Monograph. ZOOTAXA

Zootaxa 4261 (1): 001 165 http://www.mapress.com/j/zt/ Copyright 2017 Magnolia Press Monograph https://doi.org/10.11646/zootaxa.4261.1.1 http://zoobank.org/urn:lsid:zoobank.org:pub:c097c43e-d522-46c4-9d64-76d3cd4bad86 ISSN 1175-5326 (print edition) ZOOTAXA ISSN 1175-5334 (online edition) ZOOTAXA 4261 The black flies (Diptera: Simuliidae) of Vietnam HIROYUKI TAKAOKA 1,5, MOHD SOFIAN-AZIRUN 1, ZUBAIDAH YA COB 1, CHEE DHANG CHEN 1, KOON WENG LAU 1, VAN LUN LOW 2, XUAN DA PHAM 3 & PETER H. ADLER 4 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, 50603 Malaysia 2 Tropical Infectious Diseases Research and Education Centre, University of Malaya, Kuala Lumpur, 50603 Malaysia 3 National Institute of Food Control, Ministry of Health, Hanoi, Vietnam (presently, The national office in southern region, Ministry of Science and Technology, Government of Vietnam, No 31, Han Thuyen street, District 1, Ho Chi Minh city, Vietnam) 4 Entomology Program, Clemson University, Clemson, South Carolina, 29634-0310, USA 5 Corresponding author. E-mail address: takaoka@oita-u.ac.jp Magnolia Press Auckland, New Zealand Accepted by L. Hernandez-Triana: 15 Feb. 2017; published: 5 May 2017

HIROYUKI TAKAOKA, MOHD SOFIAN-AZIRUN, ZUBAIDAH YA COB, CHEE DHANG CHEN, KOON WENG LAU, VAN LUN LOW, XUAN DA PHAM & PETER H. ADLER The black flies (Diptera: Simuliidae) of Vietnam (Zootaxa 4261) 165 pp.; 30 cm. 5 May 2017 ISBN 978-1-77670-134-6 (paperback) ISBN 978-1-77670-135-3 (Online edition) FIRST PUBLISHED IN 2017 BY Magnolia Press P.O. Box 41-383 Auckland 1346 New Zealand e-mail: magnolia@mapress.com http://www.mapress.com/j/zt 2017 Magnolia Press All rights reserved. No part of this publication may be reproduced, stored, transmitted or disseminated, in any form, or by any means, without prior written permission from the publisher, to whom all requests to reproduce copyright material should be directed in writing. This authorization does not extend to any other kind of copying, by any means, in any form, and for any purpose other than private research use. ISSN 1175-5326 ISSN 1175-5334 (Print edition) (Online edition) 2 Zootaxa 4261 (1) 2017 Magnolia Press TAKAOKA ET AL.

Female adults of three black fly species, which are vectors of filariae in the Oriental Region (From top, Simulium asakoae Takaoka & Davies, Simulium nigrogilvum Summers and Simulium nodosum Puri) THE BLACK FLIES FROM VIETNAM Zootaxa 4261 (1) 2017 Magnolia Press 3

Synopsis The biodiversity of black flies (Diptera: Simuliidae), which are biting insects of medical and veterinary importance, is strikingly high in Southeast Asian countries, such as Indonesia, Malaysia, Philippines and Thailand. In 2013, we began to explore the fauna of black flies in Vietnam, which has so far been poorly studied. In this monograph, the wealth of the biodiversity of black flies in Vietnam is also confirmed on the basis of the results of our recent investigations, though limited to five provinces in the country. Morphotaxonomic studies of black flies obtained from Sapa, Lao Cai Province, northern Vietnam, in 2014 and Nghe An Province, northern Vietnam, in 2015, and reexaminations of black flies collected from Tam Dao, Vinh Phuc Province, northern Vietnam, in 2013, Thua Thien Hue Province, central Vietnam, in 2014, and Lam Dong Province, southern Vietnam, in 2014, were conducted. A total of 22 species are described as new, including one in the newly recorded subgenus Montisimulium Rubtsov, and three species are recognized as new records from Vietnam. This investigation brings the number of species of black flies known in Vietnam to 70, all of which are assigned to the genus Simulium Latreille, and are placed in four subgenera (25 in Gomphostilbia Enderlein, one in Montisimulium, seven in Nevermannia Enderlein, and 37 in Simulium Latreille s. str.). The numbers of speciesgroups recognized include seven in Gomphostilbia, three in Nevermannia and nine in Simulium, indicating a high diversity of putative phylogenetic lineages. New species include S. (G.) sanchayense sp. nov. (= the species formerly regarded as S. (G.) brinchangense Takaoka, Sofian-Azirun & Hashim), S. (S.) lowi sp. nov. (= the species formerly regarded as S. (S.) brevipar Takaoka & Davies), S. (S.) fuscicoxae sp. nov. [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)], S. (S.) suoivangense sp. nov. [= morphoform b of the S. (S.) tani Takaoka & Davies (complex)]. Newly recorded species are S. (G.) parahiyangum Takaoka & Sigit, S. (N.) maeaiense Takaoka & Srisuka, and S. (S.) doipuiense Takaoka & Choochote (complex) [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)]. The substitute name, S. (S.) huense, is given for the species that was described under the name of S. (S.) cavum from southern Vietnam. A redescription of the female, male, pupa and larva of S. (G.) asakoae Takaoka & Davies is presented, and the female and larva of S. (G.) hongthaii Takaoka, Sofian-Azirun & Ya cob are described for the first time. Keys to 10 subgenera in the Oriental Region and all 70 species recorded from Vietnam are provided for females, males, pupae and mature larvae. As investigations extend nationwide in all the provinces in Vietnam, more new species and records are expected to be discovered. It is hoped that this monograph will be useful as a baseline taxonomic reference for future studies of black flies in Vietnam and neighbouring countries. Key words: black fly, Simulium, new species, fauna, Vietnam 4 Zootaxa 4261 (1) 2017 Magnolia Press TAKAOKA ET AL.

Table of contents Frontispiece................................................................................................ 3 Synopsis.................................................................................................. 4 Introduction............................................................................................... 6 Systematics............................................................................................... 10 Genus Simulium Latreille................................................................................... 10 Keys to 10 subgenera of the genus Simulium in the Oriental Region.................................................. 11 I. Subgenus Gomphostilbia Enderlein.......................................................................... 12 Keys to 25 species of the subgenus Gomphostilbia............................................................... 13 1) Simulium asakoae species-group........................................................................... 15 Simulium (Gomphostilbia) asakoae Takaoka & Davies, 1995................................................. 15 Simulium (Gomphostilbia) chaudinhense Takaoka & Sofian-Azirun sp. nov...................................... 23 Simulium (Gomphostilbia) confertum Takaoka & Sofian-Azirun, 2015........................................... 26 Simulium (Gomphostilbia) fuscidorsum Takaoka & Ya cob, 2015............................................... 26 Simulium (Gomphostilbia) hmongense Takaoka & Ya cob sp. nov.............................................. 26 Simulium (Gomphostilbia) hongthaii Takaoka, Sofian-Azirun & Ya cob, 2014.................................... 30 Simulium (Gomphostilbia) phulocense Takaoka & Chen, 2015................................................. 33 Simulium (Gomphostilbia) quychauense Takaoka & Chen sp. nov.............................................. 34 Simulium (Gomphostilbia) sanchayense Takaoka & Lau sp. nov................................................ 38 Simulium (Gomphostilbia) tamdaoense Takaoka, Sofian-Azirun & Ya cob, 2014.................................. 42 Simulium (Gomphostilbia) unii Takaoka & Pham sp. nov.....................................................42 Simulium (Gomphostilbia) thituyenae Takaoka & Pham, 2015................................................. 49 Simulium (Gomphostilbia) vinhphucense Takaoka & Low sp. nov.............................................. 49 2) Simulium batoense species-group........................................................................... 52 Simulium (Gomphostilbia) dachaisense Takaoka & Lau, 2015................................................. 52 Simulium (Gomphostilbia) duolongum Takaoka & Davies, 1995................................................ 52 Simulium (Gomphostilbia) lamdongense Takaoka & Sofian-Azirun, 2015........................................ 52 Simulium (Gomphostilbia) longlanhense Takaoka & Ya cob, 2015.............................................. 52 Simulium (Gomphostilbia) parahiyangum Takaoka & Sigit, 1992............................................... 53 Simulium (Gomphostilbia) siamense Takaoka & Suzuki, 1984................................................. 53 3) Simulium ceylonicum species-group......................................................................... 53 Simulium (Gomphostilbia) inthanonense Takaoka & Suzuki, 1984............................................. 53 4) Simulium darjeelingense species-group....................................................................... 53 Simulium (Gomphostilbia) eshimai Takaoka & Adler sp. nov.................................................. 53 5) Simulium epistum species-group............................................................................. 60 Simulium (Gomphostilbia) montiblense Takaoka, 1983....................................................... 60 6) Simulium gombakense species-group......................................................................... 60 Simulium (Gomphostilbia) thuathienense Takaoka & Sofian-Azirun, 2015........................................ 60 7) Simulium varicorne species-group........................................................................... 61 Simulium (Gomphostilbia) breviflagellum Takaoka & Sofian-Azirun, 2015....................................... 61 II. Subgenus Montisimulium Rubtsov........................................................................... 61 Simulium (Montisimulium) nigrofilum Takaoka & Sofian-Azirun sp. nov......................................... 61 III. Subgenus Nevermannia Enderlein.......................................................................... 65 Keys to seven species of the subgenus Nevermannia from Vietnam................................................... 66 1) Simulium feuerborni species-group........................................................................... 67 Simulium (Nevermannia) bachmaense Takaoka, Sofian-Azirun & Ya cob, 2014................................... 67 Simulium (Nevermannia) langbiangense Takaoka, Sofian-Azirun & Ya cob, 2014................................. 67 Simulium (Nevermannia) maeaiense Takaoka & Srisuka, 2011................................................ 67 Simulium (Nevermannia) phami Takaoka, Sofian-Azirun & Ya cob, 2014........................................ 67 2) Simulium ruficorne species-group........................................................................... 68 Simulium (Nevermannia) aureohirtum Brunetti, 1911........................................................ 68 3) Simulium vernum species-group............................................................................. 68 Simulium (Nevermannia) laichauense Takaoka & Sofian-Azirun sp. nov.......................................... 68 Simulium (Nevermannia) tayense Takaoka & Ya cob sp. nov.................................................. 72 IV. Subgenus Simulium Latreille.............................................................................. 79 Keys to 37 species of the subgenus Simulium.................................................................... 79 1) Simulium argentipes species-group.......................................................................... 82 Simulium (Simulium) sansahoense Takaoka & Chen sp. nov.................................................. 82 2) Simulium christophersi species-group........................................................................ 90 Simulium (Simulium) atipornae Takaoka, Srisuka & Choochote, 2014........................................... 90 3) Simulium griseifrons species-group.......................................................................... 90 THE BLACK FLIES FROM VIETNAM Zootaxa 4261 (1) 2017 Magnolia Press 5

Simulium (Simulium) chungi Takaoka & Huang, 2006....................................................... 90 Simulium (Simulium) grossifilum Takaoka & Davies, 1995................................................... 90 Simulium (Simulium) laocaiense Takaoka & Lau sp. nov.....................................................90 Simulium (Simulium) maenoi Takaoka & Choochote, 2002...................................................95 Simulium (Simulium) nigrogilvum Summers, 1911.......................................................... 96 Simulium (Simulium) turgidum Takaoka & Pham sp. nov.....................................................96 Simulium (Simulium) vietnamense Takaoka, Sofian-Azirun & Chen, 2014...................................... 101 4) Simulium malyschevi species-group......................................................................... 101 Simulium (Simulium) obliquum Takaoka & Low sp. nov.....................................................101 5) Simulium multistriatum species-group....................................................................... 108 Simulium (Simulium) daoense Takaoka & Adler sp. nov..................................................... 108 Simulium (Simulium) hirtinervis Edwards, 1928........................................................... 116 Simulium (Simulium) lacduongense Takaoka & Ya cob, 2015................................................ 116 Simulium (Simulium) laui Takaoka & Sofian-Azirun, 2015.................................................. 116 Simulium (Simulium) malayense Takaoka & Davies, 1995................................................... 117 6) Simulium nobile species-group............................................................................. 117 Simulium (Simulium) nodosum Puri, 1933................................................................ 117 7) Simulium striatum species-group........................................................................... 117 Simulium (Simulium) chiangmaiense Takaoka & Suzuki, 1984............................................... 117 Simulium (Simulium) jeffreyi Takaoka & Davies, 1995...................................................... 117 Simulium (Simulium) nakhonense Takaoka & Suzuki, 1984.................................................. 117 Simulium (Simulium) quinquestriatum (Shiraki, 1935)...................................................... 118 Simulium (Simulium) thailandicum Takaoka & Suzuki, 1984................................................ 118 Simulium (Simulium) tavanense Takaoka & Sofian-Azirun sp. nov............................................ 118 Simulium (Simulium) taythienense Takaoka, Sofian-Azirun & Ya cob, 2014.................................... 125 Simulium (Simulium) xuandai Takaoka, Sofian-Azirun & Ya cob, 2014........................................ 125 8) Simulium tuberosum species-group......................................................................... 126 Simulium (Simulium) congi Takaoka & Sofian-Azirun, 2015..................................................126 Simulium (Simulium) doipuiense Takaoka & Choochote, 2005 (complex)....................................... 126 Simulium (Simulium) fuscicoxae Takaoka & Ya cob sp. nov.................................................. 127 Simulium (Simulium) rosliramlii Takaoka & Chen sp. nov.................................................... 130 Simulium (Simulium) giayense Takaoka & Lau sp. nov...................................................... 137 Simulium (Simulium) huense Takaoka & Ya cob (substitute name)............................................ 139 Simulium (Simulium) sapaense Takaoka & Low sp. nov.....................................................139 Simulium (Simulium) lowi Takaoka & Adler sp. nov........................................................ 145 Simulium (Simulium) tani Takaoka & Davies, 1995 (complex)................................................147 Simulium (Simulium) suoivangense Takaoka & Pham sp. nov................................................. 148 Simulium (Simulium) xuandei Takaoka & Pham, 2015...................................................... 153 9) Simulium variegatum species-group........................................................................ 154 Simulium (Simulium) chamlongi Takaoka & Suzuki, 1984................................................... 154 Simulium (Simulium) phuluense Takaoka & Sofian-Azirun sp. nov............................................. 154 Acknowledgements........................................................................................ 161 References............................................................................................... 161 Introduction Adult black flies (Simuliidae) are small (1.5 to 6.0 mm in length), usually brown to black, two-winged insects (hence, belonging to the order Diptera). Black flies are closely associated with clean, running water in streams and rivers, where their immature stages develop and the gravid female adults return for oviposition. There are four distinct developmental stages: egg, larva, pupa and adult. Female adults bite mammals and birds to obtain a blood for development of their eggs. Due to their biting habits, black flies are insects of medical and veterinary importance. Females of certain simuliid species can transmit viruses, protozoans and filarial parasites. The most notorious filarial parasite transmitted by black flies is Onchocerca volvulus, a causative agent of human onchocerciasis (also called river blindness ) in Africa, and Central and South America (Crosskey 1990), followed by O. dewittei japonica, a pathogen of zoonotic onchocerciasis, recently discovered in Japan (Takaoka et al. 2012). The fauna of black flies in the Oriental Region, which was poorly studied before 1970 and once ranked last 6 Zootaxa 4261 (1) 2017 Magnolia Press TAKAOKA ET AL.

among the six zoogeographical regions in number of species, has dramatically emerged as the second largest in the number of constituent species following that of the Palaearctic Region. It has 524 species (23.8 % of the total world number of extant species, 2,204; Adler and Crosskey 2016). The high biodiversity of black flies has been well documented in some countries such as Indonesia, Malaysia, Philippines and Thailand (Takaoka 1983, 2003; Takaoka and Choochote 2004b; Takaoka and Davies 1995, 1996; Takaoka and Suzuki 1984), whereas some other countries in the region remain to be fully explored. In Vietnam, the simuliid fauna has been poorly studied until recently. Only 16 species were recorded in the twentieth century (Crosskey and Howard 1997; Pham 1998, 1999). In 2013 and 2014, we conducted surveys of pupae and larvae of black flies in Tam Dao, Vinh Phuc Province (northern Vietnam); in Bach Ma and other localities, Thua Thien Hue Province (central Vietnam); and in Dalat, Lam Dong Province (southern Vietnam) (Fig. 1), and added 30 more species including 22 new species (Takaoka et al. 2014a, b, c, 2015a). Of these 46 species, which are all in the genus Simulium Latreille, 17, 4 and 25 species were placed in three subgenera: Gomphostilbia Enderlein, Nevermannia Enderlein, and Simulium Latreille s. str., respectively (Adler and Crosskey 2016; Takaoka et al. 2014a, b, c, 2015a). Three among the 46 species recorded from Vietnam S. (G.) asakoae Takaoka & Davies, S. (S.) nigrogilvum Summers and S. (S.) nodosum Puri were reported to transmit unknown filariae in Thailand (Takaoka et al. 2003, Fukuda et al. 2003, Ishii et al. 2008). One species, S. (N.) aureohirtum Brunetti, was found to be autogenous in the Ryukyu Islands, the Philippines, Malaysia, Thailand and Indonesia (Takaoka and Noda 1979; Takaoka 1989), but biting habits and other biological aspects of these and other species remain to be studied in Vietnam, though six species S. (G.) asakoae, S. (S.) chungi Takaoka & Huang, S. (S.) maenoi Takaoka & Choochote, S. (S.) grossifilum Takaoka & Davies, S. (S.) doipuiense Takaoka & Choochote (complex) (under the species name S. (S.) rufibasis Brunetti), and S. (S.) vietnamense Takaoka, Sofian-Azirun & Chen were attracted to humans in Tam Dao National Park (Takaoka et al. 2014b). We carried out surveys of larvae and pupae of black flies in Sapa, Lao Cai Province (northern Vietnam) in December, 2014, and in Nghe An Province (northern Vietnam) in December, 2015 (Fig. 1). The results of these surveys, coupled with reexaminations of black flies previously collected from Vinh Phuc Province and Lam Dong Province (Fig. 1), yielded 22 new species and three new records. All these new species are here described and illustrated. The substitute name S. (S.) huense is given for the species that originally was described under the name S. (S.) cavum Takaoka & Ya cob from southern Vietnam. A redescription of the female, male, pupa and larva of S. (G.) asakoae is presented, and the female and larva of S. (G.) hongthaii Takaoka, Sofian-Azirun & Ya cob are described for the first time. The total number of species of black flies recorded from Vietnam is now 70, all of which are placed in four subgenera of the genus Simulium (25 in Gomphostilbia; one in Montisimulium Rubtsov (a newly recorded subgenus in Vietnam); seven in Nevermannia; and 37 in Simulium). Keys to identify the 10 subgenera in the Oriental Region and all 70 species recorded from Vietnam are provided for females, males, pupae and mature larvae. The methods of collection, description and illustration, and terms for morphological features (Figs. 2 & 3) follow those of Takaoka (2003) and partially those of Adler et al. (2004). Adult black flies examined morphologically in this study were reared from pupae, and placed in 80% ethanol, together with their associated pupal exuviae and cocoons, and larvae were fixed in Carnoy s solution (one part glacial acetic acid and three parts 95% ethanol). Thoracic tissues of several adults fixed in 80% ethanol were used for a DNA sequence-based analysis. The results of DNA analysis of the Simulium tani Takaoka & Davies complex was previously published (Low et al. 2016), demonstrating the rich biodiversity of the Simulium tani complex and the discovery of more cryptic taxa in Vietnam, and delineating taxonomic boundaries for each taxon within this species complex. Larvae of the Simulium tuberosum species-group fixed in Carnoy s solutions were chromosomally analyzed, by which we also showed the wealth of biodiversity of the S. tuberosum species-group in Vietnam (Adler et al. 2016). Taxonomic treatment of each taxon was mainly based on morphological characters, referring to molecular and chromosomal data when available. Holotypes and paratypes of the new species are deposited in the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. The register numbers of University Malaya Simuliidae Research Project (abbreviated as UMSRP) are given for holotypes and paratypes. THE BLACK FLIES FROM VIETNAM Zootaxa 4261 (1) 2017 Magnolia Press 7

FIGURE 1. Map of Vietnam showing the provinces where the current and previous surveys of black flies were carried out. Nos. 1 5 show the five provinces where the current surveys were conducted (1, Lao Cai; 2, Vinh Phuc; 3, Nghe An; 4, Thua Thien Hue; 5, Lam Dong), and nos. 6 11 show the provinces where Dr. X.D. Pham collected and recorded black flies (6, Lang Son; 7, Bac Kan (a part of formerly Bac Thai); 8, Bac Giang; 9, Yen Bai; 10, Ha Tay; 11, Hoa Binh). 8 Zootaxa 4261 (1) 2017 Magnolia Press TAKAOKA ET AL.

FIGURE 2. Morphological features and their terms of adults of black flies. A, Female head (front view); B, Thorax (lateral view); C, Female legs (fore, mid and hind legs from left); D; Wing; E, Male genitalia (ventral view); F, Female terminalia (ventral view). (Reproduced from Takaoka 2003). THE BLACK FLIES FROM VIETNAM Zootaxa 4261 (1) 2017 Magnolia Press 9

FIGURE 3. Morphological features and their terms of the pupa and larva of black flies. A, Pupa in cocoon; B, Head integument of pupa spread flattened (front view); C & D, Pupal abdomens (C, dorsal view; D, ventral view); E, Mature larva (lateral view); F & G, Head capsules of larva (F, dorsal view; G, ventral view); H, Hypostoma; I, Posterior tip of larval abdomen (posterodorsal view). (Reproduced from Takaoka 2003). Systematics Genus Simulium Latreille The family Simuliidae is classified in 26 genera, of which Simulium is the largest and most widely distributed genus, with 37 subgenera (Adler and Crosskey 2016). Simulium is the only genus recognized in the Oriental 10 Zootaxa 4261 (1) 2017 Magnolia Press TAKAOKA ET AL.

Region, where a total of 524 species are recorded (Adler and Crosskey 2016), and they are further classified in 10 subgenera, i.e., three endemic subgenera (Asiosimulium Takaoka & Choochote, Daviesellum Takaoka & Adler, and Wallacellum Takaoka), one semi-endemic subgenus (Gomphostilbia), four cosmopolitan subgenera (Byssodon Enderlein, Eusimulium Roubaud, Nevermannia and Simulium), and two subgenera with a Palaearctic distribution (Montisimulium and Wilhelmia Enderlein). In the Oriental Region, the subgenus Simulium is dominant, having 232 species (44.3%), followed by Gomphostilbia with 193 component species (36.8%) and Nevermannia with 55 species (10.5%), while seven other subgenera consist of 1 to 16 species. Four subgenera (Gomphostilbia, Montisimulium, Nevermannia and Simulium) have been reported in Vietnam. Definitions of the genus Simulium and 10 subgenera follow those given by Crosskey (1969), Takaoka (2003, 2012), Takaoka and Adler (1997) and Takaoka and Choochote (2005b). The keys to all these subgenera of the genus Simulium are herein updated. The subgenera and species-groups known from Vietnam are shown in bold face type. Keys to 10 subgenera of the genus Simulium in the Oriental Region Females 1. Katepisternum haired.................................................................................. 2 Katepisternum bare.................................................................................... 3 2. Calcipala elongate, reaching apex of second tarsomere............................................... Wallacellum Calcipala medium-long, not reaching apex of second tarsomere......................................gomphostilbia 3. Pleural membrane haired................................................................................ 4 Pleural membrane bare................................................................................. 5 4. Claw without tooth.............................................................................wilhelmia Claw with small subbasal tooth.............................................. Simulium (S. ornatum species-group) 5. Postnotum with hairs.......................................................................... Eusimulium Postnotum without hairs.................................................................................6 6. Claw simple or with small subbasal tooth................................................................... 7 Claw with large or medium-sized basal tooth................................................................8 7. Paraproct with cluster of dark spines anteriorly..................................................... Daviesellum Paraproct without cluster of dark spines anteriorly........................................ Simulium (most species) 8. Basal section of radial vein bare....................................................................byssodon Basal section of radial vein haired........................................................................ 9 9. Claw with medium-sized basal tooth.......................................................... Montisimulium Claw with large basal tooth............................................................................. 10 10. Cibarium with numerous spinous processes medially............................................... Asiosimulium Cibarium bare or with minute processes near ventral margin..........................................nevermannia Males 1. Style longer than coxite................................................................................. 2 Style shorter than coxite................................................................................ 3 2. Coxite much longer than wide.................................................................. Daviesellum Coxite as long as, or slightly shorter than, wide.......................................................simulium 3. Katepisternum haired.................................................................................. 4 Katepisternum bare.................................................................................... 5 4. Calcipala elongate, reaching apex of second tarsomere............................................... Wallacellum Calcipala medium-long, not reaching apex of second tarsomere..................................... Gomphostilbia 5. Pleural membrane haired.........................................................................wilhelmia Pleural membrane bare................................................................................. 6 6. Basal section of radial vein bare................................................................... Byssodon Basal section of radial vein haired........................................................................ 7 7. Paramere without hooks...................................................................... Asiosimulium Paramere with parameral hook(s).........................................................................8 8. Hind basitarsus slender; ventral plate with median keel........................................................ 9 Hind basitarsus enlarged; ventral plate without median keel................................................... 10 9. Ventral plate narrow, with divergent long arms......................................................eusimulium Ventral plate broad, with parallel-sided short arms.......................... Nevermannia (S. ruficorne species-group) 10. Paramere with one long hook; median sclerite forked apically................. Nevermannia (S. vernum species-group) Paramere with three or more hooks; median sclerite club-shaped, not forked apically.................................................................................. Montisimulium & Nevermannia (S. feuerborni species-group) THE BLACK FLIES FROM VIETNAM Zootaxa 4261 (1) 2017 Magnolia Press 11

Pupae 1. Gill filaments with numerous minute black spots (Fig. 27H).........................................Montisimulium Gill filaments without minute black spots................................................................... 2 2. Abdominal segments 5 9 without spine-combs dorsally.......................................................3 Abdominal segments with spine-combs dorsally (at least on segment 8)........................................... 8 3. Gill with 19 33 filaments..................................................................... Asiosimulium Gill with three, four, six or eight filaments.................................................................. 4 4. Gill with three filaments..........................................................................byssodon Gill with four, six or eight filaments....................................................................... 5 5. Gill with four filaments........................................................................ Wallacellum Gill with six or eight filaments........................................................................... 6 6. Gill with eight filaments.........................................................................wilhelmia Gill with six filaments.................................................................................. 7 7. Cocoon boot-shaped, with high neck............................................................. Daviesellum Cocoon shoe-shaped, with low neck.....................................................simulium (two species) 8. Frons with three pairs of trichomes; grapnel-shaped hooklets present on last abdominal segment (except a few species)...............................................................................................gomphostilbia Frons with two pairs of trichomes; grapnel-shaped hooklets absent on last abdominal segment.........................9 9. Abdominal segments 7 and 8 (or only segment 8) with spine-combs dorsally; terminal hooks absent or small, cone-like, if present.......................................................................................... Simulium Abdominal segments 6 9 (or 5 9 or 6 8) with spine-combs dorsally; terminal hooks large, cone-like..................10 10. Gill with inflated structure................................... Nevermannia (one species of S. vernum species-group) Gill with four, five, six or eight thread-like filaments......................................................... 11 11. Gill with four filaments............. Eusimulium & Nevermannia (most species of S. vernum species-group, one species of S. feuerborni species-group & one species of S. ruficorne species-group) Gill with five, six or eight filaments...................................................................... 12 12. Gill with eight filaments................................... Nevermannia (one species of S. ruficorne species-group) Gill with five or six filaments........................................................................... 13 13. Gill with five filaments................................. Nevermannia (one species of S. feuerborni species-group) Gill with six filaments.................................. Nevermannia (most species of S. feuerborni species-group & one species of S. ruficorne species-group) Mature larvae 1. Hypostoma wide, with prominent median tooth accompanied by six small teeth on each side; posterior circlet with over 400 rows of hooklets............................................................................. Daviesellum Hypostoma with median tooth accompanied by one corner tooth and three intermediate teeth on each side; posterior circlet with fewer than 250 rows of hooklets......................................................................2 2. Lateral margins of hypostoma smooth (Fig. 7E) (except a few species); main tooth of mandibular serrations at an acute angle to mandible apically (Fig. 7D)................................................................Gomphostilbia Lateral margins of hypostoma serrated; main tooth of mandibular serrations at right or obtuse angle to mandible apically...3 3. Mandibular serrations composed of two large teeth.............eusimulium & Nevermannia (S. ruficorne species-group) Mandibular serrations composed of one large tooth and one small tooth...........................................4 4. Postgenal cleft absent or vestigial (Fig. 27R).....................................................Montisimulium Postgenal cleft small to large.............................................................................5 5. Antenna as long as stem of labral fan............................................................ Asiosimulium Antenna longer than stem of labral fan.................................................................... 6 6. Pectination of outermost primary ray of labral fan different from those of other primary rays................ Wallacellum Pectination of outermost primary ray of labral fan similar to those of other primary rays..............................7 7. Ventral papillae large.......... Nevermannia (most species of S. feuerborni species-group & S. ruficorne species-group) Ventral papillae absent or small..........................................................................8 8. Rectal organ compound, each of three lobes with secondary lobules.......................................simulium Rectal organ simple, each of three lobes without secondary lobules.............................................. 9 9. Histoblast of pupal gill with three filaments.......................................................... Byssodon Histoblast of pupal gill with eight filaments..........................................................wilhelmia * The characters of the subgenus Byssodon used above are those of S. (B.) languidum, the only species of the subgenus in the Oriental Region, and are not necessarily shared with other species of the subgenus in other regions. I. Subgenus Gomphostilbia Enderlein Twenty-five species are classified in the subgenus Gomphostilbia, and are further placed in seven species-groups (S. asakoae, S. batoense, S. ceylonicum, S. darjeelingense, S. epistum, S. gombakense and S. varicorne species- 12 Zootaxa 4261 (1) 2017 Magnolia Press TAKAOKA ET AL.

groups), defined or redefined by Takaoka (2012). The S. asakoae species-group containing 13 species is dominant, followed by the S. batoense species-group accommodating seven species, while the other five species-groups are each represented by one species. Keys to 25 species of the subgenus Gomphostilbia Females* 1. Claw with small subbasal tooth................................................................ S. dachaisense Claw with large basal tooth (Fig. 4G)...................................................................... 2 2. Hind tibia darkened except base yellow.................................................................... 3 Hind tibia darkened on apical one-third to one-half and yellow on rest............................................ 9 3. Sensory vesicle elongate, longer than 0.4 times length of third maxillary segment................................... 4 Sensory vesicle medium-long, shorter than 0.4 times length of third maxillary segment..............................6 4. Mid and hind femora yellow except apical cap darkened...........................................s. parahiyangum Mid and hind femora light to medium brown except base yellow................................................ 5 5. Sensory vesicle elongate, 0.4 times length of third maxillary segment................................ S. lamdongense Sensory vesicle elongate, 0.5 times length of third maxillary segment (Fig. 22A).....................S. eshimai sp. nov. 6. Fore coxa light brown......................................................................s. longlanhense Fore coxa yellowish...................................................................................7 7. Sternite 8 with about 24 28 stout hairs on each side................................................s. montiblense Sternite 8 with about 10 stout hairs on each side............................................................. 8 8. Frons:head ratio 1.0:5.0....................................................................... S. duolongum Frons:head ratio 1.0:4.5.........................................................................S. siamense 9. Sensory vesicle elongate, 0.6 times length of third maxillary palpal segment......................................10 Sensory vesicle short to medium-long, 0.4 or less times length of third maxillary palpal segment...................... 11 10. Frons:head ratio 1.0:5.2...................................................................... S. fuscidorsum Frons:head ratio 1.0:4.3........................................................................S. thituyenae 11. Hind tibia darkened on apical one-third...................................................................12 Hind tibia darkened on apical one-half.................................................................... 16 12. Mandible with 15 outer teeth................................................................s. thuathienense Mandible lacking outer teeth or with 1 6 outer teeth......................................................... 13 13. Outer margin of mandible without tooth........................................................... S. hongthaii Outer margin of mandible toothed........................................................................ 14 14. Outer margin of mandible with two teeth (Fig. 17B)................................................ S. unii sp. nov. Outer margin of mandible with five or six teeth (Fig. 4C)..................................................... 15 15. Sensory vesicle 0.3 0.4 times length of third maxillary palpal segment (Fig. 4A, B)......................... S. asakoae Sensory vesicle 0.2 0.3 times length of third maxillary palpal segment (Fig. 8A)................ S. chaudinhense sp. nov. 16. Tuft hairs of base of radius brown............................................................. S. inthanonense Tuft hairs of base of radius yellow................................................. S. confertum & S. phulocense Males** 1. Antenna with eight flagellomeres............................................................ S. breviflagellum Antenna with nine flagellomeres.......................................................................... 2 2. Hind basitarsus enlarged................................................................................ 3 Hind basitarsus slender, parallel-sided.................................................................... 14 3. Fore coxa darkened......................................................................s. eshimai sp. nov. Fore coxa yellowish................................................................................... 4 4. Upper-eye facets in 16 vertical columns.................................................................... 5 Upper-eye facets in 9 14 vertical columns..................................................................6 5. Hair tuft of base of radius dark................................................................s. inthanonense Hair tuft of base of radius yellow................................................................ S. hongthaii 6. Antenna yellow except apical four or five or six flagellomeres darkened.......................................... 7 Antenna darkened except scape, pedicel and base of first flagellomere........................................... 10 7. Upper-eye facets in 9 or 10 vertical columns and 11 or 12 horizontal rows......................................... 8 Upper-eye facets in 11 13 vertical columns and 13 or 14 horizontal rows.........................................9 8. Upper-eye facets in 9 vertical columns and 11 or 12 horizontal rows.......................... S. vinhphucense sp. nov. Upper-eye facets in 10 vertical columns and 12 horizontal rows.............................. S. chaudinhense sp. nov. 9. Upper-eye facets in 11 vertical columns and 13 horizontal rows......................................... S. asakoae Upper-eye facets in 13 or 14 vertical columns and 14 horizontal rows................................. S. unii sp. nov. 10. Upper-eye facets in 14 vertical columns and 14 or 15 horizontal rows........................................... 11 Upper-eye facets in 11 13 vertical columns and 13 or 14 horizontal rows........................................ 13 THE BLACK FLIES FROM VIETNAM Zootaxa 4261 (1) 2017 Magnolia Press 13

11. Paramere with setae......................................................................... S. tamdaoense Paramere without setae................................................................................ 12 12. Hind basitarsus as wide as hind femur............................................................s. confertum Hind basitarsus 0.8 times as wide as hind femur...........................................s. quychauense sp. nov. 13. Hind basitarsus yellowish on basal two-fifths and dark on rest................................ S. sanchayense sp. nov. Hind basitarsus white on little more than basal half and dark on rest....................................s. phulocense 14. Abdominal segments 2, 6 and 7 each with pair of shiny dorsolateral patches...................................... 15 Abdominal segments 2, 5, 6 and 7 each with pair of shiny dorsolateral patches.................................... 17 15. Style without terminal spine.................................................................s. parahiyangum Style with terminal spine............................................................................... 16 16. Ventral plate in ventral view with body tapered from base to middle, abruptly narrowed, then tapered posteriorly................................................................................................... S. lamdongense Ventral plate in ventral view with body nearly parallel-sided, though posterolateral corners rounded........ S. longlanhense 17. Upper-eye facets in 10 vertical columns and 10 horizontal rows...................................... S. montiblense Upper-eye facets in 15 18 vertical columns and 15 18 horizontal rows.......................................... 18 18. Upper-eye facets in 18 vertical columns and 18 horizontal rows...................................... S. dachaisense Upper-eye facets in 15 or 16 vertical columns and 15 or 16 horizontal rows....................................... 19 19. Scutum with golden-yellow short hairs.............................................................s. siamense Scutum with brassy short hairs................................................................. S. duolongum Pupae 1. Gill composed of inflated structure with eight slender filaments.....................................s. thuathienense Gill composed of eight or ten slender filaments..............................................................2 2. Gill with ten filaments...................................................................... S. lamdongense Gill with eight filaments................................................................................3 3. All gill filaments short (about 1.0 mm)..................................................................... 4 All or some gill filaments medium-long to long (more than 1.5 mm)............................................. 7 4. Antennal sheath with tubercles........................................................................... 5 Antennal sheath without tubercles........................................................................6 5. Gill filaments arranged as 3+3+2 from dorsal to ventral.......................................... S. parahiyangum Gill filaments arranged as 2+1+3+2 from dorsal to ventral......................................... S. longlanhense 6. Face with additional trichome mediolaterally on each side (Fig. 24A)............................. S. eshimai sp. nov. Face without additional trichome mediolaterally on each side........................................ S. montiblense 7. Common basal stalk longer than interspiracular trunk...............................................s. fuscidorsum Common basal stalk shorter than interspiracular trunk......................................................... 8 8. Dorsal and middle triplets of gill filaments not sharing common stalk............................................ 9 Dorsal and middle triplets of gill filaments sharing common stalk.............................................. 11 9. Gill filaments with indistinct annular ridges...................................................... S. duolongum Gill filaments with marked annular ridges................................................................. 10 10. Dorsal surface of abdominal segments 1 and 2 grayish..............................................s. dachaisense Dorsal surface of abdominal segments 1 and 2 pale yellow.............................................s. siamense 11. Dorsal surface of abdominal segments 1 and 2 (or also 3 and 4) darkened........................................ 12 Dorsal surface of abdominal segments 1 and 2 unpigmented................................................... 14 12. Cocoon with long anterodorsal projection (Fig. 9L)............................................ hmongense sp. nov. Cocoon without anterodorsal projection...................................................................13 13. Dorsal surface of abdominal segments 1 and 2 with minute tubercles (Fig. 8C)................. S. chaudinhense sp. nov. Dorsal surface of abdominal segments 1 and 2 lacking tubercles......................................... S. asakoae 14. Cocoon with anterodorsal margin produced anteromedially, appearing as letter W or with short projection (Fig. 19E)......15 Cocoon with anterodorsal margin somewhat produced anteromedially or not...................................... 18 15. Stalk of dorsal triplet against that of ventral pair of filaments at angle of 90 degrees or greater....................... 16 Stalk of dorsal triplet against that of ventral pair of filaments at angle of less than 90 degrees........................ 17 16. Dorsal triplet of filaments with secondary stalk longer than primary stalk; terminal hooks broad, plate-like, with outer serrated margin and three times length of inner margin...................................................s. breviflagellum Dorsal triplet of filaments with secondary stalk much shorter than primary stalk; terminal hooks narrow, plate-like, with outer margin 1.8 times length of inner margin (Fig. 19D)................................................S. unii sp. nov. 17. Stalk of ventral pair of filaments twice length of primary and secondary stalks of middle triplet of filaments combined................................................................................................ S. phulocense Stalk of ventral pair of filaments as long as or shorter than length of primary and secondary stalks of middle triplet of filaments combined................................................................................ S. inthanonense 18. Abdominal segment 9 without spine-combs...............................................s. quychauense sp. nov. Abdominal segment 9 with spine-combs.................................................................. 19 19. Dorsal and middle triplets with short common stalk shorter than their primary stalks.......................s. confertum Dorsal and middle triplets with short common stalk nearly as long as or longer than their primary stalks................ 20 20. Terminal hooks flat and narrow, longer than wide...................................................s. thituyenae 14 Zootaxa 4261 (1) 2017 Magnolia Press TAKAOKA ET AL.