Taxonomic notes on the genus Campiglossa Rondani (Diptera, Tephritidae, Tephritinae, Tephritini) in India, with description of three new species

Abstract Three new species of Campiglossa Rondani are described from India: adults of both sexes and third instar larvae of C. ialong David, Salini & Hancock, sp. nov. and C. sherlyae David & Hancock, sp. nov., plus an adult female of C. shaktii David, Sachin & Hancock, sp. nov., are described and illustrated. Postabdominal structures, cephalopharyngeal skeleton, and anterior and posterior spiracles of C. gemma (Hering, 1939) and C. sororcula (Wiedemann, 1830) are illustrated. DNA barcode sequences of C. ialongsp. nov., C. sherlyaesp. nov., and C. gemma were obtained and reported. Records of C. absinthii (Fabricius, 1805) and C. iracunda (Hering, 1938) are regarded as misidentifications of C. lyncea (Bezzi, 1913) and C. shaktiisp. nov., respectively, and excluded from the Indian fauna. A key to the known species of Campiglossa from India is provided. Results of preliminary phylogenetic analysis using COI revealed that C. ialongsp. nov. is paraphyletic to the Campiglossa misella group and C. C. sherlyaesp. nov. is a sister species of C. deserta.


Introduction
Campiglossa Rondani is one of the most speciose genera in the subfamily Tephritinae with nearly 200 described species Han and Ro 2019). They are characterised by an elongate proboscis, a predominantly spinulose preglans area of the phallus, and bases of the antennae widely separated by a space 0.5-1 times the width of the scape (Korneyev 1999). Campiglossa is predominantly a Palaearctic genus but has representatives in other zoogeographic regions. Most species are associated with host plants of the family Asteraceae. The Afrotropical fauna was revised by Munro (1957) and the Palaearctic fauna by Korneyev (1990) and Merz (1992). Han and Ro (2019) synonymised Homoeotricha Hering and Dioxyna Frey with Campiglossa based on their analysis employing the mtCOI marker, but study of related genera is required before precise generic limits can be determined. The Indian fauna was studied by Bezzi (1913), Agarwal et al. (1989), and Hancock and McGuire (2002). Although Agarwal and Sueyoshi (2005) listed eight species of Campiglossa from India, Hancock (2008) regarded report of C. iracunda (Hering, 1938) from India as a misidentification, while record of C. absinthii (Fabricius, 1805) is also regarded as a misidentification, as discussed below. Three new species of Campiglossa encountered in India during surveys for fruit flies are described here. Postabdominal structures and larvae of C. gemma (Hering, 1939) and C. sororcula (Wiedemann, 1830) from southern India are described and illustrated along with taxonomic notes on the four other recorded Indian species. As types of these four species were not available for study, detailed redescriptions or diagnoses are not included.

Material and methods
Specimens deposited in NBAIR were examined for the study. Following are the acronyms used in the text: NBAIR ICAR -National Bureau of Agricultural Insect Resources, Bangalore, India NPC National Pusa Collection, Indian Agricultural Research Institute, New Delhi, India ZSI Zoological Survey of India, Kolkata, India Collections were made by sweep netting and rearing infested flowers of host plants belonging to family Asteraceae. Images of specimens were taken using a Leica DFC 420 camera mounted on a Leica M205A stereozoom microscope; images of genitalia were taken using an 8 MP camera temporarily attached to a Leica DM 1000 compound research microscope. Multiple images were stacked and combined to a single image using Combine ZP (Hadley 2011). Line drawings were made using a drawing tube attached to a Leica DM 1000 compound microscope. Measurements of male and female genitalia were taken using Leica Automontage Software, LAS 3.4. Terminology adopted here follows White et al. (1999). Singular form is used for all paired organs and setae in the text (e.g., one postpronotal lobe seta means one pair of postpronotal lobe setae). Ratios have been calculated as per Han and Ro (2019).

DNA isolation and partial gene sequencing of COI
To isolate the genomic DNA, the hind and mid legs (one each) of individual insects were used and the DNA isolation was carried out using the Qiagen DNeasy Blood & Tissue Kit method following the manufacturer's protocol. After obtaining the DNA, the quality and quantity were estimated using nanodrop-BioRad. PCR amplification of partial gene sequences of mitochondrial COI gene was carried out by using the universal COI primers (Hebert et al. 2004). PCR amplification was performed for a total volume of 30 μL, containing 2 μl DNA extract (20 ng), 1 μl (2mol) of each primer, 1 μl dNTP mixture (2.5 mmol for each), 2.5 μL 10x Taq PCR reaction buffer, 3 μL 25 mM MgCl 2 + , and 1 unit of Taq DNA polymerase using a thermal cycler (BioRad iCycler) with the PCR cycle as follows: initial step at 94 °C for 1 minute and 35 cycles of the following: denaturing 95 °C for 30 seconds, annealing 51 °C for 30 seconds, extension at 72 °C for 45 seconds and 4 °C thereafter (Ball and Armstrong 2008). The PCR products size varied from 650 to 680 bp; the amplified products were confirmed by running on 1.5% agarose gel with 250 bp ladder and visualized in INGENIUS gel dock. The amplified products were purified using Qiagen PCR purification Kit by following the manufacturer's instructions and the purified samples were sequenced using Sanger's method. The sequences were annotated using NCBI Blast tools and submitted to NCBI GenBank Database where accession numbers were obtained (C. ialong sp. nov. -MT169786; C. sherlyae sp. nov. -MT019895; C. gemma -MT169785; C. sororcula -MT019889)

Construction of molecular phylogeny tree
The molecular phylogeny of Campiglossa was constructed using the software MEGAX (Kumar et al. 2018). A total of 18 DNA barcode sequences were used for this analysis including the outgroup Tephritis conura Loew, in which four were from India and another 14 were downloaded from NCBI database. Campiglossa from Oriental, Palaearctic, and Nearctic regions were included in the analysis. The evolutionary relationship was inferred using the maximum likelihood method. The General Time Reversible model (Nei and Kumar 2000) was used with uniform rate of substitution. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. Initial tree(s) for the heuristic search were obtained automatically by applying the maximum parsimony method. This analysis involved 18 nucleotide sequences. Codon positions included were 1 st + 2 nd + 3 rd . Head: Slightly higher than long (head ratio 0.83-0.86); frons fulvous (frons-head ratio 0.38-0.40), with a medial band of pruinosity from ocellar triangle to lunule leaving two dark fuscous lateral bands devoid of pruinosity; two frontal setae (three in a few specimens); two subequal orbital setae (posterior one white); well-developed proclinate ocellar seta (0.7 length of medial vertical seta); lateral vertical seta white; medial vertical seta black; paravertical seta white; postocular setae intermixed black and white. Scape, pedicel, and flagellomere concolorous with frons; pedicel plus flagellomere shorter than face; arista bare; face concave with raised epistomal margin; gena and occiput fulvous. Eye ratio 0.64-0.69; gena-eye ratio 0.13-0.18; antenna-head ratio 0.45-0.47; arista-antenna ratio 1.20-1.45.

Key to species of Campiglossa Rondani from India
Thorax: Scutum grey pollinose, with three faint stripes and well-developed chaetotaxy (all setae black); one postpronotal lobe seta, one presutural supra-alar seta, one anterior notopleural seta, one posterior notopleural seta, one dorsocentral seta near transverse suture, placed anterior of postsutural supra-alar seta and posterior notopleural seta, one presutural supra-alar seta, one postalar seta, one intra-alar seta, one prescutellar acrostichal seta. Anepisterum grey with single black anepisternal seta in line with posterior notopleural seta; anepisternum covered with tiny white setulae; thick white setulae posteriorly near phragma; anepimeron without any black setae, with thick stub- by white setulae anteriorly; katepisternum with single black seta posterior to phragma in dorsal region; anatergite and katatergite grey without any setulae; haltere pale yellow. Scutellum flat, yellow with sparse white setulae; two scutellar setae; apical scutellar seta 2/3 length of basal scutellar seta. Mediotergite grey, without setulae.  Legs: All segments unicolorous, yellowish orange; fore femur with single row of five or six stout ventral setae, two rows of dorsal setae; mid and hind femur covered with tiny black setulae. Mid tibia with four apical spines, one elongate, the others all 1/4 length of prominent spine.
Wing: Reticulate pattern, with hyaline and yellow spots; basal 1/3 hyaline with faint brown markings; apical 2/3 dark brown with numerous hyaline and yellow spots. Cell bc hyaline; cell c hyaline with two faint brown markings; pterostigma dark brown with a medial, yellow spot/patch; apex of cell r 1 and r 2+3 black without any hyaline spots; cell r 2+3 with a preapical dumbbell-shaped spot. Cell r 1 with three broad hyaline patches and irregular yellow spots or patches; cell r 2+3 hyaline only in basal portion, rest brown to black with irregular yellow spots and, broad hyaline markings that are extensions of the hyaline markings from cell r 1 and preapical dumbbell-shaped spot (separate spots in a few specimens). Cell br predominantly hyaline, with irregular brown markings; cell r 4+5 predominantly black or brown with an apical hyaline spot, two preapical spots, numerous yellow spots, and a broad basal hyaline spot. Cells bm and bcu hyaline; basal 2/3 of cell dm largely hyaline, with narrow basal and submedial brown transverse bands, apical 1/3 brown with hyaline spots; cell m with four broad irregular markings; cell cu 2 and anal lobe predominantly hyaline with irregular brown markings.
Remarks. Campiglossa ialong is most similar to C. iracunda (Hering) in appearance but with only one hyaline spot at the apex of cell R 2+3 , as in C. siamensis (Hardy 1973). However, the black posterior notopleural seta differs from C. siamensis, which has a brown or yellowish seta. As per the phylogenetic tree (Fig. 51), it is paraphyletic with the misella group. Diagnosis. Medium-sized fly (4.42-4.85 mm), body predominantly grey pollinose, with white setulae; scutum without prominent stripes; abdomen uniformly grey without submedian black markings; wing with reticulate pattern.
Legs: All segments unicolorous, yellowish orange; fore femur with single row of six or seven stout ventral setae, two rows of dorsal setae; mid and hind femur covered with tiny black setulae. Tibiae and tarsi with rows of spines; mid tibia with four apical spines, one elongate, the others all 1/4 length of prominent spine.
Wing: Reticulate pattern, with hyaline and yellow spots; cell bc hyaline with a brown spot on humeral crossvein; cell c hyaline with a single brown patch medially; pterostigma dark brown, with two round, yellow spots, the one closer to apex of vein Sc smaller compared to distal one; apex of cell r 1 and r 2+3 black, without any hyaline spots. Cell r 1 with three broad, hyaline patches and irregular yellow spots; cell r 2+3 dark basally, with two faint yellow spots or markings and with a preapical dumbbell-shaped spot. Cell br predominantly hyaline, with irregular brown markings; cell r 4+5 predominantly black or brown, with a small apical hyaline spot, three preapical spots arranged in a triangle, numerous yellow spots, and hyaline basally. Cells bm and bcu hyaline; cell dm basally broadly hyaline with three narrow, transverse, brown bands to level of r-m crossvein; apically brown with hyaline spots; cell m with diffuse hyaline markings; cell cu 2 and anal lobe predominantly hyaline with irregular brown markings.
Etymology. This species is named after its collector, Shakti Kumar Singh.
Remarks. This species is undoubtedly the 'Paroxyna' or 'Campiglossa' iracunda of previous authors (Kapoor et al. 1979;Kapoor 1993;Agarwal and Sueyoshi 2005), the identity of which was discussed by Hancock (2008) and regarded as a misidentification.
Legs: All femora with extensive black markings (0.75 of all femora with black markings), all other segments fulvous; fore femur with single row of four or five stout ventral setae, two rows of eight or nine dorsal setae; mid and hind femur covered with tiny black setulae. Tibiae and tarsi with rows of spines; mid tibia with four subequal apical spines.
Wing: Reticulate pattern with hyaline and yellow spots; cell bc hyaline with a brown streak on humeral crossvein; cell c hyaline, with a single brown band medially; pterostigma dark brown, with a single hyaline spot, apex of cell r 1 and r 2+3 without hyaline spot. Cell r 1 with three broad, hyaline patches, cell r 2+3 with three broad, hyaline markings. Cell br hyaline basally and with a broad preapical hyaline patch; cell r 4+5 with five uneven, hyaline spots (basal and subapical larger than medial and apical spot); apex of cell r 4+5 with small hyaline spot. Cells bm and bcu hyaline; cell dm predominantly hyaline with base and apex brown; cell m with a broad, hyaline mark (formed by fusion of three spots) and a preapical spot; cell cu 2 predominantly hyaline, with brown streaks and apical hyaline spot; apex of cell bcu with brown patch.
Male postabdomen: Epandrium well sclerotised, without clear delineation between epandrium and lateral surstylus; proctiger hyaline, with densely arranged setae anteriorly; surstylar flange prominent, with serrated edge; epandrium and surstyli oval in outline in posterior view; medial surstylus with well-developed apical prensisetae. Phallus, excluding glans, 1.2 mm long; glans of phallus with well-developed tubular acrophallus. Etymology. The species is named after the late Sherly Joseph, in memory of the first author's sister.
Host plant. Flowers of Sonchus sp. (Asteraceae). Remarks. This species belongs in the producta group and is known only from Karnataka. It was misidentified as C. deserta (Hering, 1939) by Hancock and McGuire (2002) and their Indian record of a female from Mudigere, Karnataka, is C. sherlyae. Other records listed by Hancock and McGuire (2002) from Thailand and Vietnam appear to have been properly identified as C. deserta, which is a species widespread in China (including Guangxi Province), Korea, and Japan. Campiglossa sherlyae is very similar to C. producta and C. deserta, differing from C. producta in possessing predominantly black or brown base of cell r 2+3 in wing with a prominent spot near crossvein r-m, and from C. deserta in lacking a hyaline base to cell r 2+3 and in having Sonchus rather than Lactuca as its host plant. The phylogenetic tree (Fig. 51) shows that this species and Korean samples of C. deserta are closely related but with a 2% divergence based on a NCBI-GenBank sequence similarity search (BLAST), along with differences in morphological characters and host plant, suggest they are distinct. Description. Medium-sized fly (male 3.24-3.92 mm; female 4.49-4.83mm) with grey pollinose body, yellow legs, and reticulate wing pattern. Head slightly higher than long; frons fulvous with two frontal setae, two orbital setae (posterior orbital seta white), postocellar and postvertical seta white, lateral vertical seta white, medial vertical seta black, ocellar seta black longer than frontal and orbital seta. Scutum grey pollinose, with postpronotal lobe and notopleuron pale yellow, and welldeveloped chaetotaxy; posterior notopleural seta white. Scutellum with two pairs of scutellar setae; apical setae as long as basal setae. Legs fulvous, without any black markings. Wing with reticulate pattern; pterostigma black, without any hyaline spot or marking; apex of cell r 2+3 and r 4+5 without hyaline spot. Abdomen grey pollinose, without any markings.
Remarks. This species is known only from Tamil Nadu and western Karnataka (Kemmangundi) in southwestern India (this study; Hancock and McGuire 2002). Although there is some slight variation in wing markings, the examined specimens are consistent with Hering's (1939) original description and most are from the type locality. In the phylogenetic tree, C. gemma is placed as a sister group to all the included Campiglossa species (Fig. 51). This might be due to the low taxon sampling or, alternatively, the species may belong to another genus, which should only be considered after a thorough study of other Campiglossa species and related groups.

Third instar larva
Host plants recorded during the study: flowers of Bidens pilosa L. and Cosmos sulphureus Cav. (Asteraceae).
Remarks. This species occurs commonly from southern Europe to Africa, Asia, and Australia, and has been introduced into Hawaii . Bezzi (1913), Hancock and McGuire (2002), Agarwal and Sueyoshi (2005), and David and Ramani (2011) recorded it from various locations in India, where it is widespread. Leg colour in many populations is variable (Hardy and Drew 1996); in India, the femora are generally yellow with a black basal patch on mid and hind femora. Vestigial apical scutellar setae have been observed in some Australian populations (Hardy and Drew 1996), but Indian specimens lack the apical pair. Bezzi, 1913 Campiglossa cribellata Bezzi, 1913: 161. Type locality: Kurseong, E. Himalayas, West Bengal, India.

Campiglossa cribellata
Remarks. This species belongs in the irrorata group and was illustrated by Bezzi (1913) and Kapoor (1993). It is known only from the eastern Himalayas in India and Nepal (Bezzi 1913;Kapoor et al. 1979b). The host plant is unknown. The holotype, deposited in ZSI, is damaged (Banerjee, D; Diptera Section, ZSI, pers. comm.) and was not available on loan; hence, a detailed diagnosis and redescription are not included here.
Remarks. This species is provisionally included in the irrorata group and was illustrated by Agarwal et al. (1989) and Kapoor (1993). It is distinguished from C. cribellata by the reduced hyaline wing markings (particularly in the pterostigma and cell r 1 ) and the more elongate wing. This species is known only from the type locality. The holotype, deposited in NPC, could not be traced and might have been lost or misplaced; hence, a diagnosis and redescription are not included here. Its unusual wing shape suggests that placement in Campiglossa requires confirmation.

Remarks.
Campiglossa lyncea is distinguished from other Indian species by its mostly black femora, white posterior notopleural seta, two hyaline marginal spots in cell r 2+3, and large, often coalesced, hyaline discal spots. This species is known only from northern India and includes the record of C. absinthii Fabricius, 1805 from Solan, Himachal Pradesh (Agarwal and Sueyoshi 2005), which was misidentified as the synonym C. parvula (Loew, 1862) by Kapoor et al. (1979a) and Kapoor (1993). The illustration of C. parvula by Kapoor (1993) closely matches C. lyncea of Bezzi (1913), whereas the figure of C. lyncea in Kapoor's (1993) publication is copied from Hardy (1973) and is neither this species nor Indian. Hence, Hardy's (1973) Vietnamese records, considered to be conspecific with Kapoor's (1993) figure of 'C. lyncea' by Hancock (2008), are also excluded. The syntypes of C. lyncea, deposited in ZSI, are damaged (Banerjee, D; Diptera Section, ZSI, pers. comm.) and were not available on loan. Hence, a detailed diagnosis and redescription are not included.
Remarks. This species was recorded from India by Hancock and McGuire (2002), based on two males and two females from Gulmarg, Kashmir. However, given the complexity of this group, additional material is required for confirmation. Elsewhere, it is widespread from Western Europe to Central Asia, including Afghanistan (Agarwal and Sueyoshi 2005).