Acta Tropica 149 (2015) 94–105

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Simulium (Gomphostilbia) (Diptera: Simuliidae) from Southern Western Ghats, India: Two new species and DNA barcoding S. Anbalagan a,∗ , V. Arunprasanna a , M. Kannan a , S. Dinakaran b , M. Krishnan a a b

Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu,India Department of Zoology, The Madura College, Madurai,Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 15 April 2015 Received in revised form 14 May 2015 Accepted 16 May 2015 Available online 18 May 2015 Keywords: Black fly New species Gomphostilbia COI gene

a b s t r a c t Two new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) are described on the basis of reared adult, pupal and larval specimens collected from Southern Western Ghats India. The morphological data of two new species S. (Gomphostilbia) panagudiense sp. n. and S. (Gomphostilbia) kottoorense sp. n. are assigned to the batoense species group in the subgenus Gomphostilbia. S. (Gomphostilbia) panagudiense sp.n. is characterized in the female by having the scutum without longitudinal vitta and arms of the genital fork wide basally and in the pupa by the stalk of ventral pair medium-long. S. (Gomphostilbia) kottoorense sp.n. is characterized by the arm of genital fork tapered near apex in the female and style in medial view 0.63 times as long as coxite in the male. Phylogeny of members in the genus Simulium was reconstructed based on DNA barcoding gene (cytochrome oxidase c subunit I). Tree analysis using new technology and maximum likelihood analyses are congruent with evidence of two new species in the subgenus Gomphostilbia and separated from other species. © 2015 Published by Elsevier B.V.

1. Introduction Simulium (Diptera: Simuliidae) is a genus of black flies, including 2177 living and 12 fossil species distributed throughout the world (Adler and Crosskey, 2015). Many species of female black flies are blood feeding behavior and they transmit the parasitic nematode Onchocerca volvulus, the causative organism of human onchocerciasis (Anbalagan et al., 2014a). Larval black flies thrive in water with slow to fast flowing streams, associated with stream substrates (bed rock, boulders and pebbles) and solid wastes submerged in water (Anbalagan et al., 2011, 2014b), and play an important role for maintaining lotic ecosystem and also used as bioindicators (Harwood and James, 1979). The fauna of black flies in the world is represented by 26 genera and 37 subgenera. Among 37 subgenera, Gomphostilbia Enderlein is a prevalent subgenus next to the subgenera of Simulium and Nevermannia. A total of 220 species in 15 species groups of Gomphostilbia are recorded in the world (Adler and Crosskey, 2015). The batoense species group had the highest number species (53 named species) in Gomphostilbia and is chiefly distributed in oriental regions (Takaoka, 2012). In India, 12 species of Gomphostilbia

∗ Corresponding author. Tel.: +91 9360525978. E-mail address: [email protected] (S. Anbalagan). http://dx.doi.org/10.1016/j.actatropica.2015.05.015 0001-706X/© 2015 Published by Elsevier B.V.

have been reported: 4 species (S. (Gomphostilbia) fidum, S. (Gomphostilbia) pattoni, S. (Gomphostilbia) peteri and S. (Gomphostilbia) takaokai) of the batoense species group, 1 species (S. (Gomphostilbia) tenuistylum) of the ceylonicum species group, 2 species (S. (Gomphostilbia) bucolium and S. (Gomphostilbia) sundaicum) of the epistum species group, 1 species (S. (Gomphostilbia) darjeelingense) of the darjeelingense species group and 4 species (S. (Gomphostilbia) litoreum), S. (Gomphostilbia) metatarsale and 2 unnamed species) of unplaced species group (Adler and Crosskey, 2015; Anbalagan et al., 2014a). Blackfly systematics using DNA sequences (12S, 18S, 28S, 5.8S, COI, COII, ITS-1, ITS-2 and ITS4) are useful for studying phylogeny and identification of species (Thanwisai et al., 2006; Conflitti et al., 2010; Hamada et al., 2010). Phylogenetic analyses of the subgenus Nevermannia and other subgenera of black flies using mitochondrial DNA gene sequences was first studied in Asia by Otsuka et al. (2007), followed by few such studies have been conducted in Thailand (Thanwisai et al., 2006; Phayuhasena et al., 2010; Pramual and Kuvangkadilok, 2012). Although Gomphostilbia is the third largest subgenus in the genus Simulium and widely distributed in the Oriental region, less attention has been received on DNA barcoding analysis (Pramual et al., 2011). Recently data acquired with identification of species by molecular techniques provides important information to resolve cryptic diversity and used to control vectors (Pramual and Kuvangkadilok, 2012). In this regard, DNA barcoding

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Table 1 Details of the members of Simulium (Gomphostilbia) including their species group, distribution and NCBI-GenBank accession numbers. Subgenus

Species group

Species

Distribution

Accession no.

Asiosimulium Daviesellum Gomphostilbia

– – Asakoae Batoense

Oblongum Pahangense Asakoae Decuplum Duolongum Kottoorense Panagudiense Parahiyangum Peteri Siamense Takaokai Curtatum Inthanonense Sheilae Trangense Angulistylum Gombakense Chumpornense Kuvangkadilokae Merga Feuerborni Fruticosum Aureohirtum Chomthongense Choochotei Baimaii Chainarongi Chaliowae Fenestratum Takense Triglobus Nobile Nodosum Chiangmaiense Quinquestriatum Rufibasis Tani Weji Chamlongi

Thailand Malaysia, Thailand Malaysia, China, Thailand, Vietnam Malaysia, India, Thailand Malaysia, Indonesia, Vietnam India India Indonesia, India, Malaysia, Thailand India Thailand, Nepal, Vietnam India Thailand Thailand, China, Vietnam Malaysia, Indonesia, Thailand Thailand Malaysia, Thailand Malaysia, Thailand Thailand Thailand Thailand Indonesia, Malaysia, Thailand Thailand India, Malaysia, Thailand, China, Nepal, Sri Lanka Thailand Thailand Thailand Thailand Thailand Indonesia, Laos, Thailand Thailand Thailand Indonesia, Malaysia, Thailand India, China, Thailand, Vietnam Thailand, China, Vietnam Taiwan, China, Thailand, Korea, Japan, Vietnam India, Burma, China, Korea, Nepal, Thailand, Vietnam Malaysia, Indonesia, Taiwan, Thailand, Vietnam Thailand Thailand, Burma, Vietnam

KF289393 KF289462 HM775266 HM775287 KF289395 KP223707 KP031495 KF289472 KM977779 HQ738690 KM985984 HM775254 HM775257 HM775275 HM775260 JQ619931 HM775253 HM775279 HM775278 KF289397 JX484833 KF289400 KF289401 KF289463 FJ477844 KF289412 JF916854 KJ649637 JF916864 JF916870 KJ649652 KF289416 KF289418 KF289422 JQ412151 KF550230 KJ636915 KF550256 KF289469

Ceylonicum

Epistum Gombakense Varicorne Montisimulium Nevermannia

– Feuerborni

Simulium

Ruficorne Vernum Griseifrons Malyschevi Multistriatum

Nobile Striatum Tuberosum

Variegatum

is an efficient tool for many needs in systematics that as linking unknown larvae, adults, identifying vector species and making quick taxonomic identifications. In the present study, we described two new black fly species in the subgenus of Gomphostilbia (Diptera: Simuliidae: Simulium) from Southern Western Ghats, India and we studied the DNA barcoding gene of cytochrome oxidase subunit I for four species of Gomphostilbia including two new species reported in South India.

2. Materials and methods 2.1. Taxa Sampling In the field, larvae and pupae were collected manually from stream substrates (leaf litter, boulders and pebbles) with a fine brush and forceps. The mature pupae were separated and placed on wet filter paper in a small plastic container for rearing. It was kept in an incubator adjusted to stream temperature for one or two days. The collected specimens were preserved separately in the field using 99% ethanol. Holotypes and paratypes were stored in 100% ethanol at −20 ◦ C and then deposited in the Insect Molecular Biology Laboratory, Department of Environmental Biotechnology, Bharathidasan University (BDU), Tiruchirappalli, Tamil Nadu province, India. In this study four species in the subgenus of Gomphostilbia recorded from peninsular India were taken for DNA barcoding analysis and other 35 species from the six subgenera available in GenBank were included as outgroups (Table 1).

2.2. DNA isolation, PCR amplification and sequencing Total genomic DNA was extracted from individual larva from each species according to the manufacture’s protocol for the QIAamp genomic DNA isolation kit (QIAGEN, Germany). The extracted genomic DNA was quantified by a spectrophotometer (Shimadzu, Japan) at 260/280 nm. The mitochondrial protein coding gene of cytochrome c oxidase subunit I (COI) was amplified by polymerase chain reaction using the primers LCO 1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO 2198 (5’TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al., 1994). The PCR reaction (20 ␮l) was conducted using the Exprime taq premix, GenetBio Inc. (Korea) and the following volumes of reagents: 2 ␮l of DNA extract, 10 ␮l of Mastermix, 1 ␮l of each primer (10 mM), and 6 ␮l of sterile water. The typical thermal cycling profile was: 5 min at 94 ◦ C and 35 cycles of 30 s at 94 ◦ C, 60 s at 58 ◦ C, and 1 min at 72 ◦ C, with a final extension at 72 ◦ C for 7 min. The amplified PCR products were identified by electrophoresis, using a 1% agarose gel and then purified using the Hiyield Gel/PCR-DNA Extraction kit (Real Biotech Corporation, Taiwan). Finally, purified PCR products were sequenced by the automated DNA sequencer Model: 3500 (Applied Biosystems, Foster City, CA, USA). The DNA sequences were aligned and analyzed using the program Chromas, v.1.41. The DNA and deduced amino acid sequences were analyzed using the NCBI-BLAST, the BOLD Identification System and EXPASY tools. Sequence alignments were performed using the Clustal W v.1.82 software (Thompson et al., 1994). The sequences of four species have been deposited in the GenBank database and their accession numbers are given in Table 1.

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Fig. 1. Female of Simulium (Gomphostilbia) panagudiense sp.n. (A) 3rd segment of right maxillary palp showing sensory vesicle (front view); (B) left hind tibia (outer view); (C) basitarsus and 2nd tarsomere of left hind leg showing calcipala and pedisulcus (outer view); (D) tarsal claw; (E) 8th sternite, ovipositor valves (ventral view); (F) genital fork (ventral view); (G & H) right paraprocts and cerci (G) ventral view; (H) lateral view); (I) spermatheca (lateral view). Scale bars. 0.01 mm for (D) 0.02 mm for (A) and (E–I) 0.1 mm for (B) and (C).

2.3. Sequence alignment and phylogenetic inference Contigs of COI assembled and edited in SEQUENCER 5.0 (Genecodes, 2011). Protein-coding sequence of COI was imported into MEGA version 6 (Tamura et al., 2013) for alignment by amino acid. After the determining of the open reading frame, sequences were translated to amino acid and then aligned with MUSCLE 3.7 (Edgar, 2004). The aligned amino acid was then back-translated to nucleotides and retaining the codon positions. Maximum parsimony analysis (MP) was calculated in TNT (Goloboff et al., 2008) using a heuristic search with TBR and the ‘new technology’ search: tree drifting, sectorial searches, tree fusing (Goloboff, 1999) and ratchet (Nixon, 1999). Gaps were taken as missing. In TNT, nodal support for the MP tree was calculated with 1000 bootstrap replications, implemented through PAUP version 4 (Swofford, 2002). In addition, Maximum likelihood (ML) was used to resolve models of evolution for individual genes and the concatenated data set under the Akaike information criteria (AIC).

3. Results 3.1. Simulium (Gomphostilbia) panagudiense Anbalagan sp. n. Type series. Holotype, ♂, INDIA: Kuthirapanchan waterfalls, Panagudi, Tirunelveli district, Tamil Nadu state, 2-II-2014, 110 m (08◦ 20 .31 N, 77◦ 32 .14 E), S. Anbalagan (Coll. S. Anbalagan, Bharathidasan University (BDU)). Paratypes, same data as holotype: 4 ♂♂, 3 ♀♀, 8 pupae, 92 larvae (Coll. S. Anbalagan, BDU). 3 ♂♂, 2 ♀♀ (Department of Environmental Biotechnology, BDU, Catalogue number: BL0016).

3.1.1. Description Female: Body length 1.5 mm. Head. Narrower than width of thorax. Frons nearly black, shiny when illuminated at certain angles, thinly yellowish pruinose, with several stout dark hairs along lateral and lower margins; frontal ratio 2.0:1.0:2.8; fronshead ratio 1.00:3.63. Fronto-ocular area well developed, narrow, directed dorso-laterally. Antenna composed of scape, pedicel and 9 flagellomeres, brown except scape, pedicel and basal 1/2 of 1st flagellomere yellow. Clypeus grayish white, shiny, thinly whitish pruinose, with dark stout hairs. Labrum 0.41 times as long as clypeus. Maxillary palp composed of 5 segments; proportional lengths of 3rd, 4th and 5th segments 1.0:1.3:3.2; 3rd segment (Fig. 1A) moderately enlarged; sensory vesicle (Fig. 1A) mediumsized (0.32 times as long as 3rd segment), with medium-sized round opening. Maxillary lacinia with 8 inner and 12 outer teeth. Mandible with 19 inner and 8 outer teeth. Cibarium bare. Thorax. Scutum dark brown except anterolateral calli dark ochreous, without longitudinal vitta; scutum shiny when illuminated at certain angles, densely covered with yellow scale-like recumbent short hairs interspersed with dark brown long upright hairs on prescutellar area. Scutellum shiny, with dark brown long upright hairs. Postnotum shiny and bare. Pleural membrane bare. Katepisternum medium to dark brown, longer than deep, shiny when illuminated at certain angles, moderately covered with fine short hairs. Legs. Foreleg: coxa yellow; trochanter yellow except apical portion somewhat darkened; femur light brown; tibia brown; tarsus brownish black, with moderate dorsal hair crest; basitarsus slightly dilated, 6.0 times as long as its greatest width. Midleg: coxa yellowish brown except posterior surface dark brown; trochanter yellow; femur and tibia brown; tarsus brownish-black except basal 1/2 of basitarsus dark yellow. Hind leg: coxa yellowish brown; trochanter

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Fig. 2. Male of Simulium (Gomphostilbia) panagudiense sp.n. (A) 3rd segment of left maxillary palp showing small sensory vesicle (frontal view); (B) left hind tibia (outer view); (C) basitarsus and 2nd tarsomere of left hind leg showing calcipala and pedisulcus (outer view); (D) coxites, styles, ventral plate and median sclerite (ventral view); (E & F) right styles ((E) medial view; (F) ventrolateral view); (G) ventral plate (ventral view); (H) ventral plate and median sclerite; (I) ventral plate (end view); (J) left paramere and aedeagal membrane (end view); (K) 10th abdominal segment and cercus (right side and lateral view). Scale bars. 0.02 mm for (A) and (D–K); 0.1 mm for (B) and (C).

yellow; femur medium brown with base yellow and apical cap dark brown; tibia (Fig. 1B) light to dark brown with basal 1/4 white, covered with brownish fine hairs on outer and posterior surfaces; tarsus brownish-black except basal 2/3 of basitarsus (though base light brown); basitarsus (Fig. 1C) narrow, nearly parallel-sided, 6.43 times as long as wide, and 0.58 and 0.51 times as wide as greatest width of tibia and femur, respectively; calcipala (Fig. 1C) slightly shorter than width at base, and 0.46 times as wide as greatest width of basitarsus. Pedisulcus (Fig. 1C) well defined. Claw (Fig. 1D) with large basal tooth 0.53 times as long as claw. Wing. Length 1.63 mm. Costa with dark spinules and hairs except hairs on basal portion yellow. Subcosta with dark hairs except near apex bare. Hair tuft on stem vein dark brown. Basal section of vein R fully haired; R1 with dark spinules and hairs; R2 with hairs only. Basal cell absent. Haltere. Light brown except apical portion white. Abdomen. Base of each segment dark brown, basal scale dark brown, with fringe of dark brown hairs, dorsal surface of abdomen brown to brownish black except basal 1/2 of segment 2 yellow, moderately covered with brown short to long hairs; tergites of segments 2 and 6–8 shiny when illuminated at certain angles. Ventral surface of segments 2 and 3 whitish-yellow except lateral side brownish yellow, and those of other segments light to dark brown; sternal plate on segment 7 undeveloped. Genitalia. Sternite 8 (Fig. 1E) well sclerotized and bare medially, covered with 5–6 long hairs and few short fine hairs on each side. Ovipositor valves tongue-like, thin, membranous, moderately covered with microsetae interspersed with

one or two short hairs; inner margins concave, somewhat sclerotized, and slightly separated from each other. Genital fork (Fig. 1F) of usual inverted-Y forms; stem slender and well sclerotized; arms of moderate width, lateral plate of each arm with thin lobe directed medioposteriorly and small stout projection directed anterodorsally. Paraproct in ventral view (Fig. 1G) concave anterolaterally, with 3 sensilla on anteromedial surface; paraproct in lateral view (Fig. 1H) somewhat produced ventrally, 0.52 times as long as wide, with about 12 medium-long to long hairs on lateral and ventral surfaces. Cercus in lateral view (Fig. 1H) moderate, rounded posteriorly, 0.51 times as long as its greatest width. Spermatheca (Fig. 1I) ellipsoidal, 1.52 times as long as its greatest width, well sclerotized except duct and small area near juncture with duct unsclerotized, and with many fissures on surface; internal setae absent; both accessory ducts slender, subequal in diameter to major one. Male: Body length 2.0 mm. Head. 0.9 times wider than thorax. Upper eye yellowish brown, consisting of 12 vertical columns and 10 horizontal rows of large facets. Face brownish black, grayishwhite pruinose. Clypeus black, whitish pruinose, densely covered with golden-yellow scale-like medium-long hairs (directed upward and lateral) interspersed with several dark brown simple longer hairs. Antenna composed of scape, pedicel and 9 flagellomeres, yellow to brown; 1st flagellomere elongate, 1.33 times as long as 2nd one. Maxillary palp light to medium brown, with 5 segments, proportional lengths of 3rd, 4th, and 5th segments 1.00:1.46:3.55;

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Fig. 3. Pupa and larva of Simulium (Gomphostilbia) panagudiense sp.n. (A & B) pupa; (C–E) larva. (A) left gill filaments (outer view); (B) terminal hooks (end view); (C) right mandible (lateral view); (D) hypostoma (ventral view); (E) head capsule showing postgenal cleft and hypostoma (ventral view). Scale bars. 0.01 for (B, C and E) 0.2 mm for (A and D).

3rd segment (Fig. 2A) widened apically; sensory vesicle (Fig. 2A) globular, small (0.27 times as long as 3rd segment), and with small opening. Thorax. Scutum slightly darker than female and short hairs on scutum golden yellow. Legs. Foreleg: coxa yellow; trochanter yellow with some portions light brown; femur light brown except apical cap brown; tibia brown with median 2/3 light brown and covered with dark brown hairs; tarsus brown to dark brown; basitarsus moderately dilated 7.1 times as long as its greatest width. Midleg: coxa yellowish brown; trochanter yellow to brown; femur yellow except apical 1/4 brown; tibia medium brown to dark brown; tarsus dark brown to brownish-black except anterior surface of little less than basal 1/2 of basitarsus dark yellow to light brown. Hind leg: coxa dark yellow to brown; trochanter yellow; femur light brown except apical 1/2 dark brown; tibia (Fig. 2B) brown except basal and apical dark brown; tarsus medium to dark brown except basal 1/2 (or little less) of basitarsus whitish-yellow and little less than basal 1/3 of 2nd tarsomere white; basitarsus (Fig. 2C) slender, parallel-sided, 6.16 times as long as wide, and 0.51 and 0.42 times as wide as greatest width of tibia and femur, respectively; calcipala (Fig. 2C) nearly as long as wide, and 0.43 times as wide as greatest width of basitarsus. Pedisulcus (Fig. 2C) well defined. Wing. Length 1.63 mm. Costa with dark brown spinules as well as dark brown hairs except basal portion with patch of yellowish hairs. Subcosta bare. Hair tuft on stem vein dark brown. Basal portion of radius fully haired; R1 with dark spinules and hairs and R2 with hairs only. Basal cell absent. Haltere. Yellowish brown except outer surface ochreous, basal stem darkened and apex white. Abdomen. Basal scale dark brown, with fringe of light to medium brown hairs. Dorsal surface of abdomen dark brown except segment 2 light brown (though posterior 1/4 of dorsal surface brown), covered with dark brown short to long hairs; segments 2–7 each with shiny dorsolat-

eral or lateral patches; ventral surface of segment 2 yellow, those of segments 3 and 4 yellow except sternites medium brown, and those of other segments medium to dark brown. Genitalia. Coxite in ventral view (Fig. 2D) nearly rectangle, 1.1 times as long as its greatest width. Style in ventral view (Fig. 2D) bent inward, slightly tapered from base toward middle, then nearly parallelsided, rounded apically and with apical spine; style in medial view (Fig. 2E) shorter than coxite (0.83 times as long as coxite), gently bent inward, nearly parallel-sided, with apical spine; style in ventro-lateral view (Fig. 2F) very slightly tapered toward apical 3/4, with rounded apex. Ventral plate in ventral view (Fig. 2G) with body transverse, 0.45 times as long as wide, slightly widened posteriorly, with anterior margin produced anteromedially, and posterior margin convex medially, densely covered with microsetae on ventral surface; basal arms of moderate length, directed forward, then slightly convergent apically; ventral plate in lateral view (Fig. 2H) moderately produced ventrally; ventral plate in end view (Fig. 2I) concave ventrally, densely covered with microsetae on posterior surface. Median sclerite (Fig. 2D,H) thin, plate-like, wide. Paramere (Fig. 2J) of moderate size, with 2 distinct long and stout hooks and several smaller ones. Aedeagal membrane (Fig. 2J) moderately setose, slightly sclerotized at base but dorsal plate not well defined. Ventral surface of abdominal segment 10 with 6–8 distinct hairs near posterior margin. Cercus in lateral view (Fig. 2K) small, rounded, with 10–12 hairs. Pupa: Body length 1.9–2.1 mm. Head. Integument dark yellow, sparsely covered with small round tubercles; antennal sheath with protuberance; face with pair of simple very long trichomes with uncoiled apices, and frons with 2 pairs of simple very long trichomes with coiled or uncoiled apices; 3 frontal trichomes on each side arising close together, subequal in length to one another and

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slightly longer than facial one. Thorax. Integument yellow, moderately covered with round tubercles, with 4 simple very long dorsomedial trichomes with uncoiled apices, 1 simple very long anterolateral trichome with uncoiled apex, 1 simple very long mediolateral trichome with uncoiled apex, and 1 simple ventrolateral trichome with uncoiled apex. Gill (Fig. 3A) composed of 8 slender thread-like filaments, arranged as [(1 + 2) + (1 + 2)] + 2 filaments from dorsal to ventral, with short common basal stalk having somewhat swollen transparent organ ventrally at base; common basal stalk 0.39 times length of interspiracular trunk; both primary and secondary stalks of dorsal triplet short, primary and secondary stalks of middle triplet medium-long; length of primary and secondary stalks of middle triplet combined slightly shorter than stalk of ventral pair; stalk of ventral pair medium-long, 1.7–1.75 times length of common basal stalk and 0.65–0.68 times length of interspiracular trunk; stalk of ventral pair 1.25–1.28 times as thick as primary stalk of middle triplet, and 1.1–1.2 times as thick as primary stalk of dorsal triplet; primary stalk of dorsal triplet lying against stalk of ventral pair at angle of 40–50 degrees or little more when viewed laterally; all filaments yellowish brown, gradually tapered toward apex; entire length of filaments (measured from base of gill to tips of filaments) based on one pupa as follows: 0.8–2.3 mm for dorsal triplet, 2.4–2.5 mm for middle triplet and 2.6–2.7 mm for ventral paired filaments; cuticle of all filaments with well-defined annular ridges and furrows though gradually becoming indistinct from middle to apex, densely covered with minute tubercles. Abdomen. Dorsally, segments 1 and 2 pigmented with tubercles; segment 1 with 1 simple slender medium-long hairlike seta on each side; segment 2 with 1 simple medium-long and 3 short hair like setae on each side; segments 3 and 4 each with 4 hooked spines and 1 short hair like seta on each side; segment 5 lacking spine–combs and 1 short hair like seta on each side; segments 6–9 each with spine-combs in transverse row (though those on segment 9 slightly smaller than those on segment 8) and comb-like groups of minute spines on each side; segment 9 with pair of triangular flat terminal hooks, of which outer margin is 4 times as long as inner margin and crenulated (Fig. 3B). Ventrally, segments 4 and 5 with 1 simple hook and segment 4 with few simple slender very short setae on each side; segments 5 with a pair of bifid hooks on each side; segments 6 and 7 each with bifid inner and unbranched outer teeth on each side; segments 4–8 with comb-like groups of minute spines. Each side of segment 9 with 3 grapnel-shaped hooklets. Cocoon. Wall pocket-shaped, thinly and moderately woven, widely extended ventrolaterally; anterior margin somewhat thickly woven, with dorsal portion slightly produced anteriorly when viewed dorsally; posterior 1/2 with floor roughly or moderately woven; individual threads visible; 2.3 mm long by 1.7 mm wide. Mature larva: Body length 3.8–4.5 mm. Body creamy to color markings as follows: ventral surface of thoracic segment 1 encircled with ochreous broad transverse band (though disconnected ventrally), proleg grayish, thoracic segments 2 and 3 grayish dorsally and each with distinct ochreous wide areas ventrally, abdominal segments 1–4 each encircled with yellowish brown broad band, abdominal segments 5–8 almost entirely covered by yellowish brown transverse broad band on dorsal and dorsolateral surfaces; abdominal segments 5 and 6 each with W-shaped broad transverse band on dorsolateral surfaces of posterior 1/2 of each segment; abdominal segment 7 and 8 with transverse yellowish brown band on ventral surface; Cephalic apotome yellowish brown, and sparsely covered with simple minute setae; head spots indistinct. Lateral surface of head capsule yellowish brown except eye-spot region yellow, and very sparsely covered with simple minute setae; spots indistinct. Ventral surface of head capsule yellowish brown except somewhat darkened area near posterior margin on each side of postgenal cleft, and very sparsely covered

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with simple minute setae. Antenna composed of 3 segments and apical sensillum, somewhat longer than stem of labral fan; proportional lengths of 1st, 2nd, and 3rd segments 1.00:0.91:1.2. Labral fan with 32 main rays. Mandible (Fig. 3C) with 3 comb-teeth decreasing in length from 1st to 3rd; mandibular serration composed of 2 teeth (1 medium-sized and 1 small); major tooth at acute angle against mandible on apical side; supernumerary serrations absent. Hypostoma (Fig. 3D) with row of 9 apical teeth; median and each corner tooth prominent (though median tooth slightly longer than corner teeth) and much longer than 3 intermediate teeth on each side; lateral margin smooth; 4 hypostomal bristles per side lying parallel to lateral margin. Postgenal cleft (Fig. 3E) lanceolate, 2.1 times as long as postgenal bridge. Cervical sclerite composed of 2 very pale small pieces, not fused to occiput, widely separated medially from each other. Thoracic cuticle bare. Abdominal cuticle almost bare except few posterior segments sparsely to moderately covered with simple minute setae dorsally and dorsolaterally and last segment densely covered with colorless simple setae on each side of anal sclerite. Rectal scales absent. Rectal papilla compound, each of 3 lobes with 5 finger-like secondary lobules. Anal sclerite of usual Xform, with anterior arms little shorter (0.9 times as long as posterior arms) than posterior ones, broadly sclerotized at base; accessory sclerite absent. Last abdominal segment expanded ventrolaterally forming double bulges on each side, visible as large conical ventral papilla when viewed from side. Posterior circlet with 63 rows of up to 11 hooklets per row. Etymology: The species is named after the place of collection, Panagudi. Habitat: S. (Gomphostilbia) panagudiense sp. n. larvae and pupae are mainly found on aquatic weed. 3.2. Simulium (Gomphostilbia) kottoorense Anbalagan sp.n. (Figs. 3–6) Type series. Holotype, ♂, INDIA: Anchu Nazhika river, Kottoor, Tiruvananthapuram district, Kerala state, 4-II-2014, 275 m (08◦ 33 .95 N, 77◦ 08 .49 E), S. Anbalagan (Coll. S. Anbalagan, Bharathidasan University (BDU)). Paratypes, same data as holotype: 6 ♂♂, 5 ♀♀, 9 pupae, 143 larvae (Coll. S. Anbalagan, BDU). 4 ♂♂, 3 ♀♀ (Department of Environmental Biotechnology, BDU, Catalogue number: BL0017). 3.2.1. Description Female: Body length 1.6 mm. Head. Narrower than width of thorax. Frons black, thinly whitish pruinose with several stout dark hairs along lateral and lower margins; frontal ratio 1.3:1.0:1.8; frons-head ratio 1.0:2.8. Fronto-ocular area well developed, narrow, directed dorso-laterally. Antenna composed of scape, pedicel and 9 flagellomeres, brown except scape, pedicel and basal 1/2 of 1st flagellomere yellow. Clypeus yellowish brown, shiny, thinly whitish pruinose. Labrum 0.6 times as long as clypeus. Maxillary palp composed of 5 segments; proportional lengths of 3rd, 4th and 5th segments 1.0:1.1:2.4; 3rd segment (Fig. 4A) enlarged; sensory vesicle (Fig. 4A) medium-sized (0.47 times as long as 3rd segment), with small-sized round opening. Maxillary lacinia with 12 inner and 19 outer teeth. Mandible with 32 inner and 12 outer teeth. Cibarium medially forming sclerotized plate folded forward from posterior margin, with moderately sclerotized medial longitudinal ridge. Thorax. Scutum dark brown except anterolateral calli dark ochreous, with 3 brownish-black longitudinal vittae (1 narrow median and 2 lateral), median vitta united anteriorly to anterior calli, lateral vittae united posteriorly to brownish-black prescutellar area; scutum shiny when illuminated at certain angles, densely covered with yellow scale-like recumbent short hairs interspersed with dark brown long upright hairs on prescutellar area. Scutellum brown, shiny when illuminated at certain angles, cov-

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Fig. 4. Female of Simulium (Gomphostilbia) kottoorense sp.n. (A) 3rd segment of right maxillary palp showing sensory vesicle (front view); (B) left hind tibia (outer view); (C) basitarsus and 2nd tarsomere of left hind leg showing calcipala and pedisulcus (outer view); (D) tarsal claw; (E) 8th sternite, ovipositor valves (ventral view); (F) genital fork (ventral view); (G & H) right paraprocts and cerci ((G) ventral view; (H) lateral view); (I) spermatheca (lateral view). Scale bars. 0.01 mm for (D) 0.02 mm for (A) and (E–I) 0.1 mm for (B and C).

ered with yellow short hairs and dark brown long upright hairs along posterior margin. Postnotum shiny and bare. Pleural membrane bare. Katepisternum medium to dark brown, longer than deep, shiny when illuminated at certain angles, moderately covered with fine short hairs. Legs. Foreleg: coxa yellow; trochanter yellow except apical portion somewhat darkened; femur light brown; tibia brown; tarsus brownish black with moderate dorsal hair crest; basitarsus slightly dilated, 4.5 times as long as its greatest width. Midleg: coxa yellowish brown except posterior surface dark brown; trochanter yellow; femur and tibia brown; tarsus brownish-black except basal 1/2 of basitarsus dark yellow. Hind leg: coxa yellowish brown except posterior surface dark brown; trochanter yellow; femur medium brown with base yellow and apical cap dark brown; tibia (Fig. 4B) light to dark brown with basal 1/4 white, covered with brownish fine hairs on outer and posterior surfaces; tarsus brown to dark brown except basal 2/3 of basitarsus (though base white); basitarsus (Fig. 4C) narrow, nearly parallel-sided, 7.1 times as long as wide, and 0.50 and 0.43 times as wide as greatest width of tibia and femur, respectively; calcipala (Fig. 4C) slightly shorter than width at base, and 0.41 times as wide as greatest width of basitarsus. Pedisulcus (Fig. 4C) well defined. Claw (Fig. 4D) with large basal tooth 0.66 times as long as claw. Wing. Length 1.75 mm. Costa with dark spinules and hairs except hairs on basal portion yellow. Subcosta with dark hairs except near apex bare. Hair tuft on stem vein dark brown. Basal section of vein R fully haired; R1 with dark spinules and hairs; R2 with hairs only. Basal cell absent. Haltere. Yellowish brown except apical light brown. Abdomen. Basal scale ochreous, with fringe of dark brown hairs. Dorsal surface of abdomen brown to brownish black except basal 1/2 of segment 2 yellow, moderately covered with brown short to long hairs; tergites of segment 2 and 6-8 shiny when illuminated at certain angles. Ventral surface of segments 2 and 3 whitish-yellow except lateral side brownish yellow, and those of other segments light to dark brown; sternal plate on segment 7 undeveloped. Genitalia. Sternite 8 (Fig. 4E) well sclerotized and bare medially, covered

with 7 long hairs and few short fine hairs on each side. Ovipositor valves tongue-like, thin, membranous, moderately covered with microsetae interspersed with four short hairs; inner margins concave, somewhat sclerotized, and slightly separated from each other. Genital fork (Fig. 4F) of usual inverted-Y forms; stem slender and well sclerotized; arms of moderate width, lateral plate of each arm with thin lobe directed medioposteriorly and small stout projection directed anterodorsally. Paraproct in ventral view (Fig. 4G) concave anterolaterally, with 3 sensilla on anteromedial surface; paraproct in lateral view (Fig. 4H) somewhat produced ventrally, 0.66 times as long as wide, with about 16 medium-long to long hairs on lateral and ventral surfaces. Cercus in lateral view (Fig. 4H) moderate, rounded posteriorly, 0.86 times as long as its greatest width. Spermatheca (Fig. 4I) ellipsoidal, 1.9 times as long as its greatest width, well sclerotized except duct and small area near juncture with duct unsclerotized, and with many fissures on surface; internal setae absent; both accessory ducts slender, subequal diameter to major one. Male: Body length 1.9 mm. Head. 0.7 times wider than thorax. Upper eye medium brown, consisting of 20 vertical columns and 12 horizontal rows of large facets. Face brownish black, grayishwhite pruinose. Clypeus black, whitish pruinose, densely covered with golden-yellow scale-like medium-long hairs (directed upward and lateral) interspersed with several dark brown simple longer hairs. Antenna composed of scape, pedicel and 9 flagellomeres, yellow to brown; 1st flagellomere elongate, 1.75 times as long as 2nd one. Maxillary palp light to medium dark brown, with 5 segments, proportional lengths of 3rd, 4th, and 5th segments 1.00:1.36:2.27; 3rd segment (Fig. 5A) widened apically; sensory vesicle (Fig. 5A) globular, small (0.33 times as long as 3rd segment), and with small opening. Thorax. Scutum darker than female and short hairs on scutum golden yellow. Legs. Foreleg: coxa yellow; trochanter yellow with some portions brown; femur light brown except apical cap brown; tibia brown with median 2/3 light brown and covered with dark brown hairs; tarsus brown to dark brown; basitarsus moder-

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Fig. 5. Male of Simulium (Gomphostilbia) kottoorense sp.n. (A) 3rd segment of left maxillary palp showing small sensory vesicle (frontal view); (B) left hind tibia (outer view); (C) basitarsus and 2nd tarsomere of left hind leg showing calcipala and pedisulcus (outer view); (D) coxites, styles, ventral plate and median sclerite (ventral view); (E & F) right styles ((E) medial view; (F) ventrolateral view); (G) ventral plate (ventral view); (H) ventral plate and median sclerite; (I) ventral plate (end view); (J) 10th abdominal segment and cercus (right side and ventrolateral view). Scale bars. 0.02 mm for (A) and (D–J) 0.1 mm for (B and C).

ately dilated, 6.8 times as long as its greatest width. Midleg: coxa yellowish brown; trochanter yellow to brown; femur yellow except apical 1/4 brown; tibia medium brown to dark brown; tarsus dark brown to brownish-black except anterior surface of little less than basal 1/2 of basitarsus dark yellow to light brown. Hind leg: coxa dark yellow to brown; trochanter yellow; femur light brown except apical 1/2 dark brown; tibia (Fig. 5B) brown except basal yellow and apical dark brown; tarsus medium to dark brown except basal 1/2 (or little less) of basitarsus whitish-yellow and little less than basal 1/3 of 2nd tarsomere white; basitarsus (Fig. 5C) slender, parallelsided, 4.8 times as long as wide, and 0.63 and 0.71 times as wide as greatest width of tibia and femur, respectively; calcipala (Fig. 5C) nearly as long as wide, and 0.33 times as wide as greatest width of basitarsus. Pedisulcus (Fig. 5C) well defined. Wing. Length 1.82 mm. Costa with dark brown spinules as well as dark brown hairs except basal portion with patch of yellowish hairs. Subcosta bare. Hair tuft on stem vein dark brown. Basal portion of radius fully haired; R1 with dark spinules and hairs and R2 with hairs only. Basal cell absent. Haltere. Yellowish brown except outer surface ochreous, basal stem darkened and apical brown. Abdomen. Basal scale dark brown, with fringe of light to medium brown hairs. Dorsal surface of abdomen dark brown except segment 2 light brown (though posterior 1/4 of dorsal surface brown), covered with dark brown short to long hairs; segments 2–7 each with shiny dorsolateral or lateral patches; ventral surface of segment 2 yellow, those of segments 3 and 4 yellow except sternites medium brown, and those of other segments medium to dark brown. Genitalia. Coxite in ventral view (Fig. 5D) nearly rectangle, 1.1 times as long as its greatest width. Style in ventral view (Fig. 5D) bent inward, slightly tapered from base toward apex, apical portion rounded and with apical spine; style in medial view (Fig. 5E) shorter than coxite (0.63 times as long as coxite), gently bent inward, nearly parallel-sided, with apical spine; style in ventro-lateral view (Fig. 5F) very slightly tapered toward apical 3/4, with rounded apex. Ventral plate in ventral view (Fig. 5G) with body transverse, 0.93 times as long as wide, narrowed

posteriorly, with anterior margin produced anteromedially, and posterior margin convex medially, densely covered with microsetae on ventral surface; basal arms of moderate length, directed forward, then straight apically; ventral plate in lateral view (Fig. 5H) moderately produced ventrally; ventral plate in end view (Fig. 5I) concave ventrally, densely covered with microsetae on posterior surface. Median sclerite (Fig. 5D, H) thin, plate-like, wide. Paramere of moderate size, with 2 distinct long and stout hooks and several smaller ones. Aedeagal membrane moderately setose, slightly sclerotized at base but dorsal plate not well defined. Ventral surface of abdominal segment 10 with 9 distinct hairs near posterior margin. Cercus in ventrolateral view (Fig. 5J) small, rounded, with 9 or 12 hairs. Pupa: Body length 2.1–2.3 mm. Head. Integument dark yellow, sparsely covered with small round tubercles; antennal sheath with protuberance; face with pair of simple very long trichomes with uncoiled apices, and frons with 3 pairs of simple very long trichomes with coiled or uncoiled apices; 3 frontal trichomes on each side arising close together, subequal in length to one another and slightly longer than facial one. Thorax. Integument yellow, moderately covered with round tubercles, with 3 simple very long dorsomedial trichomes with coiled or uncoiled apices, 2 simple very long anterolateral trichomes (1 with coiled apex, 1 with uncoiled apex), 1 simple very long mediolateral trichome with uncoiled apex, and 3 simple ventrolateral trichomes with uncoiled apices. Gill (Fig. 6A) composed of 8 slender thread-like filaments, arranged as [(1 + 2) + (1 + 2)] + 2 filaments from dorsal to ventral, with short common basal stalk having somewhat swollen transparent organ ventrally at base; common basal stalk 0.36 times length of interspiracular trunk; dorsal and middle triplet sharing short common stalks; both primary and secondary stalks of dorsal triplet short, primary and secondary stalks of middle triplet short and long respectively; length of primary and secondary stalks of middle triplet combined longer than stalk of ventral pair; stalk of ventral pair short, 0.7–0.8 times length of common basal stalk and

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Fig. 6. Pupa and larva of Simulium (Gomphostilbia) kottoorense sp.n. (A & B) pupa; (C–E) larva. (A) left gill filaments (outer view); (B) terminal hooks (end view); (C) right mandible (lateral view); (D) hypostoma (ventral view); (E) head capsule showing postgenal cleft and hypostoma (ventral view). Scale bars. 0.01 for (B, C and E) 0.1 mm for (D) 0.2 mm for (A).

0.28–0.31 times length of interspiracular trunk; stalk of ventral pair 1.17–1.19 times as thick as primary stalk of middle triplet, and 1.33–1.35 times as thick as primary stalk of dorsal triplet; primary stalk of dorsal triplet lying against stalk of ventral pair at angle of 40–50 degrees or little more when viewed laterally; all filaments yellowish brown, gradually tapered toward apex; entire length of filaments (measured from base of gill to tips of filaments) based on one pupa as follows: 1.3–1.4 mm for dorsal triplet, 1.8–1.9 mm for middle triplet and 2.0–2.1 mm for ventral paired filaments; cuticle of all filaments with well-defined annular ridges and furrows though gradually becoming indistinct from middle to apex, densely covered with minute tubercles. Abdomen. Dorsally, segments 1 and 2 pigmented, covered with tubercles; segment 1 with 1 simple slender medium-long hair-like seta on each side; segment 2 and 3 with 1 simple slender medium-long hair-like seta; segments 3 and 4 with 4 hooked spines and 1 short hair like seta on each side; segment 5 lacking spine–combs; segments 6–9 each with spine-combs in transverse row (though those on segment 9 slightly smaller than those on segment 8) and comb-like groups of minute spines on each side; segment 9 with pair of triangular flat terminal hooks of which outer margin is 2.3–2.5 times as long as inner margin and crenulated (Fig. 6B). Ventrally, segments 4 and 5 with 1 simple hook and few simple slender very short setae on each side; segments 6 and 8 with pair of bifid inner and simple outer hooks somewhat spaced from each other and few very short simple slender setae on each side; segments 4–8 with comb-like groups of minute spines. Each side of segment 9 with 3 grapnel-shaped hooklets. Cocoon. Wall pocket-shaped, thinly and moderately woven, widely extended ventrolaterally; anterior margin somewhat thickly woven, with dorsal portion slightly produced anteriorly when viewed dorsally;

posterior 1/2 with floor roughly or moderately woven; individual threads visible; 2.8 mm long by 1.8 mm wide. Mature larva: Body length 4.1–4.6 mm. Body creamy to color markings as follows: ventral surface of thoracic segment 1 encircled with ochreous broad transverse band (though disconnected ventrally), proleg grayish, thoracic segments 2 and 3 brownish dorsally and each with distinct ochreous wide areas ventrally, abdominal segments 1–4 each encircled with yellowish brown broad band, abdominal segments 5–8 almost entirely covered by yellowish brown transverse broad band on dorsal and dorsolateral surfaces; abdominal segments 5 and 6 each with W-shaped broad transverse band on dorsolateral surfaces of posterior 1/2 of each segment; abdominal segment 7 and 8 with transverse yellowish brown band on ventral surface; Cephalic apotome yellowish brown, and sparsely covered with simple minute setae; head spots indistinct. Lateral surface of head capsule yellowish brown except eye-spot region yellow, and very sparsely covered with simple minute setae; spots indistinct. Ventral surface of head capsule yellowish brown except somewhat darkened area near posterior margin on each side of postgenal cleft, and very sparsely covered with simple minute setae. Antenna composed of 3 segments and apical sensillum, somewhat longer than stem of labral fan; proportional lengths of 1st, 2nd, and 3rd segments 1.00:2.31:1.78. Labral fan with 27 main rays. Mandible (Fig. 6C) with 3 comb-teeth decreasing in length from 1st to 3rd; mandibular serration composed of 2 teeth (1 medium-sized and 1 small); major tooth at acute angle against mandible on apical side; supernumerary serrations present. Hypostoma (Fig. 6D) with row of 9 apical teeth; median and each corner tooth prominent (though median tooth slightly longer than corner teeth) and much longer than 3 intermediate teeth on

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Fig. 7. Strict consensus of the parsimonious trees of the members of the genus Simulium found in TNT (Boot strap values are above the nodes).

each side; lateral margin smooth; 4 hypostomal bristles per side lying parallel to lateral margin. Postgenal cleft (Fig. 6E) lanceolate, 1.7 times as long as postgenal bridge. Cervical sclerite composed of 2 dark small pieces, not fused to occiput, widely separated medially from each other. Thoracic cuticle bare. Abdominal cuticle almost bare except few posterior segments sparsely to moderately covered with simple minute setae dorsally and dorsolaterally and last segment densely covered with colorless simple setae on each side of anal sclerite. Rectal scales absent. Rectal papilla compound, each of 3 lobes with 5 finger-like secondary lobules. Anal sclerite of usual Xform, with anterior arms little shorter (0.8 times as long as posterior arms) than posterior ones, broadly sclerotized at base; accessory sclerite absent. Last abdominal segment expanded ventrolaterally forming double bulges on each side, visible as large conical ventral papilla when viewed from side. Posterior circlet with 64 rows of up to 12 hooklets per row. Etymology: The species is named after the place of collection, Kottoor.

Habitat: S. (Gomphostilbia) kottoorense sp.n. larvae were associated with leaf litter and woody debris. 3.3. DNA barcoding In the subgenus Gomphostilbia, COI gene sequences of four species (S. (Gomphostilbia) panagudiense sp. n., S. (Gomphostilbia) kottoorense sp. n., S. (Gomphostilbia) peteri and S. (Gomphostilbia) takaokai) and 35 outgroup species were taken for studying phylogenetic relationships. The COI sequences from 39 species (Table 1) had high AT content (65.7%), with an average nucleotide composition of A = 28.1%, C = 17.7%, G = 16.6%, and T = 37.6%. The two recovered topologies (TNT and ML) were almost identical, differing in the relative position of Gomphostilbia members. The ML tree reconstructed had a score of −5405.87 and the consistency index (CI) was 0.324. The strict consensus of TNT with bootstrap support (BSS) is shown in Fig. 7. Both methods (TNT and ML) recovered similar topologies with some clearly resolved and well-supported branches.

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The TNT tree revealed five main clades. Five species from three subgenera (S. (A.) oblongum, S. (S.) chiangmaiense, S. (S.) quinquestriatum, S. (Gomphostilbia) trangense, and S. (N.) chomthongense) formed distinct clades. The subgenera Nevermannia and Montisimulium formed a third clade. The subgenus Gomphostilbia was paraphyletic because its clade included a species of the subgenus Daviesellum. The first and second clades are the species of the subgenus Simiulium except S. (S.) nobile and S. (S.) chamlongi formed in a separate clade with 100% BSS. S. (Gomphostilbia) panagudiense sp. n. was the sister to S. (Gomphostilbia) peteri and S. (Gomphostilbia) takaokai with 95% BSS. S. (Gomphostilbia) kottoorense sp. n. was a separate clade. Both TNT and ML analyses recovered a highly supported relationship of S. (Gomphostilbia) panagudiense and S. (Gomphostilbia) kottoorense within the batoense species group.

panagudiense sp. n. and S. (Gomphostilbia) kottoorense sp. n.) is congruent with evidence of the batoense species group in the subgenus Gomphostilbia. The result of TNT and ML analyses are evident to the phylogenetic relationship using COI and ITS1 genes (Thanwisai et al., 2006; Phayuhasena et al., 2010; Pramual et al., 2011; Pramual and Adler, 2014) and morphological assignment (Takaoka, 2012; Adler and Crosskey, 2015) of black flies in the oriental region. However, the phylogenetic relationship of Simulium members using COI gene sequences indicates that the S. (G.) panagudiense sp. n. and S. (G.) kottoorense sp. n. are distinguished from the members of Gomphostilbia. Both the morphological and molecular data confirmed to S. (G.) panagudiense sp. n. and S. (G.) kottoorense sp. n. as new species. Acknowledgements

4. Discussion In the genus Simulium, the diagnostic characters of subgenus Gomphostilbia proposed by Takaoka (2003) there are two major key characters that (a) female claws (usually bear a large basal tooth, but rarely have a minute to medium-long subbasal tooth or lack any tooth) and (b) presence of terminal hooks in the pupa (small and cone-like, or small and flat, with round apex, or widened outwardly). The characters of large basal tooth in female and the presence of terminal hooks in the pupa of S. (Gomphostilbia) panagudiense sp. n. and S. (Gomphostilbia) kottoorense sp. n. are confirmed the subgenus Gomphostilbia. Among 15 species groups proposed in Gomphostilbia (Takaoka, 2003), S. (Gomphostilbia) panagudiense sp. n. and S. (Gomphostilbia) kottoorense sp. n. are assigned to the batoense species-group by having the following characters: antenna with scape, pedicel and nine flagellomeres; pleural membrane bare; katepisternum haired; hair tuft on the base of radial vein dark brown; mid and hind tibiae darkened except the base yellowish in the female and male; eight gill filaments and grapnel-like hooklets in the pupa. S. (Gomphostilbia) panagudiense, new species, is characterized by the female the scutum without longitudinal vitta and arms of the genital fork which are wide basally, in the male by the coxite 1.1 times as long as its greatest width, in the pupa by the mediumlong primary and secondary stalks of middle triplet, and in the larva by the postgenal cleft 2.1 times as long as postgenal bridge. S. (Gomphostilbia) panagudiense sp. n. is very similar to those of S. (Gomphostilbia) peteri and S. (Gomphostilbia) takaokai in many characteristics including the medium-sized sensory vesicle (Fig. 1A) and tarsal claw with large basal tooth (Fig. 1D) in the female, upper eye large facets in 12 vertical columns in the male and medium-long stalk of ventral pair in the pupa (Fig. 3A). S. (Gomphostilbia) kottoorense sp. n. is morphologically similar to S. (Gomphostilbia) pattoni and S. (Gomphostilbia) peteri in sharing the scutum with three brownish-black longitudinal vittae in the female, the slender, parallel-sided male hind basitarsus (Fig. 5C) and short common basal stalk of pupal respiratory filaments (Fig. 6A). However, this new species is distinguished from S. (Gomphostilbia) pattoni by the tarsal claw with large basal tooth (Fig. 1D) in the female, upper eye large facets in 20 vertical columns in the male and small stalk of ventral pair (Fig. 6A) in the pupa. This new species is distinguished from S. (Gomphostilbia) peteri by the arm of genital fork tapered near apex (Fig. 4F) in the female, style in medial view 0.63 times as long as coxite (Fig. 5E) in the male, stalk of middle triplet longer (Fig. 6A) in the pupa, and in the larva supernumerary serrations present in mandible (Fig. 6C). The phylogenetic analysis of the species from six subgenera of the genus Simulium using COI gene showed more divergence with the existing morphological classification (Adler and Crosskey, 2015). In the present study, two new species (S. (Gomphostilbia)

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