Taxonomic status and additional description of White’s Stalked-eyed Fly Cyrtodiopsis whitei (Curran, 1936) (Diptera: Diopsidae) from India with a key to the allied species and note on its habitat

Systematics and ecology of Cyrtodiopsis whitei (Curran, 1936), initially described in brief from a tropical dry deciduous forest in eastern India as Diopsis whitei, remained obscure for want of the type specimens. Recent collections of male and female specimens of this species from a new locality in the northeastern part of India, the country of the type locality, has allowed a fresh appraisal of its morphology, taxonomic position and habitat ecology in the background of several studies done on Diopsidae. Herein are included some new characters, hitherto unknown in the species of Cyrtodiopsis, a taxonomic key to the separation of four species, considered monophyletic under the genus, and a note on the habitat of the species.


INTRODUCTION
Worldwide, Stalked-eyed Flies of the family Diopsidae Bilberg, 1820 are known by 189 species from 12 genera (Roskov et al. 2015).These include reports of eight species in five genera from India (Mitra et al. 2014).Baker et al. (2001), Meier & Baker (2002), and Földvári et al. (2007) considered species of Teleopsis Rondani, 1875 and Cyrtodiopsis Frey, 1928 to be congeneric based on molecular affinity provided by partial nucleotides alignments of three mitochondrial and three nuclear genes.Feijen (2011), however, disputed the single clade phylogenetic hypothesis by Baker et al. (2001) and preferred Teleopsis and Cyrtodiopsis to be paraphyletic and that is being widely followed in diopsid taxonomy and biology till date (Roskov et al. 2015).
The original description of Cyrtodiopsis whitei under the genus Diopsis by Curran from India in 1936 had inadequate morphometrical details and illustrations of diagnostic characters.The whereabouts of the type specimens (male holotype and a female allotype) collected on 2 May 1935 and originally deposited in the American Museum of Natural History (New York) is uncertain, and Shillito (1940) based his study of the species on a single specimen by the identical name collected on 21 October 1920 from a location in 'Jungle of Assam', northeastern India.Földvári et al. (2007), based on laboratory culture specimens from Malaysia, provided another brief description of the species without sufficient illustration of diagnostic characters, and without reference to the original description provided by Curran (1936).It is, therefore, doubtful if the Malaysian specimens are really C. whitei.Extensive uses of laboratory-reared C. whitei in behavioral (Lorch et al. 1993;Wilkinson et al. 1998;Al-Khairulla et al. 2003), physiological (Burkhardt & de la Motte 1983;Buschbeck & Hoy 2005) and genetic studies (Wilkinson et al. 1997;Wilkinson & Sanchez 2001) has made this species name well known as an experimental object for molecular and behavioral studies of stalked-eyed flies in general.
Therefore, in view of the recent collection of both the sexes of C. whitei from a new location in northeastern India, the country of the type locality, it became necessary to provide accurate description of the species based on biometric data and supported by photographs and line drawings which include descriptions of new morphological characters previously unnoticed in this species.Another objective of this study is to provide a better understanding of C. whitei in its area of distribution and its relation to other species so as to ensure its accurate identification and a better taxonomic appraisal of the genus.To that end, an identification key to the known species of Cyrtodiopsis, sensu Feijen (2011), and a note on the habitat have also been provided.

METHODS AND MATERIALS
Live specimens of C. whitei were collected from the wild habitat using insect nets and these were transferred to killing jars.Biotic and abiotic features of the habitat of occurrence were recorded on each occasion of specimen collection.Dead and dry specimens were brought to the laboratory and kept in relaxation boxes for 36 hours to allow softening of external parts.Individual insects were spread to their natural posture and mounted on paper tips, pinned, labeled, and studied under Leica M205C zoom stereoscopic microscope fitted with Leica DFC295 digital camera.Biometry to the accuracy of 0.01mm and microphotographs were taken using Leica Application 3.8.0version software.Images, so acquired, were transferred in Microsoft power point slides to write the names of characters.Abdomens of six males and a female were dissected for genitalia study.These were individually subjected to heating in glass vials at 60 0 C, first in 10% KOH solutions for 10 minutes for maceration, then for five minutes each at increasing concentrations of ethyl alcohol (70%, 80%, 90% and 99.99%) for dehydration.Dehydrated specimens were boiled for five minutes in the saturated solution of choral phenol for softening of cuticle and sclerotised structures (Feijen & Feijen 2011).Finally, the abdomens with the ventral side up were mounted individually on clean glass slides with the help of fine tip needles under the Carl Zeiss Stemi 2000-C microscope and studied under Carl Zeiss AXIO Lab.1 microscope under 10X and 40X objectives and for Camera Lucida drawings.Specimens of this study are deposited with identical accession numbers, as used in Tables 1 and 2, in the Insect Biodiversity Laboratory, Department of Zoology of Tripura University.

Genus Cyrtodiopsis Frey, 1928
Diagnosis: Thorax with a pair of infra-alar spines, supra-alar spines absent, scutellar spines slightly to strongly curved outward, hairy, and each spine with a terminal bristle; fore femora conspicuously constricted on inner side at apex with incrassate surface; a tubercle is present at the base of inner margins of fore tibia that seems to fit into the constricted apex of fore femora when the fly is in rest.Taxonomic status of the genus: Frey (1928) proposed Cyrtodiopsis from a collection of the Stalk-eyed Flies of Philippines on the basis of a distinct "peg and hollow structure of forelegs in certain males" with dalmanni (Weidmann) as the type species.Shillito (1940) provided the first illustrated account of the family with a key to the identification of eight genera with particular reference to five species of the genus Cyrtodiopsis.Shillito (1940) distinguished Cyrtodiopsis from its nearest taxonomic relative Teleopsis in the wings without an alula, the thorax with infra-alar spines but without supra-alar spines, scutellar spines strongly curved, hairy and with a terminal long bristle and, most important, fore femora with constricted apex ventrally and fore tibia with rounded tubercles present at the base of the ventral side.Földvári et al. (2007) added a new species Teleopsis thaii, which is considered to be a Cyrtodiopsis species in the present work.Habitat: The habitat of C. whitei in the Dhalai District of Tripura province is an evergreen primary virgin forest of low hills of 'Longtharai' (local name meaning 'deep valley') and is the catchment area of two rivers, each of which traverses through valleys forming wide and narrow to very narrow streams lined by rocky banks and verticals.Stalk-eyed flies were found to be active in the sunshine hours in decomposing mixed vegetation dominated with banana leaves that was either floating in streams or in organic mass formed near the bank of stream.Atmospheric temperature and humidity of the habitat at the collection sites were recorded to be 24.2-25.6 0C and 74-77 %, respectively, and that of the microhabitat within 10cm of aerial distance of the collection points were found to be 21.4-22.8 0C and 81-84 %, respectively.

Description of additional characters
Male: Length 4.56-5.69mm; coloration generally brownish, head with antennae yellowish-brown to brown, thorax shining brown, scutellum darker than prescutellum or scutum; coxae and femora reddish-brown, tibiae and bases of tarsi reddish-brown to deep brown; wings pale brown with outer margins dark; abdomen with basal three segments pale brown and distal segments dark brown (Image 1).Head (Images 2,3; Fig. 1 ): sub-triangular, dorsum of central part yellowish brown (Image 2), conspicuously raised and bears three ocelli, one bigger in the front and two smaller on sides, with a pair of deep brown bristles having pointed apices (Fig. 1), about 0.14-0.18mm long; frons brownish, humped, projected forward, with a dorsal curved deep brown band and a distinct mid-suture, face concolorous with dorsum of head, slightly protruding, bear several long blackish bristles with pointed apices, 1-2 of these with bifid apices, about 0.20-0.24mm long; vertex yellowish brown to brown, narrower in front, broader at base and with rounded edges, bearing long hairs of bifid apices on the posterior edges (Image 3a); eye stalks yellowishbrown, smooth, bears a row of sparse, thin hairs, facing outward, with pointed apices, curved gently to strongly, these gradually decrease in lengths from the origin of stalks in the central part of the head to the bases of antennae (Image 2), the longest ones at the base of eye stalk 0.12-0.14mm long, 0.07-0.0.10 mm long in the middle of eye stalks, and 0.06-0.0.08 mm long at the bases of antennae, thus the longest ones at the base of eye stalk, on average, are 1.50-2.33times longer than the shortest ones near the base of antenna; inner vertical bristles (IVB) and outer vertical bristles (OVB) at low tubercles, 0.51-0.66mm and 0.25-0.37mm long, respectively (Table 1), and these 4.31-5.90times  2); eye span 3.98-7.52mm long and 0.87-1.32times the body length; antennae light brown, 3-segmented (Image 2), 0.32-0.36mm long, the shortest basal segment with a dark bristle on inner side, called scape, 0.07-0.10mm long, the middle segment, called pedicel, 0.15-0.19mm long (Table 1) and about 2-3 times longer than the basal segment (Table 2), bear 2-3 dark bristles, and the third and last segment, called first flagellomere, nearly bulbous, densely covered with small hairs, 0.09-0.12mm long, about twice the length of the middle segment, and bears a thick, long bristlelike structure with pointed apex on a raised base, called arista (Fig. 2), 0.70-0.90mm long (Table 1), and 1.30-1.52times and 1.89-2.57times the lengths of IVB and OVB, respectively (Table 2).Thorax (Image 3): collar glossy brown, V-shaped, scutum glossy brown, bi-lobed, smooth; scutellum shorter than wide, 0.18-0.25 mm long and 0.44-0.61mm wide, with dorsum broad, glossy brown, densely pollinose in the centre of pronotum and mesonotum (Image 3b), bears many short thin hairs with pointed apices only and a few long and prominent hairs with pointed or bifid apices, the longest ones about 0.25-0.39mm long; infra-alar spines yellowish, short, dorso-ventrally flattened, and with blunt apices (Image 3c), these about 0.24-0.32mm long (Table 1); scutellar spines dark, slightly curved inward, 0.69-1.12mm long, 3.75-4.73times the length of scutellum, each spine bears on its inner side 2-3 small hairs with pointed apices and 3-4 longer hairs with bifid apices (Image 3d), the longest ones about 0.26-0.35mm long, and a long apical bristle, about 0.44-0.49mm long (Table 1).Wings (Image 4): 3.16-4.30mm long, bases of fore wings leathery, rest membranous, dorsal surface densely covered with minute hairs; four distinct pale brown to brown bands present from base to the apex, the basalmost band paler than the other three bands, covers anal cell, discal cell, radial cell and subcostal-radial cell from lower to upper parts of the wing; the second band from the wing base broadest and darkest between R 2+3 and R 4+5 , and pale between costa and R 1 ; the sub-apical third band brownish, irregular, widest in the middle or radialmedial cell, and the fourth apical-most band narrowest, pale brown to brown in different specimens, extending from R 2+3 to M 1+2 from the apex and projects slightly to prominently towards the subapical band in the median cell; subapical and apical bands separated by three pale spots from apex to base of wings, with the median semicircular hazy spot in comparison to pale but distinct anterior and posterior circular spots; hind wings leathery, stump-like with a short stalk and attached to the raised bases.Legs: conspicuously hairy, longer hairs with bifid apices; fore coxae 0.78-0.85mm long, swollen in the middle; fore femora much wider (0.33-0.37 mm) than mid-(0.13-0.16mm) and hind femora (0.10-0.12 mm), basal ¼ part of inner margins smooth, rest ¾ margins incrassate, with a shallow constriction near the joint with tibia (Image 5); fore tibia with a low, rounded and dark tubercle in the apex that seems to fit in the constricted part of the fore femur on each side when the fly is in rest; tibiae dark, sparsely hairy on margins; tarsi 5-segmented, first segment the darkest and the longest, 0.66-0.74mm long, densely hairy on posterior margins, next four segments paler, decreasingly smaller in sizes, the apical-most segment the smallest, 0.10-0.12mm long, about 6.16-7.20 times the first tarsal segment, bear two dark, curved divergent claws.Abdomen black, clavate shaped (Image 1), first three segments fused, fuscus, with sparse long and thin hairs having pointed apices, tergites mildly pollinose, fourth, fifth and sixth segments with distinct inter-segmental sutures, wider than first three segments, gently deflexed ventrally, tergites and pleurites with hairs all over, segments 7 to 10 narrow to very narrow, condensed, covered with microtrichia and a few sparsely distributed long hairs; sub-anal plate triangular, heart-shaped; cerci clubshaped, apically rounded, about twice the length at base.Genitalia (Figs. 3-5): In ventral view, epandrium rounded with sclerotised and smooth margins, with 18 pairs of long setae counted when mounted in slide; surstyli pale, broad basally with thin margins but brown, bulbous apically, bulbous ends sclerotised and proximate in the middle, covered with microtrichia, with four pairs of long setae, two pairs originate from the inner margins of the base and other two pairs originate from the outer margins of bulbous apex (Fig. 3); cerci    large, with broad base and nearly conical apices, about twice as long as broad at the base, with thin margins and five pairs of hairs, two pairs of smaller hairs projected outwards and three pairs of longer ones projected inwards; hypandrium flat brown, with smooth margins, hypandrial bridge glabrous, in ventral view smooth, pale brown, bridge brown, with rough surface (Fig. 4); aedeagal apodeme elongated, brown, connected basally to hypandrium, in lateral view aedeagus with somewhat rounded end, sclerotic, and with a well developed ejaculatory apodeme (Fig. 5).
Female: The single female in the collection is similar to males except in longer body (6.49mm), longer OVB (0.39mm), longer antennae (0.43mm), longer hairs on head and eye stalks (Table 1), longer scutellum (0.32mm) and scutellar spines (1.12mm), fore coxae, fore femora and wings (Table 1).Genitalia parts were damaged in course of slide mounting.
Taxonomic status: Curran (1936) described Diopsis whitei from a tropical dry deciduous forest in Jharkhand in eastern India.That description lacked illustrations or drawings of distinguishing characters of the species.Shillito (1940) transferred the species to Cyrtodiopsis because of the presence of characteristic apical incrassate constriction infore femora and low rounded tubercles in the fore tibia.Since then six males and one female specimen of this species from a tropical evergreen forest in northeastern parts of India have become available from the country of its type locality.These show strong similarities with C. whitei in the structure of wings, the pollinose pattern of the scutellum, and general description of body parts sensu Curran (1936) and Shillito (1940).Hans Feijen (pers. comm. 10.07.2015) found our specimens to show similarity with the single C. whitei specimen in his possession from Meghalaya in northeastern India.Detail examination of specimens used in this study, however, warranted description of new characters not described earlier; these include some of the hairs present on dorsal surfaces of head, thorax, scutellar spines and on femora and tibiae with bifid apices, presence of a row of progressively gently to strongly curved frontal hairs of decreasing lengths from the base of the eyestalk to the base of antenna, and in the structure of male genitalia which were not adequately described.None of existing literature on Diopsidae mention the occurrence of hairs with bifid apices and curved frontal hairs of eye stalks that were noted in specimens of C. whitei of this study.It is possible that earlier workers might have missed these characters in whitei or other taxa of Diopsidae, therefore, it may be premature to conclude that these characters are unique to C. whitei or that the sample of this study might represent a new population of a distinct species.The author was not able to access the type specimens of whitei or specimens of other species of Diopsidae from valid sources.Therefore, at this point, the specimens of this study from moist evergreen forests of northeastern India are considered to represent possibly a part of widely distributed populations of whitei complex in its geographic range extending from the dry deciduous forest of eastern India (Jharkhand, the type locality) to moist evergreen forest in northeastern India, and, possibly, further east in Southeast Asia (Malaysia included), and it is assumed that populations of whitei might show habitat/area-specific variations and this position may be maintained until such time future study reveals more information.

DISCUSSION AND CONCLUSIONS
Feijen (2011) considered Cyrtodiopsis to be a weakly defined genus from the oriental region due to inclusion of several unrelated species at different times but preferred its distinct identity sensu Shillito (1940) in view of distinctive morphological attributes (chiefly in having prominent incrassate constrictions on apex of fore femora and low tubercles on the inner bases of fore tibiae) absent in the species of other genera under the family.Earlier, cladistic study using mitochondrial genes made by Baker et al. (2001), Meier & Baker (2002) and Földvári et al. (2007) revealed phylogenic relationship between Teleopsis and Cyrtodiopsis but molecular distinctions between the two genera based on four marker genes, particularly between T. thaii and T. breviscopium, were not conclusive (Földvári et al. 2007).To date, the presence of sharp incrassate apical constriction in fore femora and corresponding rounded tubercles on the basal parts of the fore tibia are the most robust and unique characters to the species of Cyrtodiopsis.Also, supra-alar spines, characteristic of all Teleopsis species, are absent in Cyrtodiopsis species.
The presence of several hairs with bifid or split tips on the body of C. whitei could possibly be another unique character of this genus till further study reveals its presence in other species of Cyrtodiopsis and possibly in other genera of Diopsidae.Despite prominent differences in morphometry between the two populations of whitei from geographically isolated locations of India and Malaysia, as evident from the taxonomic key above, we do not describe these specimens as a new species, because both populations share fundamental similarities in characters of fore wings, pollinosity distribution in thorax, body coloration, and general structure of genitalia.The observed differences in morphometry and presence of some of the body hairs with bifid tips in whitei from northeast India, among others, might represent the influence of differences in environments of the two habitats separated by several hundred miles.This study has founded the basis of future study to ascertain the prevalence of these characters when more specimens become available from these or nearby areas and also from the original type locality.

Table 2 . Ratio of bivariate characters of C. whitei*
Specimens are provided with respective accession numbers in parentheses. *