- Email: [email protected]
A new species of the Simulium (Gomphostilbia) (Diptera: Simuliidae) from Thailand, with its phylogenetic relationships with related species in the Simulium asakoae species-group
Acta Tropica 197 (2019) 105043
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
A new species of the Simulium (Gomphostilbia) (Diptera: Simuliidae) from Thailand, with its phylogenetic relationships with related species in the Simulium asakoae species-group Wichai Srisukaa, Hiroyuki Takaokab, Masako Fukudac, Yasushi Otsukad, Atiporn Saeunge,
T
⁎
a
Entomology Section, Queen Sirikit Botanic Garden, P.O. Box 7, Maerim, Chiang Mai 50180, Thailand Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Kuala Lumpur 50603, Malaysia c Institute for Research Promotion, Oita University, Idaigaoka 1-1, Hasama, Yufu City, Oita 879-5593, Japan d Research Center for the Pacific Islands, Kagoshima University, Korimoto 1-21-24, Kagoshima City, Kagoshima 890-8580, Japan e Center of Insect Vector Study, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand b
A R T I C LE I N FO
A B S T R A C T
Keywords: Aquatic insects Taxonomy Biodiversity Oriental Region
A new species of black fly, Simulium (Gomphostilbia) rampae, is described, based on adult male, its pupal exuviae and mature larvae collected from Doi Inthanon National Park, northern Thailand. This new species is placed in the Simulium asakoae species-group, and is characterized in the male by the high number of upper-eye facets in 17 vertical columns and 18 horizontal rows, in the pupa by the gill with a long common basal stalk, cone-shaped terminal hook, and cocoon with an anterodorsal projection, and in the larva by the medium-long postgenal cleft. A DNA analysis using COI gene supported its assignment to the S. asakoae species-group and showed its close relationship to S. (G.) udomi Takaoka & Choochote and S. (G.) chiangdaoense Takaoka & Srisuka. This is the fourth member of the S. asakoae species-group recorded from Thailand.
1. Introduction
2. Material and methods
The fauna of black flies (Diptera: Simuliidae) in Thailand is characterized by its richness in species number and high lineage diversity. It consists of 110 species, of which 85 species are assigned in two dominant subgenera (34 in Gomphostilbia Enderlein and 51 in Simulium Latraille), and remaining 25 species are in four other small subgenera in the genus Simulium Latreille (Takaoka et al., 2019). The species in these two dominant subgenera, Gomphostilbia and Simulium, are placed in eight and eleven species-groups, respectively, showing a high diversity in phylogenetic lineage (Adler and Crosskey, 2018; Takaoka, 2012, 2017). Recently, we collected one undescribed species of the subgenus Gomphostilbia from Doi Inthanon National Park, northern Thailand. It is described as a new species, and a COI gene sequence-based analysis on its assignment to species group and phylogenetic relationship to related species are carried out.
2.1. Description of a new species One adult male reared from a pupa, its associated pupal exuviae and six mature larvae collected from Doi Inthanon National Parks, Chiang Mai, Thailand, were used. The methods of description and illustration, as well as terms for morphological features used here, follow those of Takaoka (2003) and partially those of Adler et al. (2004). The holotype and paratypes of the new species are deposited at Entomology Section of the Queen Sirikit Botanic Garden, Chiang Mai, Thailand. 2.2. DNA analysis Two mature larvae of the new species and a female of S. (G.) chiangdaoense Takaoka & Srisuka were used. The procedures for DNA extraction, PCR amplification, and sequencing follow those of Low et al. (2015) with slight modification. DNA was extracted from each specimen with a DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). PCR amplification of the partial mitochondrial COI gene region was
⁎
Corresponding author. E-mail addresses: [email protected] (W. Srisuka), [email protected] (H. Takaoka), [email protected] (M. Fukuda), [email protected] (Y. Otsuka), [email protected] (A. Saeung). https://doi.org/10.1016/j.actatropica.2019.105043 Received 30 April 2019; Received in revised form 29 May 2019; Accepted 29 May 2019 Available online 30 May 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
carried out in a final volume of 50 μL containing 50 ng of DNA, 1 × Ex Taq buffer including 2 mM Mg2+, 200 μM each of dNTPs, 1.25 units of Ex Taq Polymerase (TAKARA BIO INC., Kusatsu, Japan), and 10 pmol of each forward (LCO1490) and reverse primer (HCO2198) (Folmer et al., 1994). Purified PCR products were directly sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems). The sequences determined were deposited in DDBJ/ EMBL/GenBank under the following accession numbers LC472507–LC472509. The sequences of the new species were aligned with those of nine related species (six spp. including S. (G.) chiangdaoense of the S. asakoae species-group and three of the S. ceylonicum species-group) and two other species as outgroup (S. (S.) tani Takaoka & Davies and S. (Nevermannia) feuerborni Edwards. Using this alignment, sequences were compared and a neighbor-joining tree was constructed by MEGA6 (Tamura et al., 2013) based on 586 positions. The Kimura 2-parameter method was used to estimate evolutionary distances in the tree.
brownish black. Hind leg: coxa dark brown though apical small portion yellow; trochanter yellow; femur medium brown with base yellow and apical cap dark brown (though apical tip yellow); tibia medium to dark brown except basal two-fifths yellow to ochreous (though posterior surface somewhat darkened) and apical cap brownish black; tarsus (Fig. 1C) medium brown except basal two-fifths of basitarsus (border not well defined) and basal one-fourth of second tarsomere grayish yellow; basitarsus (Fig. 1C) enlarged, 3.8 times as long as wide, and 0.9 and 1.1 times as wide as greatest width of tibia and femur, respectively; calcipala (Fig. 1C) slightly shorter than basal width, and 0.32 times as wide as greatest width of basitarsus; pedisulcus (Fig. 1C) well defined. Wing. Length 2.5 mm. Costa with dark spinules and hairs. Including patch of hairs at base. Subcosta with 14 or 15 hairs. Hair tuft on humeral plate black. Hair tuft on base of radial vein (= stem vein) yellow. Basal portion of radius fully haired; R1 with dark spinules and hairs; R2 with hairs only. Basal cell absent. Halter. Grayish except basal stem darkened. Abdomen. Basal scale dark brown, with fringe of light brown hairs. Dorsal surface of abdomen brownish black to black, covered with dark-brown short to long hairs; segments 2 and 5–7 each with pair of shiny dorsolateral or lateral patches. Genitalia. Coxite in ventral view (Fig. 1D) nearly rectangular, 1.8 times as long as its greatest width. Style in ventral view (Fig. 1D) bent inward, bluntly rounded apically and with apical spine; style in ventrolateral view (Fig. 1E) slightly tapered toward apex, and 0.85 times as long as coxite. Ventral plate in ventral view (Fig. 1D) with body transverse, 0.46 times as long as wide, with anterior margin produced anteromedially, and posterior margin slightly concave medially, lateral margins slightly widened posteriorly, and densely covered with micro-setae on ventral surface; basal arms of moderate length, directed forward, then convergent apically; ventral plate in lateral view (Fig. 1F) moderately produced ventrally; ventral plate in end view (Fig. 1G) rounded ventrally, densely covered with microsetae on posterior surface except portion near each dorsolateral tip narrowly bare. Median sclerite (Fig. 1H) thin, plate-like, wide. Paramere (Fig. 1I) of moderate size, each with four distinct stout hooks. Aedeagal membrane (Fig. 1J) densely setose, not sclerotized at base; dorsal plate not defined. Ventral surface of abdominal segment 10 (Fig. 1K and L) with anterior portion well sclerotized and brownish, and with one distinct hair or without hairs near posterior margin. Cercus in lateral view (Fig. 1K and L) small, rounded, with 12–16 hairs. Pupa. Body length 2.4 mm. Head. Integument yellow, moderately covered with small round tubercles except antennal sheaths and ventral surface almost bare; antennal sheath without any protuberances; face with pair of unbranched long trichomes with coiled apices, and frons with three pairs of unbranched 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, and with three long dorsomedial trichomes with coiled or uncoiled apices, two long anterolateral trichomes (one with coiled apex, one with uncoiled apex), one medium-long mediolateral trichome with uncoiled apex, and three ventrolateral trichomes with uncoiled apices (one medium-long, two short) on each side; all unbranched. Gill (Fig. 2A and B) composed of seven or eight slender thread-like filaments, arranged as 3 + 2+(2 + 1) or 2 + 2+(2 + 1) from dorsal to ventral, with long common basal stalk having somewhat swollen transparent basal fenestra at base; common basal stalk 1.3–1.5 times length of interspiracular trunk; all filaments light brown, gradually tapered toward apex; cuticle of all filaments with well-defined annular ridges though becoming less marked apically, densely covered with minute tubercles, of which relatively larger ones on ridges. Abdomen. Dorsally, all segments light yellow and without tubercles; segment 1 with one unbranched slender short hair-like seta on each side; segment 2 with one unbranched slender short hair-like seta (though much longer than seta on segment 1) and five minute setae submedially on each side; segments 3 and 4 each with four hooked spines and one or two minute setae on each side; segment 5 lacking spine-combs on each side;
2.3. Ethical clearance This study was approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chiang Mai University (Protocol Number 46/2561), Chiang Mai province, Thailand. 3. Results and discussion 3.1. Description of a new species 3.1.1. Simulium (Gomphostilbia) rampae Takaoka, Srisuka & Saeung sp. nov. Male. Body length 2.5 mm. Head. Somewhat wider than thorax. Upper eye medium brown, consisting of large facets in 17 vertical columns and 18 horizontal rows. Clypeus brownish black, whitish pruinose, densely covered with golden-yellow scale-like medium-long hairs (mostly directed upward) interspersed with several dark-brown longer hairs. Antenna composed of scape, pedicel and nine flagellomeres, medium to dark brown except scape, basal half of pedicel yellow (though ventral suface of pedicel entirely yellow) and base of first flagellomere yellow; first flagellomere elongate, 1.8 times length of second one. Maxillary palp light brown except third palpomere dark brown, with five palpomeres, proportional lengths of third, fourth, and fifth palpomeres 1.0:1.2:2.4; third palpomere (Fig. 1A and B) slender; sensory vesicle (Fig. 1A and B) ellipsoidal, small (0.2 times length of third palpomere), and with opening of small or medium-size. Thorax. Scutum brownish black to black without longitudinal vittae, shiny and thinly gray pruinose on shoulders, on wide area along each lateral margin and on prescutellar area when illuminated at certain angles, densely covered with golden-yellow scale-like recumbent hairs. Scutellum brownish black, covered with yellow short hairs and dark-brown long upright hairs along posterior margin. Postnotum brownish black, shiny and white pruinose when illuminated at certain angles, and bare. Pleural membrane ochreous and bare. Katepisternum dark brown, longer than deep, shiny when illuminated at certain angles, moderately covered with fine pale and dark short hairs. Legs. Foreleg: coxa yellow; trochanter light brown; femur light brown with apical cap medium brown (though apical tip yellowish); tibia light to medium brown except base dark yellow, little less than apical one-third brownish black and median large portion of outer surface white; outer surface of basal four-fifths of tibia covered with white hairs and sheeny when illuminated at certain angles; tarsus brownish black; basitarsus slightly dilated, 8.8 times as long as its greatest width. Midleg: coxa dark brown except posterolateral surface brownish black; trochanter light brown except base yellow; femur light brown with posterior surface of base somewhat yellow and apical cap medium brown (though apical tip yellow); tibia brownish black except basal one-third yellow; tarsus 2
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
Fig. 1. Male of S. rampae sp. nov. A & B, third palpomeres of maxillary palps with sensory vesicles (front view; A, right side; B, left side); C, basitarsus with calcipala and second tarsomere with pedisulcus of left hind leg (outer view); D, coxites, styles and ventral plate (ventral view); E, right style (ventrolateral view); F, ventral plate and median sclerite (lateral view); G, ventral plate (caudal view); H, median sclerite (caudal view); I, right paramere (dorsal view); J, aedeagal membrane (caudal view); K & L, abdominal segment 10 and cerci (K, lateral view; L, caudal view). Scale bars. 0.1 mm for C; 0.02 mm for A, B and D–L.
thoracic segment 1 encircled with ochreous band (though disconnected ventromedially), thoracic segments 2 and 3 ochreous on ventral surface and partially light ochreous on dorsal surface; abdominal segment 4 with reddish-brown band (though disconnected ventromedially and no band in three larvae); abdominal segments 5 encircled by reddishbrown transverse band (though disconnected or narrowly connected ventromedially), abdominal segments 6–9 each covered with reddish brown colored pigments to varying extent on dorsal and dorsolateral surface, but completely faded out in three larvae); abdominal segments 6 and 7 with two reddish-brown spots and wide transverse band, respectively, on ventral surface (though completely faded out in three larvae). Head. Head capsule whitish yellow to yellow except eye-spot region whitish, sparsely covered with minute setae (though moderately to densely on dorsal surface); head spots indistinct or faintly positive (though moderately positive in one larva); eyebrow distinct. Antenna composed of three articles and apical sensillum, longer than stem of labral fan; proportional lengths of first, second, and third articles
segments 6–9 each with spine-combs in transverse row, and segments 5–9 each with comb-like groups of micro- spines on each side; segment 5 with four minute setae near posterior margin on each side; segments 6–8 each with two minute setae on each side; segment 9 with pair of triangular terminal hooks (Fig. 2C). Ventrally, segment 4 with one unbranched hook (subequal in size to those on segments 5–7) and few minute setae on each side; segment 5 with pair of bifid or trifid hooks submedially and few minute setae on each side; segments 6 and 7 each with pair of bifid inner and unbranched outer hooks somewhat spaced from each other and few minute setae on each side; segments 4–8 each with comb-like groups of micro-spines. Each side of segment 9 with three grapnel-shaped hooklets. Cocoon (Fig. 2D). Wall-pocket-shaped, thinly and moderately woven, somewhat extended ventrolaterally; with anterodorsal projection; individual threads invisible; 3.2 mm long by 2.2 mm wide. Mature larva. Body length 5.0–6.0 mm. Body white except abdominal segments 1–4 light greenish, with following color markings: 3
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
Fig. 2. Pupa and larva of S. rampae sp. nov. A–D, pupa; E–H, larva. A & B, gill filaments (outer view; A, left side; B, right side); C, terminal hooks (caudal view); D, cocoon (dorsal view); E, apical portion of mandible; F, hypostoma; G, head capsule showing postgenal cledt (ventral view); H, postgenal cleft (ventral view). Scale bars. 1.0 mm for D; 0.1 mm for A, B, G & H; 0.02 mm for C & F; 0.01 mm for E.
present. Rectal organ compound, each of three lobes with 9–11 fingerlike secondary lobules. Anal sclerite of usual X-form, with anterior arms 1.1–1.2 times as long as posterior ones, broadly sclerotized at base; accessory sclerite absent. Last abdominal segment with pair of large conical ventral papillae. Posterior circlet with 83–87 rows of hooklets with up to 13 or 14 hooklets per row. Female. Unknown. Type specimens. Holotype. Male (with its associated pupal exuviae and cocoon) (preserved in 80% ethanol), reared from a pupa collected from a small stream (width 0.6 m, depth 0.1 m, bottom sandy, 14.5 °C, pH 6.6, partially shaded, elevation 1685 m, N18˚31′15.4″, E98˚29′59.4″), stream before Check point 2, Doi Inthanon National Park, Chiang Mai, Thailand, 20-XII-2018, by W. Srisuka. Paratypes. Three mature larvae, same data as the holotype. Biology. The pupa and larvae of this new species were collected from trailing grass and fallen leaves in the stream. The associated species were S. (G.) chiangdaoense, S. (G.) inthanonense, S. (Simulium) chamlongi, S. (S.) doipuiense and S. (S.) yuphae.
1.0:0.7–0.8:0.7–0.8. Labral fan with 28–42 primary rays. Mandible (Fig. 2E) with three comb-teeth decreasing in length from first tooth to third; mandibular serration composed of two teeth (one medium-sized, one small); major tooth at acute angle against mandible on apical side; supernumerary serrations absent. Hypostoma (Fig. 2F) with row of nine apical teeth, of which median tooth longer than each corner tooth; lateral margin smooth; five or six hypostomal bristles per side lying slightly divergent posteriorly from lateral margin. Postgenal cleft (Fig. 2G and H) rounded anteriorly (though somewhat angulate in one larva) and medium-long (0.9–1.2 times length of postgenal bridge). Cervical sclerites composed of pair of small yellow rod-like pieces. Thorax and Abdomen. Thoracic cuticle and abdominal cuticle sparsely covered with unbranched unpigmented minute setae on dorsal surface except abdominal segments 6–8 moderately covered with unpigmented minute setae on dorsal and dorsolateral surfaces; last abdominal segment densely covered with unbranched unpigmented minute setae on dorsolateral and lateral surfaces of each side of anal sclerite and on each lateral surface even down to base of ventral papilla. Rectal scales 4
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
Fig. 3. Neighbor-joining phylogenetic tree of the Simulium asakoae species-group based on COI gene sequences. Numbers at the nodes are the bootstrap confidence values after 500 replicates. The bootstrap values above 50% are shown. The scale bar indicates the distance in substitutions per nucleotide.
having the pupal gill with an elongated common basal stalk, terminal hook small and narrow, and larval postgenal cleft medium-sized. However it is distinguished from this new species by the male upper-eye facets in 15 or 16 vertical columns and 15 or 16 horizontal rows, pupal gill with eight filaments arranged as (3 + 3)+2 from dorsal to ventral, cocoon without an anterodorsal projection, and larval postgenal cleft lanceolate, 2.3 times as long as the postgenal bridge (Takaoka et al., 2011). Simulium (G.) longitruncum Takaoka & Davies from Peninsular Malaysia, and S. (G.) atratoides Takaoka & Davies from Java, Indonesia, both of the S. ceylonicum species-group, have the pupal gill with an elongated common basal stalk, but are distinguished from this new species by the male upper-eye facets in 11 or 13 vertical columns and 14 horizontal rows, ventral plate narrowed posteriorly when viewed ventrally, pupal gill with eight filaments arranged as 3 + 4+1 or 3+(3 + 2) from dorsal to ventral, or with 10 filaments arranged as 6 + 4 from dorsal to ventral, and cocoon without an anterodorsal projection (Takaoka and Davies, 1995, 1996). Simulium (G.) serratum Takaoka from Sulawesi, Indonesia, described only from a single pupa and unplaced in species-group, has also an elongated common basal stalk of the pupal gill, but is distinguished from this new species by the terminal hooks much widened and with crenulated outer margins (Takaoka, 2003). Three members of the S. asakoae species-group recorded from Thailand, i.e., S. (G.) asakoae Takaoka & Davies, S. (G.) chiangdaoense
Etymology. The species name rampae is in honor of Dr. Rampa Rattanarithikul, who is a supervisor of W. S. Taxonomic notes. This new species is assigned to the Simulium asakoae species-group of the subgenus Gomphostilbia, defined by Takaoka (2012), by having the yellow hair tuft on the base of the radial vein, yellow fore coxae, and enlarged hind basitarsi (Fig. 1C) in the male. It is unlikely that this new species belongs to the S. cylonicum species-group or the S. darjeelingense species-group, both of which have, though, the enlarged male hind basitarsi, because the males of these two species-groups have the darkened hair tuft on the base of the radial vein and ventral plate narrowed posteriorly when viewed ventrally (Takaoka, 2012). Notably, the ventral plate of this new species with lateral margins somewhat widened posteriorly (Fig. 1D), slightly differing from the lateral margins of the ventral plate of other species in the S. asakoae species-group, which are emarginated medially when viewed ventrally. This new species is characterized in the male by the high number of upper-eye facets in 17 vertical columns and 18 horizontal rows, in the pupa by the gill with a long common basal stalk, terminal hook coneshaped (Fig. 2C) and cocoon with an anterodorsal projection (Fig. 2D), and in the larva by the medium-long postgenal cleft (Fig. 2G). A combination of these characters easily separates this new species from all the 36 species of the asakoae species-group. Simulium (G.) sofiani Takaoka & Hashim from Peninsular Malaysia, a member of the S. asakoae species-group, is similar to this new species by 5
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
Distinguished Research Professor Grant, Thailand Research Fund (grant number DPG6280002) and Khon Kaen University Grant to W. Maleewong (subproject to A. Saeung).
and S. (G.) udomi Takaoka & Choochote, have the pupal gill with a medium-long common basal stalk of the gill, terminal hooks wide, plate-like, and cocoon with a short anterodorsal bulge or an extremely elongated anterodorsal projection, all these characters differing from those of this new species (Saeung et al., 2017; Takaoka and Choochote, 2006; Takaoka and Srisuka, 2009). In addition, S. (G.) udomi has the pupal gill with six filaments differing from those of most other species of the S. asakoae species-group (Takaoka, 2012). This is the fourth member of the S. asakoae species-group recorded from Thailand.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.actatropica.2019. 105043. References
3.2. COI gene sequence-based analysis Adler, P.H., Crosskey, R.W., 2018. World Blackflies (Diptera: Simuliidae): A Comprehensive Revision of the Taxonomic and Geographical Inventory [2018]. 134 pp., http://entweb.clemson.edu/biomia/pdfs/blackflyinventory.pdf. (Accessed on January 30, 2019). . Adler, P.H., Currie, D.C., Wood, D.M., 2004. The Black Flies (Simuliidae) of North America. xv+941 pp.. Cornell University Press, Ithaca, New York, USA. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Low, V.L., Takaoka, H., Adler, P.H., Ya’cob, Z., Norma-Rashid, Y., Chen, C.D., SofianAzirun, M., 2015. A multi-locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages. Med. Vet. Entomol. 29, 330–337. Saeung, A., Srisuka, W., Maleewong, W., Low, V.L., Takaoka, H., 2017. Descriptions of the female and larva of Simulium (Gomphostilbia) udomi from Thailand, and their transfer to theSimulium asakoae species-group. Acta Trop. 172, 14–19. Takaoka, H., 2003. The Black Flies (Diptera: Simuliidae) of Sulawesi, Maluku and Irian Jaya. xxii + 581 pp.. Kyushu University Press, Fukuoka. Takaoka, H., 2012. Morphotaxonomic revision of Simulium (Gomphostilbia) (Diptera: Simuliidae) in the Oriental Region. Zootaxa 3577, 1–42. Takaoka, H., 2017. Morphotaxonomic revision of species-groups of Simulium (Simulium) (Diptera: Simuliidae) in the Oriental Region. Zootaxa 4353, 425–446. Takaoka, H., Choochote, W., 2006. A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from northern Thailand. Med. Entomol. Zool. 57, 229–233. Takaoka, H., Davies, D.M., 1995. The Black Flies (Diptera: Simuliidae) of West Malaysia. viii + 175 pp.. Kyushu University Press, Fukuoka, Japan. Takaoka, H., Davies, D.M., 1996. The Black Flies (Diptera: Simuliidae) of Java, Indonesia. viii + 81 pp., Bishop Museum Bulletin in Entomology 6. Bishop Museum Press, Honolulu, U.S.A. Takaoka, H., Sofian-Azirun, M., Hashim, R., 2011. Simulium (Gomphostilbia) sofiani, a new species of black fly (Diptera: Simuliidae) from Peninsular Malaysia. Trop. Biomed. 28, 389–399. Takaoka, H., Srisuka, W., 2009. Simulium (Gomphostilbia) chiangdaoense sp. nov. (Diptera: Simuliidae) from northern Thailand. Med. Entomol. Zool. 60, 269–276. Takaoka, H., Srisuka, W., Seung, A., 2019. Checklist and keys for the black flies (Diptera: Simuliidae) in Thailand. Med. Entomol. Zool (in press). Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.
Our phylogenetic analysis shows that S. (G.) rampae sp. nov. (sample 1) and S. rampae sp. nov. (sample 2) fall in the S. asakoae species-group, rather than the S. ceylonicum species-group (Fig. 3), supporting its assignment to the S. asakoae species-group by morphological characters. Among nine species of the S. asakoae species-group compared, this new species is most closely related to S. (G.) udomi and S. (G.) chiangdaoense (Supplementary file 1), from which this new species is, though, quite different morphologically, as already noted. The intraspecific variation of S. (G.) rampae sp. nov. was 0.17%, and the interspecific variation among S. (G.) rampae sp. nov., S. (G.) udomi, and S. (G.) chiangdaoense was 0.34–1.02%. The low interspecific variations of the COI gene among these three species are not unexpected. The limitation of the COI gene in differentiating species among the S. asakoae speciesgroup has been reported for S. (G.) lurauense and S. (G.) sofiani, though both species were resolved by the 28S rRNA gene (Low et al., 2015). Further investigation is necessary for resolving power of other gene loci to genetically separate these three species. Acknowledgements Our thanks are due to Mr. Manachai Tabutr and Ms. Thapanat Pankan, Queen Sirikit Botanic Garden for their assistance in the field surveys. This work was supported by a research grant from University of Malaya (RP021A/16SUS) to H. Takaoka, and the Thailand Research Fund (TRF) and Office of the Higher Education Commission (OHEC) through the Research Grant for New Scholar (grant number MRG5980101), and Chiang Mai University through the Center of Insect Vector Study to A. Saeung. This work was co-supported by
6
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
A new species of the Simulium (Gomphostilbia) (Diptera: Simuliidae) from Thailand, with its phylogenetic relationships with related species in the Simulium asakoae species-group Wichai Srisukaa, Hiroyuki Takaokab, Masako Fukudac, Yasushi Otsukad, Atiporn Saeunge,
T
⁎
a
Entomology Section, Queen Sirikit Botanic Garden, P.O. Box 7, Maerim, Chiang Mai 50180, Thailand Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Kuala Lumpur 50603, Malaysia c Institute for Research Promotion, Oita University, Idaigaoka 1-1, Hasama, Yufu City, Oita 879-5593, Japan d Research Center for the Pacific Islands, Kagoshima University, Korimoto 1-21-24, Kagoshima City, Kagoshima 890-8580, Japan e Center of Insect Vector Study, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand b
A R T I C LE I N FO
A B S T R A C T
Keywords: Aquatic insects Taxonomy Biodiversity Oriental Region
A new species of black fly, Simulium (Gomphostilbia) rampae, is described, based on adult male, its pupal exuviae and mature larvae collected from Doi Inthanon National Park, northern Thailand. This new species is placed in the Simulium asakoae species-group, and is characterized in the male by the high number of upper-eye facets in 17 vertical columns and 18 horizontal rows, in the pupa by the gill with a long common basal stalk, cone-shaped terminal hook, and cocoon with an anterodorsal projection, and in the larva by the medium-long postgenal cleft. A DNA analysis using COI gene supported its assignment to the S. asakoae species-group and showed its close relationship to S. (G.) udomi Takaoka & Choochote and S. (G.) chiangdaoense Takaoka & Srisuka. This is the fourth member of the S. asakoae species-group recorded from Thailand.
1. Introduction
2. Material and methods
The fauna of black flies (Diptera: Simuliidae) in Thailand is characterized by its richness in species number and high lineage diversity. It consists of 110 species, of which 85 species are assigned in two dominant subgenera (34 in Gomphostilbia Enderlein and 51 in Simulium Latraille), and remaining 25 species are in four other small subgenera in the genus Simulium Latreille (Takaoka et al., 2019). The species in these two dominant subgenera, Gomphostilbia and Simulium, are placed in eight and eleven species-groups, respectively, showing a high diversity in phylogenetic lineage (Adler and Crosskey, 2018; Takaoka, 2012, 2017). Recently, we collected one undescribed species of the subgenus Gomphostilbia from Doi Inthanon National Park, northern Thailand. It is described as a new species, and a COI gene sequence-based analysis on its assignment to species group and phylogenetic relationship to related species are carried out.
2.1. Description of a new species One adult male reared from a pupa, its associated pupal exuviae and six mature larvae collected from Doi Inthanon National Parks, Chiang Mai, Thailand, were used. The methods of description and illustration, as well as terms for morphological features used here, follow those of Takaoka (2003) and partially those of Adler et al. (2004). The holotype and paratypes of the new species are deposited at Entomology Section of the Queen Sirikit Botanic Garden, Chiang Mai, Thailand. 2.2. DNA analysis Two mature larvae of the new species and a female of S. (G.) chiangdaoense Takaoka & Srisuka were used. The procedures for DNA extraction, PCR amplification, and sequencing follow those of Low et al. (2015) with slight modification. DNA was extracted from each specimen with a DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). PCR amplification of the partial mitochondrial COI gene region was
⁎
Corresponding author. E-mail addresses: [email protected] (W. Srisuka), [email protected] (H. Takaoka), [email protected] (M. Fukuda), [email protected] (Y. Otsuka), [email protected] (A. Saeung). https://doi.org/10.1016/j.actatropica.2019.105043 Received 30 April 2019; Received in revised form 29 May 2019; Accepted 29 May 2019 Available online 30 May 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
carried out in a final volume of 50 μL containing 50 ng of DNA, 1 × Ex Taq buffer including 2 mM Mg2+, 200 μM each of dNTPs, 1.25 units of Ex Taq Polymerase (TAKARA BIO INC., Kusatsu, Japan), and 10 pmol of each forward (LCO1490) and reverse primer (HCO2198) (Folmer et al., 1994). Purified PCR products were directly sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems). The sequences determined were deposited in DDBJ/ EMBL/GenBank under the following accession numbers LC472507–LC472509. The sequences of the new species were aligned with those of nine related species (six spp. including S. (G.) chiangdaoense of the S. asakoae species-group and three of the S. ceylonicum species-group) and two other species as outgroup (S. (S.) tani Takaoka & Davies and S. (Nevermannia) feuerborni Edwards. Using this alignment, sequences were compared and a neighbor-joining tree was constructed by MEGA6 (Tamura et al., 2013) based on 586 positions. The Kimura 2-parameter method was used to estimate evolutionary distances in the tree.
brownish black. Hind leg: coxa dark brown though apical small portion yellow; trochanter yellow; femur medium brown with base yellow and apical cap dark brown (though apical tip yellow); tibia medium to dark brown except basal two-fifths yellow to ochreous (though posterior surface somewhat darkened) and apical cap brownish black; tarsus (Fig. 1C) medium brown except basal two-fifths of basitarsus (border not well defined) and basal one-fourth of second tarsomere grayish yellow; basitarsus (Fig. 1C) enlarged, 3.8 times as long as wide, and 0.9 and 1.1 times as wide as greatest width of tibia and femur, respectively; calcipala (Fig. 1C) slightly shorter than basal width, and 0.32 times as wide as greatest width of basitarsus; pedisulcus (Fig. 1C) well defined. Wing. Length 2.5 mm. Costa with dark spinules and hairs. Including patch of hairs at base. Subcosta with 14 or 15 hairs. Hair tuft on humeral plate black. Hair tuft on base of radial vein (= stem vein) yellow. Basal portion of radius fully haired; R1 with dark spinules and hairs; R2 with hairs only. Basal cell absent. Halter. Grayish except basal stem darkened. Abdomen. Basal scale dark brown, with fringe of light brown hairs. Dorsal surface of abdomen brownish black to black, covered with dark-brown short to long hairs; segments 2 and 5–7 each with pair of shiny dorsolateral or lateral patches. Genitalia. Coxite in ventral view (Fig. 1D) nearly rectangular, 1.8 times as long as its greatest width. Style in ventral view (Fig. 1D) bent inward, bluntly rounded apically and with apical spine; style in ventrolateral view (Fig. 1E) slightly tapered toward apex, and 0.85 times as long as coxite. Ventral plate in ventral view (Fig. 1D) with body transverse, 0.46 times as long as wide, with anterior margin produced anteromedially, and posterior margin slightly concave medially, lateral margins slightly widened posteriorly, and densely covered with micro-setae on ventral surface; basal arms of moderate length, directed forward, then convergent apically; ventral plate in lateral view (Fig. 1F) moderately produced ventrally; ventral plate in end view (Fig. 1G) rounded ventrally, densely covered with microsetae on posterior surface except portion near each dorsolateral tip narrowly bare. Median sclerite (Fig. 1H) thin, plate-like, wide. Paramere (Fig. 1I) of moderate size, each with four distinct stout hooks. Aedeagal membrane (Fig. 1J) densely setose, not sclerotized at base; dorsal plate not defined. Ventral surface of abdominal segment 10 (Fig. 1K and L) with anterior portion well sclerotized and brownish, and with one distinct hair or without hairs near posterior margin. Cercus in lateral view (Fig. 1K and L) small, rounded, with 12–16 hairs. Pupa. Body length 2.4 mm. Head. Integument yellow, moderately covered with small round tubercles except antennal sheaths and ventral surface almost bare; antennal sheath without any protuberances; face with pair of unbranched long trichomes with coiled apices, and frons with three pairs of unbranched 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, and with three long dorsomedial trichomes with coiled or uncoiled apices, two long anterolateral trichomes (one with coiled apex, one with uncoiled apex), one medium-long mediolateral trichome with uncoiled apex, and three ventrolateral trichomes with uncoiled apices (one medium-long, two short) on each side; all unbranched. Gill (Fig. 2A and B) composed of seven or eight slender thread-like filaments, arranged as 3 + 2+(2 + 1) or 2 + 2+(2 + 1) from dorsal to ventral, with long common basal stalk having somewhat swollen transparent basal fenestra at base; common basal stalk 1.3–1.5 times length of interspiracular trunk; all filaments light brown, gradually tapered toward apex; cuticle of all filaments with well-defined annular ridges though becoming less marked apically, densely covered with minute tubercles, of which relatively larger ones on ridges. Abdomen. Dorsally, all segments light yellow and without tubercles; segment 1 with one unbranched slender short hair-like seta on each side; segment 2 with one unbranched slender short hair-like seta (though much longer than seta on segment 1) and five minute setae submedially on each side; segments 3 and 4 each with four hooked spines and one or two minute setae on each side; segment 5 lacking spine-combs on each side;
2.3. Ethical clearance This study was approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chiang Mai University (Protocol Number 46/2561), Chiang Mai province, Thailand. 3. Results and discussion 3.1. Description of a new species 3.1.1. Simulium (Gomphostilbia) rampae Takaoka, Srisuka & Saeung sp. nov. Male. Body length 2.5 mm. Head. Somewhat wider than thorax. Upper eye medium brown, consisting of large facets in 17 vertical columns and 18 horizontal rows. Clypeus brownish black, whitish pruinose, densely covered with golden-yellow scale-like medium-long hairs (mostly directed upward) interspersed with several dark-brown longer hairs. Antenna composed of scape, pedicel and nine flagellomeres, medium to dark brown except scape, basal half of pedicel yellow (though ventral suface of pedicel entirely yellow) and base of first flagellomere yellow; first flagellomere elongate, 1.8 times length of second one. Maxillary palp light brown except third palpomere dark brown, with five palpomeres, proportional lengths of third, fourth, and fifth palpomeres 1.0:1.2:2.4; third palpomere (Fig. 1A and B) slender; sensory vesicle (Fig. 1A and B) ellipsoidal, small (0.2 times length of third palpomere), and with opening of small or medium-size. Thorax. Scutum brownish black to black without longitudinal vittae, shiny and thinly gray pruinose on shoulders, on wide area along each lateral margin and on prescutellar area when illuminated at certain angles, densely covered with golden-yellow scale-like recumbent hairs. Scutellum brownish black, covered with yellow short hairs and dark-brown long upright hairs along posterior margin. Postnotum brownish black, shiny and white pruinose when illuminated at certain angles, and bare. Pleural membrane ochreous and bare. Katepisternum dark brown, longer than deep, shiny when illuminated at certain angles, moderately covered with fine pale and dark short hairs. Legs. Foreleg: coxa yellow; trochanter light brown; femur light brown with apical cap medium brown (though apical tip yellowish); tibia light to medium brown except base dark yellow, little less than apical one-third brownish black and median large portion of outer surface white; outer surface of basal four-fifths of tibia covered with white hairs and sheeny when illuminated at certain angles; tarsus brownish black; basitarsus slightly dilated, 8.8 times as long as its greatest width. Midleg: coxa dark brown except posterolateral surface brownish black; trochanter light brown except base yellow; femur light brown with posterior surface of base somewhat yellow and apical cap medium brown (though apical tip yellow); tibia brownish black except basal one-third yellow; tarsus 2
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
Fig. 1. Male of S. rampae sp. nov. A & B, third palpomeres of maxillary palps with sensory vesicles (front view; A, right side; B, left side); C, basitarsus with calcipala and second tarsomere with pedisulcus of left hind leg (outer view); D, coxites, styles and ventral plate (ventral view); E, right style (ventrolateral view); F, ventral plate and median sclerite (lateral view); G, ventral plate (caudal view); H, median sclerite (caudal view); I, right paramere (dorsal view); J, aedeagal membrane (caudal view); K & L, abdominal segment 10 and cerci (K, lateral view; L, caudal view). Scale bars. 0.1 mm for C; 0.02 mm for A, B and D–L.
thoracic segment 1 encircled with ochreous band (though disconnected ventromedially), thoracic segments 2 and 3 ochreous on ventral surface and partially light ochreous on dorsal surface; abdominal segment 4 with reddish-brown band (though disconnected ventromedially and no band in three larvae); abdominal segments 5 encircled by reddishbrown transverse band (though disconnected or narrowly connected ventromedially), abdominal segments 6–9 each covered with reddish brown colored pigments to varying extent on dorsal and dorsolateral surface, but completely faded out in three larvae); abdominal segments 6 and 7 with two reddish-brown spots and wide transverse band, respectively, on ventral surface (though completely faded out in three larvae). Head. Head capsule whitish yellow to yellow except eye-spot region whitish, sparsely covered with minute setae (though moderately to densely on dorsal surface); head spots indistinct or faintly positive (though moderately positive in one larva); eyebrow distinct. Antenna composed of three articles and apical sensillum, longer than stem of labral fan; proportional lengths of first, second, and third articles
segments 6–9 each with spine-combs in transverse row, and segments 5–9 each with comb-like groups of micro- spines on each side; segment 5 with four minute setae near posterior margin on each side; segments 6–8 each with two minute setae on each side; segment 9 with pair of triangular terminal hooks (Fig. 2C). Ventrally, segment 4 with one unbranched hook (subequal in size to those on segments 5–7) and few minute setae on each side; segment 5 with pair of bifid or trifid hooks submedially and few minute setae on each side; segments 6 and 7 each with pair of bifid inner and unbranched outer hooks somewhat spaced from each other and few minute setae on each side; segments 4–8 each with comb-like groups of micro-spines. Each side of segment 9 with three grapnel-shaped hooklets. Cocoon (Fig. 2D). Wall-pocket-shaped, thinly and moderately woven, somewhat extended ventrolaterally; with anterodorsal projection; individual threads invisible; 3.2 mm long by 2.2 mm wide. Mature larva. Body length 5.0–6.0 mm. Body white except abdominal segments 1–4 light greenish, with following color markings: 3
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
Fig. 2. Pupa and larva of S. rampae sp. nov. A–D, pupa; E–H, larva. A & B, gill filaments (outer view; A, left side; B, right side); C, terminal hooks (caudal view); D, cocoon (dorsal view); E, apical portion of mandible; F, hypostoma; G, head capsule showing postgenal cledt (ventral view); H, postgenal cleft (ventral view). Scale bars. 1.0 mm for D; 0.1 mm for A, B, G & H; 0.02 mm for C & F; 0.01 mm for E.
present. Rectal organ compound, each of three lobes with 9–11 fingerlike secondary lobules. Anal sclerite of usual X-form, with anterior arms 1.1–1.2 times as long as posterior ones, broadly sclerotized at base; accessory sclerite absent. Last abdominal segment with pair of large conical ventral papillae. Posterior circlet with 83–87 rows of hooklets with up to 13 or 14 hooklets per row. Female. Unknown. Type specimens. Holotype. Male (with its associated pupal exuviae and cocoon) (preserved in 80% ethanol), reared from a pupa collected from a small stream (width 0.6 m, depth 0.1 m, bottom sandy, 14.5 °C, pH 6.6, partially shaded, elevation 1685 m, N18˚31′15.4″, E98˚29′59.4″), stream before Check point 2, Doi Inthanon National Park, Chiang Mai, Thailand, 20-XII-2018, by W. Srisuka. Paratypes. Three mature larvae, same data as the holotype. Biology. The pupa and larvae of this new species were collected from trailing grass and fallen leaves in the stream. The associated species were S. (G.) chiangdaoense, S. (G.) inthanonense, S. (Simulium) chamlongi, S. (S.) doipuiense and S. (S.) yuphae.
1.0:0.7–0.8:0.7–0.8. Labral fan with 28–42 primary rays. Mandible (Fig. 2E) with three comb-teeth decreasing in length from first tooth to third; mandibular serration composed of two teeth (one medium-sized, one small); major tooth at acute angle against mandible on apical side; supernumerary serrations absent. Hypostoma (Fig. 2F) with row of nine apical teeth, of which median tooth longer than each corner tooth; lateral margin smooth; five or six hypostomal bristles per side lying slightly divergent posteriorly from lateral margin. Postgenal cleft (Fig. 2G and H) rounded anteriorly (though somewhat angulate in one larva) and medium-long (0.9–1.2 times length of postgenal bridge). Cervical sclerites composed of pair of small yellow rod-like pieces. Thorax and Abdomen. Thoracic cuticle and abdominal cuticle sparsely covered with unbranched unpigmented minute setae on dorsal surface except abdominal segments 6–8 moderately covered with unpigmented minute setae on dorsal and dorsolateral surfaces; last abdominal segment densely covered with unbranched unpigmented minute setae on dorsolateral and lateral surfaces of each side of anal sclerite and on each lateral surface even down to base of ventral papilla. Rectal scales 4
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
Fig. 3. Neighbor-joining phylogenetic tree of the Simulium asakoae species-group based on COI gene sequences. Numbers at the nodes are the bootstrap confidence values after 500 replicates. The bootstrap values above 50% are shown. The scale bar indicates the distance in substitutions per nucleotide.
having the pupal gill with an elongated common basal stalk, terminal hook small and narrow, and larval postgenal cleft medium-sized. However it is distinguished from this new species by the male upper-eye facets in 15 or 16 vertical columns and 15 or 16 horizontal rows, pupal gill with eight filaments arranged as (3 + 3)+2 from dorsal to ventral, cocoon without an anterodorsal projection, and larval postgenal cleft lanceolate, 2.3 times as long as the postgenal bridge (Takaoka et al., 2011). Simulium (G.) longitruncum Takaoka & Davies from Peninsular Malaysia, and S. (G.) atratoides Takaoka & Davies from Java, Indonesia, both of the S. ceylonicum species-group, have the pupal gill with an elongated common basal stalk, but are distinguished from this new species by the male upper-eye facets in 11 or 13 vertical columns and 14 horizontal rows, ventral plate narrowed posteriorly when viewed ventrally, pupal gill with eight filaments arranged as 3 + 4+1 or 3+(3 + 2) from dorsal to ventral, or with 10 filaments arranged as 6 + 4 from dorsal to ventral, and cocoon without an anterodorsal projection (Takaoka and Davies, 1995, 1996). Simulium (G.) serratum Takaoka from Sulawesi, Indonesia, described only from a single pupa and unplaced in species-group, has also an elongated common basal stalk of the pupal gill, but is distinguished from this new species by the terminal hooks much widened and with crenulated outer margins (Takaoka, 2003). Three members of the S. asakoae species-group recorded from Thailand, i.e., S. (G.) asakoae Takaoka & Davies, S. (G.) chiangdaoense
Etymology. The species name rampae is in honor of Dr. Rampa Rattanarithikul, who is a supervisor of W. S. Taxonomic notes. This new species is assigned to the Simulium asakoae species-group of the subgenus Gomphostilbia, defined by Takaoka (2012), by having the yellow hair tuft on the base of the radial vein, yellow fore coxae, and enlarged hind basitarsi (Fig. 1C) in the male. It is unlikely that this new species belongs to the S. cylonicum species-group or the S. darjeelingense species-group, both of which have, though, the enlarged male hind basitarsi, because the males of these two species-groups have the darkened hair tuft on the base of the radial vein and ventral plate narrowed posteriorly when viewed ventrally (Takaoka, 2012). Notably, the ventral plate of this new species with lateral margins somewhat widened posteriorly (Fig. 1D), slightly differing from the lateral margins of the ventral plate of other species in the S. asakoae species-group, which are emarginated medially when viewed ventrally. This new species is characterized in the male by the high number of upper-eye facets in 17 vertical columns and 18 horizontal rows, in the pupa by the gill with a long common basal stalk, terminal hook coneshaped (Fig. 2C) and cocoon with an anterodorsal projection (Fig. 2D), and in the larva by the medium-long postgenal cleft (Fig. 2G). A combination of these characters easily separates this new species from all the 36 species of the asakoae species-group. Simulium (G.) sofiani Takaoka & Hashim from Peninsular Malaysia, a member of the S. asakoae species-group, is similar to this new species by 5
Acta Tropica 197 (2019) 105043
W. Srisuka, et al.
Distinguished Research Professor Grant, Thailand Research Fund (grant number DPG6280002) and Khon Kaen University Grant to W. Maleewong (subproject to A. Saeung).
and S. (G.) udomi Takaoka & Choochote, have the pupal gill with a medium-long common basal stalk of the gill, terminal hooks wide, plate-like, and cocoon with a short anterodorsal bulge or an extremely elongated anterodorsal projection, all these characters differing from those of this new species (Saeung et al., 2017; Takaoka and Choochote, 2006; Takaoka and Srisuka, 2009). In addition, S. (G.) udomi has the pupal gill with six filaments differing from those of most other species of the S. asakoae species-group (Takaoka, 2012). This is the fourth member of the S. asakoae species-group recorded from Thailand.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.actatropica.2019. 105043. References
3.2. COI gene sequence-based analysis Adler, P.H., Crosskey, R.W., 2018. World Blackflies (Diptera: Simuliidae): A Comprehensive Revision of the Taxonomic and Geographical Inventory [2018]. 134 pp., http://entweb.clemson.edu/biomia/pdfs/blackflyinventory.pdf. (Accessed on January 30, 2019). . Adler, P.H., Currie, D.C., Wood, D.M., 2004. The Black Flies (Simuliidae) of North America. xv+941 pp.. Cornell University Press, Ithaca, New York, USA. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Low, V.L., Takaoka, H., Adler, P.H., Ya’cob, Z., Norma-Rashid, Y., Chen, C.D., SofianAzirun, M., 2015. A multi-locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages. Med. Vet. Entomol. 29, 330–337. Saeung, A., Srisuka, W., Maleewong, W., Low, V.L., Takaoka, H., 2017. Descriptions of the female and larva of Simulium (Gomphostilbia) udomi from Thailand, and their transfer to theSimulium asakoae species-group. Acta Trop. 172, 14–19. Takaoka, H., 2003. The Black Flies (Diptera: Simuliidae) of Sulawesi, Maluku and Irian Jaya. xxii + 581 pp.. Kyushu University Press, Fukuoka. Takaoka, H., 2012. Morphotaxonomic revision of Simulium (Gomphostilbia) (Diptera: Simuliidae) in the Oriental Region. Zootaxa 3577, 1–42. Takaoka, H., 2017. Morphotaxonomic revision of species-groups of Simulium (Simulium) (Diptera: Simuliidae) in the Oriental Region. Zootaxa 4353, 425–446. Takaoka, H., Choochote, W., 2006. A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from northern Thailand. Med. Entomol. Zool. 57, 229–233. Takaoka, H., Davies, D.M., 1995. The Black Flies (Diptera: Simuliidae) of West Malaysia. viii + 175 pp.. Kyushu University Press, Fukuoka, Japan. Takaoka, H., Davies, D.M., 1996. The Black Flies (Diptera: Simuliidae) of Java, Indonesia. viii + 81 pp., Bishop Museum Bulletin in Entomology 6. Bishop Museum Press, Honolulu, U.S.A. Takaoka, H., Sofian-Azirun, M., Hashim, R., 2011. Simulium (Gomphostilbia) sofiani, a new species of black fly (Diptera: Simuliidae) from Peninsular Malaysia. Trop. Biomed. 28, 389–399. Takaoka, H., Srisuka, W., 2009. Simulium (Gomphostilbia) chiangdaoense sp. nov. (Diptera: Simuliidae) from northern Thailand. Med. Entomol. Zool. 60, 269–276. Takaoka, H., Srisuka, W., Seung, A., 2019. Checklist and keys for the black flies (Diptera: Simuliidae) in Thailand. Med. Entomol. Zool (in press). Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.
Our phylogenetic analysis shows that S. (G.) rampae sp. nov. (sample 1) and S. rampae sp. nov. (sample 2) fall in the S. asakoae species-group, rather than the S. ceylonicum species-group (Fig. 3), supporting its assignment to the S. asakoae species-group by morphological characters. Among nine species of the S. asakoae species-group compared, this new species is most closely related to S. (G.) udomi and S. (G.) chiangdaoense (Supplementary file 1), from which this new species is, though, quite different morphologically, as already noted. The intraspecific variation of S. (G.) rampae sp. nov. was 0.17%, and the interspecific variation among S. (G.) rampae sp. nov., S. (G.) udomi, and S. (G.) chiangdaoense was 0.34–1.02%. The low interspecific variations of the COI gene among these three species are not unexpected. The limitation of the COI gene in differentiating species among the S. asakoae speciesgroup has been reported for S. (G.) lurauense and S. (G.) sofiani, though both species were resolved by the 28S rRNA gene (Low et al., 2015). Further investigation is necessary for resolving power of other gene loci to genetically separate these three species. Acknowledgements Our thanks are due to Mr. Manachai Tabutr and Ms. Thapanat Pankan, Queen Sirikit Botanic Garden for their assistance in the field surveys. This work was supported by a research grant from University of Malaya (RP021A/16SUS) to H. Takaoka, and the Thailand Research Fund (TRF) and Office of the Higher Education Commission (OHEC) through the Research Grant for New Scholar (grant number MRG5980101), and Chiang Mai University through the Center of Insect Vector Study to A. Saeung. This work was co-supported by
6