhand and of Datura suaveolens (Humb. & Bonpl. exWilld.) Bercht. & J. Presl (Solanaceae) in Darjeeling, West Bengal. Until now, Lordiphosa flies were known for breeding on herbage plants, and their larvae feeding on decayed tissues of leaves and stems (Kimura et al. 1977, Shorrocks 1982, Toda et al. 1984 or living tissue of leaves as leaf minors (Okada and Sasakawa 1956). This paper describes the four new species, and reviews all known Indian species of Lordiphosa with supplementary and revised descriptions for some species, and provides a key to all Indian species of Lordiphosa.

Materials and methods
Specimens used for the present study were collected from four different hill stations of the Himalayan region in India: Chopta (2,700 m a.s.l.; 30°29'N, 79°10'E) in Rudraprayag district; Ayarpatha (2,278 m a.s.l.; 29°23'N, 79°27'E) in Nainital district; Kurseong subdivision of Darjeeling hills (1,458 m a.s.l.; 26°53'N, 88°17'E) in West Bengal; and HNBGU Forestry nursery (560 m a.s.l.; 30°13'N, 78°47'E) at Chauras Srinagar Garhwal in Uttarakhand. These localities are covered with dense mixed-deciduous subtropical forests, under extremely moist condition due to heavy rainfall during the summer monsoon season. The temperature ranges approximately from 3°C to 24°C. Specimens were collected by net sweeping or directly from flowers of Hedychium spicatum and Datura suaveolens by an aspirator, and preserved in 70% ethanol. In addition, some specimens collected from China were examined to give supplementary and revised descriptions for some known species.
External morphology of adult flies was examined under a stereomicroscope and metric characters were measured with an ocular micrometer. To observe detailed structures, the male and female terminalia and some other organs were detached from the body, cleared by warming in 10% KOH solution at approximately 100°C for several minutes, mounted in a droplet of glycerin on a cavity slide, examined under a light microscope, and some samples imaged using a DinoLite® Digital Eyepiece Camera.
The morphological terminology and the definition of measurements and indices mostly follow McAlpine (1981), Zhang and Toda (1992) and Hu and Toda (2001). All the holotypes and some paratypes of new species are deposited in the Department of Zoology, H.N.B Garhwal University, Chauras Campus, Srinagar-Garhwal, Uttarakhand, India (DZHNBGU), some paratypes in Museum of Zoological Survey of India, Kolkata, India (MZSIK) and the remaining paratypes in the Systematic Entomology, Hokkaido University Museum, Hokkaido University, Sapporo, Japan (SEHU).

Systematic accounts
Genus Lordiphosa Basden Lordiphosa Basden, 1961: 186 (as a subgenus of Drosophila); Laštovka and Máca 1978: 404;Okada 1984: 571. Type species: Drosophila fenestrarum Fallén, 1823. Lordiphosa: Grimaldi, 1990: 121 (new status as genus); De and Gupta 1996: 131;Bächli et al. 2004: 250. Diagnosis. Prementum thicker in ventral than in dorsal portion from lateral view ( Remarks. Okada (1967) proposed the denticeps group as a new species group of Hirtodrosophila (a subgenus of Drosophila at that time), including two species so far described, denticeps Sasakawa, 1956 andtripartita Okada, 1966. However, it had been noticed that these two species have aberrant morphological characters inconsistent with the definition of Hirtodrosophila. Three more species were subsequently added to this species group (Okada 1971, Singh andGupta 1981). Then Okada (1990) transferred the members of this species group to Lordiphosa (a subgenus of Drosophila at that time), but considered that the denticeps group was synonymous with the nigricolor group proposed by Laštovka and Máca (1978). Then, Grimaldi (1990) elevated Lordiphosa to the generic rank, and Zhang (1993b) resurrected and redefined the denticeps group as a species group independent from the nigricolor group in the genus Lordiphosa.
Description (supplementary and revised). Adult male. Head. Eye with interfacetal setulae. Approximately 15 supracervical setae thin, apically more or less curved and pointed; postocular setae approximately 18; occipital setae 30-31, including medial tiny ones. Dorsolateral arms of tentorial apodeme divergent, nearly straight, reaching to fronto-orbital plate; dorsomedial arm 1/3 as long as dorsolateral arm. Interspace between antennal sockets narrower than half of socket width; first flagellomere with only one small invaginated pouch ("sacculus" called by earlier taxonomists : Ferris 1965); arista with 4-5 dorsal and one ventral branches in addition to terminal fork. Facial carina slightly elevated, narrower and shorter than first flagellomere, without setulae below. Subvibrissal seta distinctly shorter than vibrissa; additional row of oral setulae present above marginal row on anterior portion. Palpus with one prominent terminal and several short, subapical to lateroventral setae, without setulae on basal lobe. Cibarium (Fig. 1A) thickened on anterior margin, not dilated laterad in anterior portion; anterolateral projections shorter than half width of anterior margin; dorsal sclerite pear-shaped in dorsal view, anteriorly convex in lateral view; anterior sensilla two pairs, widely arranged in square behind anterior margin of hypopharynx; 23-26 medial sensilla arranged in anteriorly slightly convergent rows; two sensilla campaniformia; posterior sensilla very long, trichoid, gently curved forward, approximately 17 arranged in anteriorly divergent rows; somewhat sclerotized, thickened (in lateral view), anterior portion of hypopharynx shorter than 1/5 length of cibarium. Prementum ventrally slightly expanded. Labellum with five pseudotracheae per side.
Thorax. Postpronotal lobe with two prominent setae. Posterior dorsocentral seta situated nearer to anterior margin of scutellum than to anterior dorsocentral seta. Prescutellar setae absent. One or a few acrostichal setulae in lines with and anterior to dorsocentral setae thicker and longer than others. Mid katepisternal seta shorter than anterior katepisternal seta; anterior katepisternal seta thicker than aristal branches; no setula present anteriorly to anterior katepisternal seta.
Legs. Foreleg tarsus with neither tuft of dense, soft hairs nor long setae. Foreleg tarsomere I as long as three succeeding tarsomeres together; midleg one slightly longer than three succeedings together; hindleg one slightly longer than rest together.
Remarks. This species was first described by Singh and Gupta (1981) based on three male specimens collected from Darjeeling, West Bengal, India. Later, Zhang (1993b) reported this species from southwestern China, based on some male and female specimens collected from Kunming, Yunnan, but did not describe the female characters. Here, the description of the female is provided, with a supplementary and revised description for male based on the specimens collected from southwestern China.  Description (not referring to characters commonly seen in the foregoing species, L. neokurokawai). Adult male. Head. Eye with dense, interfacetal setulae. Supracervical setae 16-18 ( Fig. 2A); postocular setae approximately 19; occipital setae 21-25. Dorsolateral arms of tentorial apodeme divergent, apically curved outward; dorsomedial arm half as long as dorsolateral arm ( Fig. 2A). Occiput orange yellow, medially dark brown; ocellar triangle and fronto-orbital plates glossy, orange yellow; frontal vittae mat, greyish orange. Pedicel greyish orange yellow; first flagellomere grey; arista with 3-4 dorsal and one ventral branches in addition to terminal fork. Face orange yellow. Gena orange yellow but dark brown on anteroventral margin. Clypeus orange brown. Palpus yellow. Cibarium: anterolateral projections longer than half width of anterior margin; medial sensilla 20-21; posterior sensilla approximately 20. Prementum ventrally slightly expanded, thicker in ventral than in dorsal portion from lateral view (Fig. 2B, C). Labellum with five pseudotracheae per side (Fig. 2B).

Lordiphosa curva
Thorax. Postpronotal lobe grey yellow, with two prominent setae: upper one 0.8 as long as lower one. Scutum and scutellum grey yellow but grey brown medially. Thoracic pleura grey yellow, with dark grey patches. Acrostichal setulae in six rows. Basal scutellar setae divergent; apicals cruciate.
Wing. C 1 setae two, subequal in size. Halter opaque white. Legs grey yellow; tarsomere Vs of all legs darker. Foreleg femur with 4-6 long setae in two rows on outer side. Fore-and mid-leg tarsomere Is longer than three succeeding tarsomeres together; hindleg one longer than rest together. Preapical, dorsal setae present on tibiae of all legs; apical setae on tibiae of fore-and mid-legs.
Remarks. This species closely resembles L. neokurokawai in having only one ventral branch of arista, the long, apically pointed paramere curved ventrad medially and outward apically, and the epandrium and the surstylus nearly identical in morphology, but can be clearly distinguished from it by the diagnostic characters.
Legs. Foreleg femur with approximately nine long setae in two rows on outer side; tarsus with neither tuft of dense, soft hairs on ventral side nor long setae.
Description. Adult male. Head. Eye dark red, with sparse, interfacetal setulae. Supracervical setae 15-18, thin, apically more or less curved and pointed; postocular setae 16-19; occipital setae approximately 18, including medial tiny ones. Dorsolateral arms of tentorial apodeme divergent, nearly straight, reaching to fronto-orbital plate; dorsomedial arm half as long as dorsolateral arm. Occiput, ocellar triangle and frontoorbital plates black; frontal vittae mat, light orange. Interspace between antennal sockets narrower than half of socket width; pedicel yellowish brown; first flagellomere grey, with only one small invaginated pouch; arista with 3-4 dorsal and two ventral branches in addition to terminal fork. Facial carina slightly elevated, narrower and shorter than first flagellomere, without setulae below. Gena and clypeus light brown. Subvibrissal seta distinctly shorter than vibrissa; additional row of oral setulae present above marginal row on anterior portion. Palpus with one prominent terminal and several short, subapical to lateroventral setae, without setulae on basal lobe. Cibarium thickened on anterior margin, not dilated laterad in anterior portion; anterolateral corners almost not projected; dorsal sclerite pear-shaped in dorsal view, anteriorly convex in lateral view; anterior sensilla two pairs, widely arranged in square behind anterior margin of hypopharynx; 32-33 medial sensilla arranged in mostly parallel but anteriorly convergent rows; sensilla campaniformia two; posterior sensilla very long, trichoid, gently curved forward, approximately 22, arranged in anteriorly divergent rows; somewhat sclerotized, thickened (in lateral view), anterior portion of hypopharynx shorter than 1/5 length of cibarium. Prementum slightly thicker in ventral than in dorsal portion from lateral view (Fig. 4A), nearly parallel-sided in posterior view (Fig. 4B). Labellum with five pseudotracheae per side (Fig. 4A).
Legs light brown; last two tarsomeres of all legs darker. Foreleg femur with approximately eight long setae in two rows on ventral and outer surfaces; tarsus without any sexual ornamentation. Foreleg tarsomere I as long as three succeeding tarsomeres together; mid-leg one slightly longer than three succeeding tarsomeres together; hindleg one slightly shorter than rest together. Preapical, dorsal setae present on tibiae of all legs; apical setae on tibiae of fore-and mid-legs.
Abdomen. Tergites I to IV medially, widely yellow, laterally brown; V and VI nearly entirely dark brown; each tergite with small setae in approximately three rows and large setae on posterior margin. Sternites light brown; setigerous VI present.
Measurements ( Etymology. Pertaining to type locality. Distribution. India (Uttarakhand). Flower visitation. Adult flies were collected from flowers of Hedychium spicatum (local name: Haldu, Kapur Kachri or Sand harlika; English common name: Spiked Ginger Lily; Fig. 5A), a smallish, hardy, perennial herb, belonging to the family Zingiberaceae, with fleshy rhizomes, green, broadly lanceolate leaves, straight stem (up to approximately 1 m high) and large orange and white flowers. It grows throughout subtropical Himalaya in the Indian states of Assam, Arunachal Pradesh and Uttarakhand, with an altitudinal range of 1,000 m to 3,000 m.
Thorax light brownish yellow. Posterior dorsocentral seta situated nearer to anterior dorsocentral seta than to anterior margin of scutellum.
Wing. Veins greyish yellow. Legs light brownish yellow. Foreleg femur with approximately five long setae in two rows on ventral and outer surfaces.
Abdomen. Tergites nearly entirely yellow; each tergite with small setae in approximately two rows and large setae on posterior margin. Sternites off-white.
Etymology. Partaining to "Makaibari tea estates". Makaibari was the first tea factory in the world, established in 1859, in Kurseong, Darjeeling, West Bengal.
Distribution. India (West Bengal). Flower visitation. Adult flies of this species were collected from flowers of Datura suaveolens (local name: Dhokrey; English common name: Angel trumpet or Devils trumpet; Fig. 5B, C), an exotic plant belonging to the Solanaceae. It was introduced from South America and is now found growing along riverbeds or forest edges at moist places almost all over India.
Remarks. This species closely resembles the foregoing species, L. ayarpathaensis, in having the large flap on caudosubapical margin of epandrium, the oviscapt medially broad and humped in lateral view and distally narrowing and curved ventrad, and the large, sclerotized perineal plate present between oviscapts, but can be distinguished from it by the smaller size and paler color of the body and the diagnostic characters.

Lordiphosa srinagarensis
Description. Adult male. Head. Eye red, with sparse interfacetal setulae (Fig. 7A). Occiput glossy, dark brown in dorsal half, grey yellow in ventral half. Supracervical setae 14-19, thin, apically more or less curved and pointed; postocular setae 14-16; occipital setae 12-13, including tiny medial ones. Dorsolateral arms of tentorial apodeme divergent, apically curved outwards, reaching to fronto-orbital plate; dorsomedial arm half as long as dorsolateral arm. Frons grey yellow except for dark brown upper portion of fronto-orbital plate and medial portion of ocellar triangle. Interspace between antennal sockets narrower than half of socket width; pedicel grey brown; first flagellomere grey, fringed with sparse, somewhat curved and long hairs on distal, outer margin, with only one small invaginated pouch; arista with 6-7 dorsal and 3-4 ventral branches in addition to terminal fork (Fig. 7A). Face grey-yellow; carina only slightly elevated, without setulae below (Fig. 7A). Gena grey-yellow, with dark brown, medial patch and ventral margin; subvibrissal seta distinctly shorter than vibrissa; additional row of oral setulae present above marginal row on anterior portion. Clypeus greybrown. Palpus dark grey, with one prominent terminal and several short subapical to lateromedian setae, without setulae on basal lobe. Cibarium thickened on anterior margin, not dilated laterad in anterior portion; anterolateral corners almost not projected; dorsal sclerite pear-shaped in dorsal view, anteriorly convex in lateral view; anterior sensilla two pairs, widely arranged in square behind anterior margin of hypopharynx; 24-29 medial sensilla arranged in anteriorly convergent rows; sensilla campaniformia two; posterior sensilla long, trichoid, nearly straight, approximately 27, arranged in nearly parallel rows; somewhat sclerotized, thickened (in lateral view) anterior portion of hypopharynx 1/4 as long as cibarium. Labellum with five pseudotracheae.
Legs grey yellow. Foreleg femur with approximately six long setae in two rows on outer side; tarsus without any sexual ornamentation. Foreleg tarsomere I as long as three succeeding tarsomeres together; mid-and hind-leg ones as long as rest together. Preapical, dorsal setae present on tibiae of all legs; apical setae on tibiae of fore-and mid-legs.
Abdomen. Tergites entirely glossy, brownish black, each with setae arranged in roughly four transverse rows: those in last row longest. Sternites pale to dark grey; setigerous VI present.
Measurements ( Remarks. This species closely resembles Lordiphosa penicilla (Zhang, 1993) from southwestern China in the morphology of male terminalia, but can be distinguished from it by the diagnostic characters: in L. penicilla, surstylus medially pubescent and with several stout setae on outer surface; sclerotized ventral branches of parameres symmetric in length; gonopod with single medial ridge in lateral view (Zhang 1993a: "Figs 4−6").

Discussion
In this study, it was found that L. neokurokawai has a special type of sex comb composed of thick setae of approximately 15 TBRs along the entire length of tarsomere I of male foreleg (Fig. 1B), which was overlooked in its original description by Singh and Gupta (1981). This finding is important for considering the evolution of sex comb in the genus Lordiphosa. The sex comb is a male-specific morphological structure composed of thickened setae ("teeth") that develops on the foreleg tarsus of adult male in the Drosophilidae. This male-specific character is seen only in Sophophora and Lordiphosa (Hu and Toda 2001), and is used variously in tactile interactions between males and females during courtship and mating behavior (Spieth 1952; see also Kopp 2011 for a review of sex comb functions). Likely in relation to its use as an important component of mating behavior, the sex comb varies in structure even between closely related species, implying that its rapid diversification would have been driven by sexual selection (Markow et al. 1996, Kopp 2011. Three major patterns are recognized in the sex comb structure: (i) "transverse" sex comb comprising TBR(s) of thickened setae on the distal portion of tarsomere; (ii) "oblique" one of row(s) more or less rotated and moderate in length on the distal portion of tarsomere; and (iii) "longitudinal" one aligned along the nearly entire length of tarsomere (Kopp andTrue 2002, Atallah et al. 2009). All four known species of the Lordiphosa miki species group have extended "longitudinal" sex combs of the last type (Laštovka and Máca 1978, Okada 1984, Kopp 2011. The phenotypically identical "longitudinal" sex combs are present in the melanogaster and obscura species groups of the subgenus Sophophora (Kopp 2011, Atallah et al. 2012, explaining why members of the miki group had once been assigned to the subgenus Sophophora (Kikkawa and Peng 1938, Okada 1956, Lee 1959, Bock and Wheeler 1972. Species of the L. denticeps group possess the "transverse" sex combs on the foreleg tarsomeres I to III (Kopp 2011, Atallah et al. 2012. However, probably because the sex comb teeth of the denticeps group are less prominent than those of Sophophora, this structure had been overlooked in earlier descriptions of denticeps-group species until Zhang (1993b) first recognized it. The two other species groups, i.e., fenestrarum and nigricolor ones, of Lordiphosa lack sex combs. Interestingly, the Neotropical Sophophora comprising the saltans and the willistoni groups, which is the sister clade of Lordiphosa (Gao et al. 2011), has no sex comb either. This character distribution pattern across Lordiphosa and Sophophora suggests two hypotheses for the evolution of sex comb. One is the "single-origin" hypothesis: the sex comb was acquired in the common ancestor of Lordiphosa and Sophophora, and secondarily lost in several lineages. The other is the "multiple-origin" hypothesis: the sex comb evolved independently on several lineages. To date, any approach from the phylogenetic analysis has not succeeded in distinguishing between these two hypotheses. Another possible way is to elucidate the real homology of similar phenotypes by studying the molecular processes underlying their development. Recent evo-devo studies have succeeded in revealing that similar phenotypic structures in sex comb result from different developmental mechanisms (Atallah et al. 2009, 2012, Tanaka et al. 2009, Kopp 2011. For instance, the "longitudinal" sex combs seen in the melanogaster and obscura groups develop, under similar regulation by the same key genes, through different cellular mechanisms. In some species, such as Drosophila rhopaloa Bock & Wheeler, 1972 of the melanogaster group and Drosophila guanche Monclus, 1976 of the obscura group, the "longitudinal" sex comb originates from one or a few distal, transverse rows of bristle-precursor cells that are homologous to those for female TBRs but subsequently rotate 90° and align to form a longitudinal row (Tanaka et al. 2009, Atallah et al. 2012. In species of the montium subgroup and Drosophila ficusphila Kikkawa & Peng, 1938 of the melanogaster group, however, the sex comb arises from male-specific precursor cells aligned in a longitudinal row on the presumptive region (Tanaka et al. 2009, Atallah et al. 2012. Furthermore, Atallah et al. (2012) found the third developmental mode of "longitudinal" sex comb in Lordiphosa magnipectinata (Okada, 1956) of the miki group: the sex comb development starts from the ancestral, sexually monomorphic arrangement of TBR precursor cells; then, most of such short, transverse rows of precursors rotate independently of each other and eventually assemble into a contiguous, longitudinal row. In relation to this developmental process of "longitudinal" sex comb in the miki group, the sex comb of L. neokurokawai of the denticeps group, which consists of multiple transverse combs arranged along the entire length of tarsomere I, may represent an intermediate stage, i.e., before rotation of TBRs, of the sex comb development in the miki group. Taken together these results support a common origin for sex combs in Lordiphosa.