Simulium phurueaense Tangkawanit, Wongpakam & Pramual, 2018

Tangkawanit, Ubon, Wongpakam, Komgrit & Pramual, Pairot, 2018, A new black fly (Diptera: Simuliidae) species of the subgenus Asiosimulium Takaoka & Choochote from Thailand, Zootaxa 4388 (1), pp. 111-122 : 113-119

Morphological description of Simulium phurueaense Tangkawanit, Wongpakam & Pramual sp. nov.

Simulium phurueaense sp. nov. was assigned into subgenus Asiosimulium according to the diagnostic characters of this subgenus described by Takaoka and Choochote (2005) and keys to subgenera of genus Simulium in the Oriental region ( Takaoka et al. 2017) as follows; in both sexes, katepisternum and pleural membrane bare, basal portion of the radial vein haired. In female, cibarium with numerous spines, claws with large basal tooth. In male, style much shorter than coxite, ventral plate transverse, paramere without hooks. In pupa, gill with 30–33 filaments. In larva with deep postgenal cleft.

Female (n = 5). Body length 3.0–4.0 mm, Head narrower than thorax. Frons brownish black, thinly whitishgrey pruinose, not shiny, densely covered with yellowish white hairs (except median longitudinal portion bare) interspersed with several dark longer and stouter hairs along each lateral margin; median suture on lower portion absent, Frontal ratio 1.16–1.8: 1: 1.14–1.67. Frons-head ratio 1.0:4.0–4.5; fronto-ocular area well developed, triangular, directed laterally. Clypeus dark brown, whitish-grey pruinose; densely covered with yellowish white hairs except narrow portion near upper margin, long dark hairs and stout hairs along lateral margins and near lower margin. Labrum 0.6–0.7X as long as clypeus. Antenna ( Fig. 1A ) composed of scape, pedicel, and 9 flagellomeres, scape and pedicel yellowish white, nine of flagella segments brownish black. Maxillary palp ( Fig. 1B ) composed of 5 segments, 1 st segment whitish gray; 2nd segment with anterior surface dark brown, posterior surface whitish gray; 3rd segment blackish brown; 4th segment, anterior surface dark brown, posterior surface whitish gray, 5th whitish gray; proportional lengths of 3rd, 4th, and 5th segments 1.0:1.0:1.4; sensory vesicle ( Fig. 1C ) ellipsoidal, 1.5 times as long as wide, and 0.32X as long as 3rd segment, with large opening, Lacinia with 10 or 11 inner and 12–14 outer teeth. Mandible with 20 inner and 12–13 outer teeth. Cibarium ( Fig. 1D ) spinose, bearing ~ 92–115 minute pointed processes elaborately arranged on anterior surface. Thorax. Scutum medium to dark brown with 3 brownish black longitudinal vittae (1 median, 2 submedian), shiny at certain angle of light, densely covered with yellowish white short hairs mixed with several dark upright long hairs on prescutellar area. Scutellum medium brown, with many dark upright long hairs as well as yellowish white short hairs. Postnotum dark brown, slightly shiny at certain angle of light, bare. Pleural membrane bare. Katepisternum longer than deep, dark brown, shiny at certain angle of light, bare. Furcasternum with distinct ventrally directed apodemes on internal dorsal arms. Legs, foreleg; dark brown except coxa yellowish brown, trochanter medium brown; midleg, coxa and trochanter yellowish brown, femur medium brown, tibia, tarsus and pretarsus dark brown; hind leg ( Fig. 1E ), coxa medium brown, trochanter light brown, femur medium brown, tibia and tarsus dark brown. Fore basitarsus moderately dilated, about 6.0X as long as its greatest width, with moderate dorsal hair crest. Hind basitarsus nearly parallelsided on basal 2/3, then somewhat narrowed toward apex, 6.2–6.5X as long as its greatest width, 0.7X and 0.5X as wide as the greatest widths of tibia and femur respectively; calcipala well developed, slightly shorter than its width at base, and about 0.35X as wide as apical portion of basitarsus, pedisulcus well developed. All claws each with large basal tooth ( Fig. 1F ) 0.35X as long as claw. Wing. Length 2.2–3.0 mm. Costa with 2 parallel rows of dark short spines as well as dark hairs, subcosta with dark hairs along horizontal vein. Hair tuft on stem vein dark. Basal portion of radius fully haired. Basal cell, and basal median cell absent. Abdomen. Basal scale medium to dark brown, with fringe of yellowish long hairs. Dorsal surface of abdomen medium to dark brown, densely covered with yellowish hairs interspersed with dark hairs; ventral surface dark ocherous on segments 2–4, light to medium brown on other posterior segments; segment 7 with large sternal plate medially. Genitalia. Sternite 8 ( Fig. 1G ) wide, bare medially but laterally with 16–18 short and medium–long hairs on each side. Ovipositor valve nearly triangular, thin, membranous except inner margin narrowly sclerotized, densely covered with microsetae (except narrow portion along posterior margin bare) interspersed with 9–12 short hairs; inner margins nearly straight, narrowly separated from each other. Genital fork ( Fig. 1H ) inverted-Y shaped, with well sclerotized stem and wide arms, and with a narrow deep incision between arms; each arm with a distinct projection directed anterodorsally on dorsal surface; paraproct ( Fig. 1I –J ) slightly protruding ventrally (in lateral view), with numerous short and medium–long hairs on ventral and lateral surfaces, and with 14 or 15 spinous colorless sensilla on sclerotized anteromedial surface; cercus elongate, much longer than paraproct, rounded outwardly (in ventral view), and with numerous short and medium-long hairs; cercus in lateral view gradually narrowed posteriorly, with rounded apex, about twice as long as its width at base. Spermatheca ( Fig. 1K ) ovoid, strongly sclerotized (except area around its junction with duct unsclerotized), with distinct reticulate surface pattern, and with internal setae; accessory ducts subequal in diameter to each other and slightly larger in diameter than that of main duct.

Male (n = 3). Body length 2.5–3.0 mm. Head Slightly wider than thorax. Holoptic. Upper eye consisting of large facets in 19 vertical columns and 20 horizontal rows. Clypeus brownish black, white pruinose, and moderately covered with dark simple long hairs interspersed with pale shorter hairs (median portion widely bare), Antenna composed of scape, pedicel, and 9 flagellomeres; dark brown to brownish black, except scape and pedicel pale yellow; maxillary palp composed of 5 segments, the color nearly as in female, proportional lengths of 3rd, 4th, and 5th segments 1.0:1.0:1.5; sensory vesicle ( Fig. 2A ) globular or slightly ellipsoidal, about 0.35X times as long as 3rd segment, Thorax, Nearly as in female. Legs, foreleg; dark brown except coxa trochanter and femur median brown; midleg, dark brown except trochanter, femur and tibia median brown; Hind leg, coxa median brown, trochanter light brown, femur median brown, tibia and tarsus dark brown. Fore basitarsus moderately dilated, about 7.0X as long as its greatest width. Hind basitarsus ( Fig. 2B ) spindle shaped, 5.0X as long as its greatest width, 0.8X and 0.75X as wide as the greatest widths of tibia and femur, respectively; calcipala well developed, slightly shorter than its width at base and about 0.30X as wide as apical portion of barsitarsus, pedisulcus well developed. Wing. Length 2.3–3.0 mm. Other characters as in female except horizontal vein bare. Abdomen. Basal scale brownish black with fringe of long dark hairs. Dorsal surface of abdomen entirely brownish black, and moderately covered with dark simple hairs Genitalia. Coxite ( Fig. 2C–D ) subquadrate, much longer than wide. Style short, 0.65X as long as coxite, tapered apically, bent inward, rounded apically, and with a spine at apex when viewed ventrally; style broad on basal 1/2, then abruptly tapered apically when viewed posterolaterally. Ventral plate ( Fig. 2E–F ) lamellate, much shorter than wide, well sclerotized (especially anterior margin and arms), rounded posterolaterally, with posterior margin concave medially when viewed ventrally, and moderately covered with fine short setae on ventral and posterior surfaces (except each lateral area on ventral surface bare); arm of moderate length, slender, directed forwardly, with apex bent inwardly. Paramere ( Fig. 2G ) narrow, without parameral hook. Median sclerite ( Fig. 2H ) narrow basally, then widened apically. Aedeagal membrane with simple minute spines, and with weakly sclerotized broad dorsal plate basally. Ventral surface of 10th segment without hair. Cercus small, very low in lateral view, encircled with 9–11 simple hairs.

Pupa (n = 3). Body length 3–3.5 mm. Head. Integument dark yellow moderately covered with tubercles of various size, frons with 2 simple trichomes on each side, face with 1 long simple trichome on each side. Thorax. Integument dark yellow moderately covered with tubercles; 9 simple trichomes on each side; with 2 mediodorsally, 2 mediolaterally, 1 posterolaterally, and 3 ventrolaterally. Gill ( Fig. 3A ) composed of 30–32 slender filaments, number and configurations of filaments different in individual pupae. Arrangement from dorsal to ventral for pupa with 30 filament is (4+3) +(1+2)+2+2+[1+(1+2)]+(1+2) +(1+2)+(2+1+1+2), with 31 filaments is (2+2)+2]+(2+1)+2+[2+2+1]+(1+2)+(2+1+2)+ [(1+2)+(2+2)], and for pupa with 32 filaments is [(2+2)+2]+(2+1)+2+[2+(2+1+1+2+1)]+ [2+(2+1+1)]+[2+(2+2)]. Abdomen. Dorsally, all segments moderately sclerotized and yellowish; segment 1 and 2 covered with small tubercles, with one short slender seta on each side; segments 3 and 4 each with four stout hooks and one short spinous seta on each side; segments 5–7 each with comb-like groups of minute spines, segments 8–9 each with 2 pairs of short stout hooks on each side. Segment 9 with pair of small cone-shaped terminal hooks. Laterally, segments 2–4 each with three short spinous setae on each side; segment 9 with two grapnel-shaped hooklets on each side in male pupae. Ventrally, all segments unpigmented except segment 9 yellowish; segment 4 with pair of unbranched dark hooklets and few short setae on each side; segments 5–7 each with pair of unbranched stout dark hooks and few short setae on each side; segments 4–8 each with comb like groups of minute spines on each side. Cocoon ( Fig. 3B ). Wall-pocket shaped, moderately woven, without anterodorsal margin, slightly extended ventrolaterally. Individual threads visible; 4.0– 4.5 mm long by 1.5– 2.0 mm wide.

Mature larva (n = 5). Body length 6.5– 6.7 mm. Body color greyish brown to brownish; abdomen gradually widened toward segment 6 then narrowed posteriorly. Cephalic apotome whitish on anterior 1/2, whitish yellow on posterior 1/2 and darkened brown along posterior margin; head-spots distinct, medium dark brown except anterior spot of posterolateral spots on each side light brown; posterior spot of posterolateral on each side merged in medium brown background and posterior of posteromedial longitudinal spots connected to dark background color. Lateral surface of head capsule somewhat yellowish brown except eye-spot region and anterior portion whitish or whitish yellow; lateral surface of head capsule yellowish brown, except area above and posterior of eyespot light to median brown, eyebrow moderately defined; 2 large and 1 small spots near posterior margin and 2 small spots just below eye-spot region prominent, though 2 large spots sometimes merged in dark background color; ventral surface of head capsule light brown, with dark basal area on each side of postgenal cleft; horizontal and round spots on each side of postgenal cleft distinctly positive, cervical sclerites composed of 2 small elliptical pieces, not fused to occiput, very widely separated medially from each other. Antenna consisting of 3 articles and apical sensillum, subequal in length to stem of labral fan; proportional lengths of lst, 2nd, and 3rd segments 1.0:1.2:1.0. Labral fan with 42–45 main rays, mandible ( Fig. 4A ) with mandibular serrations consisting of 2 teeth (1 large, 1 small); large tooth at a right angle to mandible on apical side; comb-teeth composed of 3 teeth decreasing in size from lst to 3rd; supernumerary serrations absent; mandibular phragma extended to maxilla base. Hypostoma ( Fig. 4B ) with 9 apical teeth in row; median and corner teeth well developed; median tooth of 3 intermediate teeth on each side smallest; 8 hypostomal bristles per side, lying slightly divergent posteriorly from lateral margin. Postgenal cleft ( Fig. 4C ) deep, leaving narrow bridge up to posterior margin of hypostoma, almost reaching posterior border of hypostoma nearly parallel-sided on basal 2/3, then converging apically forming a round extension, postgenal cleft with pigmented subesophageal ganglion. Prothoracic proleg with dorsolateral sclerite well defined. Abdominal cuticle bare except both sides of anal sclerite moderately covered with simple colorless setae. Rectal scales not discernible. Rectal organ compound, each of 3 lobes with 9–11 finger-like secondary lobules, anal sclerite ( Fig.

4D) X-shaped, with anterior arms 0.6–0.7X as long as posterior ones; anterior arms broadened, and space between arms widely sclerotized basally; 10–15 colorless sensilla on posterior to basal junction area; accessory sclerite absent. Last abdominal segment somewhat expanded ventrally forming ventral bulge, visible as a very small ventral papilla when viewed from side, Posterior circlet with ca. 80 rows of up to 15 hooklets per row.

Type specimens. HOLOTYPE. Male, adult with associated pupal exuviae and cocoon: THAILAND, Loei Province, Phu Ruea District, Phu Ruea Mountain (17° 30′ 20′′ N 101° 20′ 30′′E), collected from small (width = 1.5 m), temporary stream with moderate flow (0.59 m /s) in open area, 1,220 m, 28. V. 2017. leg. U. Tangkawanit, P. Pramual, K. Wongpakam. Deposited in the Department of Biology, Mahasarakam University, Thailand.

PARATYPES. Adults: 2 males, 3 females (preserved in 80% ethanol). Pupae: 9; mature larvae (20) (preserved in Carnoy’s solution and then transferred to 80% ethanol), same data as those of holotype.

Biological notes. Immature stages (i.e. larva and pupa) of this species co-exist with S. (Nevermannia) aureohirtum Brunetti, 1911 and S. (Nevermannia) feuerborni . The biting habit of this species has not yet been confirmed but females are attracted to humans. In the area near the type locality, about 20 females were attracted to the head and face but they did not land. The observation times were 7.00–8.00 am and 11.00–12.00 am. The ecological conditions in which the immature stages (i.e. larva and pupa) of this species were found are shown in Table 1. The new species occurs in small streams with a wide range of stream velocity ( Table 1) in both forest and open areas. However, we have found more larvae in open habitats than in the forest areas. This new species is restricted to high elevations (> 1,100 m).

Etymology. The species name phurueaense refers to the locality name, Phu Ruea, where this new species was collected.

Mitochondrial DNA sequences analyses. Recognition of the new species, S. phurueaense sp. nov. is well supported by genetic distance and phylogenetic analyses. A total of 11 sequences for S. phurueaense sp. nov. and nine sequences for S. oblongum were included in genetic variation analysis ( Table 2 ). Simulium phurueaense shows slightly lower diversity compared to the closely related species, S. oblongum . Maximum intraspecific genetic distance based on COI sequences for S. oblongum is 2.44% with an average of 1.33% compared to 1.38% with mean of 0.74% for S. phurueaense sp. nov. These two species show distinct genetic differentiation with minimum genetic distance of 3.51% ( Table 3).

Phylogenetic analyses support the genetic distance between these species as two well-supported clades were recovered ( Fig. 5 ). All specimens of S. phurueaense formed a monophyletic clade (clade I) with strong support (>90%). Members of S. oblongum formed another monophyletic clade (clade II) although the node support varied between phylogenetic methods ( Fig. 5 ).

Figures

FIGURE 1. Female of Simulium (Asiosimulium) phurueaense sp. nov. (A) Antenna (B) Head part showing the maxillary palp (anterior view) (C) Third segment of maxillary palpshowing sensory vesicle (right side; front view) (D) Cibarium (E)Hide leg (F) Claw showing large basal tooth (G) Sternite 8 and ovipositor valves (H) Genital fork (ventral view). (I-J) Paraprocts and cerci (I, lateral view, J, ventral view) (K) Spermatheca.Scale bars.I = 0.1 mm, D = 0.05 mm, A–C and E–K = 0.02 mm.

FIGURE 2. Male of Simulium (Asiosimulium) phurueaense sp. nov. (A) 3rd maxillary palp with sensory vescicle (B) hind basitarsus (C) coxites, styles and ventral plates (ventral view) (D) coxites and styles (lateral view) (E) ventral plate (end view) (F) ventral plate with median sclerite (lateral view) (G) paramere (left side, lateral view) (H) mediansclerite (end view). Scale bar.B = 0.05 mm, A, C–D and E–H = 0.02 mm.

FIGURE 4. Larva of Simulium (Asiosimulium) phurueaense sp. nov. (A) Mandible. (B) Hypostoma. (C) Head capsule showing postgenal cleft (ventral view). (D) Anal sclerite (top view). Scale bar.A = 0.01 mm, B–D = 0.1mm.

FIGURE 5. Neighbor-joining tree of Simulium phurueaense sp. nov. and S. oblongum based on mitochondrial cytochrome c oxidase I sequences. Bootstrap values for neighbor-joining and posterior probability of maximum likelihood and Bayesian analysis are shown above or near the branches—denotes bootstrap support less than 50%.Scale bar represents 0.009 substitutions per nucleotide position.

FIGURE3. Pupaof Simulium (Asiosimulium) phurueaensesp.nov. (A) pupal gill filaments (leftside, outer view).(B) Cocoon (lateral view).Scale bar. A = 0.2 mm, B = 1 mm.

Tables

TABLE 2. Details of specimens of Simulium (Asiosimulium) phurueaense sp. nov. and Simulium (Asiosimulium) oblongum used for molecular analyses.

Species	Location	Code1	GenBank Accession Number
S. phurueaense sp. nov.	Unnamed stream 3 along the road to	AS4464L	MF616451
	Hin Sam Chan waterfall, Loei Province	AS4465L	MF616452
		AS4466P	MF616453
		AS4467P	MF616454
		AS4468P	MF616455
		AS4469A	MF616456
		AS44610A	MF616457
	Unnamed stream 1 along the road to	Asio4201	MF616458
	Lert Pop waterfall	Asio4202	MF616459
		Asio4203	MF616460
		Asio4204	MF616461
S. oblongum	Kuchinarai 1, Kalasin Province	OB1961	KF289391 2
	Huai Rai, Nong Sung, Mukdahan	OB1971	KF289392 2
	Phupakud 1, Mukdahan	OB1991	KF289393 2
	Kuchinarai 2, Kalasin Province	OB2331	MF616445
		OB2332	MF616446
	Phupakud 2, Mukdahan	OB2341	MF616447
		OB2342	MF616448
	Phupakud 3, Mukdahan	OB2351	MF616449
		OB2352	MF616450

1Code of specimen as shown in FIGURE 5 , 2 Sequences are from the study of Pramual & Adler (2014); all other sequences are from the current study.