Limited differentiation among black flies in the Simulium multistriatum species group (Diptera: Simuliidae) in Thailand: cryptic species, homosequential species and homosequential cryptic species

Limited differentiation among black flies in the Simulium multistriatum species group (Diptera:... Abstract The high degree of morphological homogeneity in the Simuliidae often necessitates an integrated approach to the systematics of the family. We used cytological and molecular approaches to examine species delimitation and evolutionary relationships of black flies in the Simulium multistriatum species group in Thailand. A total of 664 larvae of eight nominal morphospecies (Simulium bullatum, Simulium chainarongi, Simulium chaliowae, Simulium daoense, Simulium fenestratum, Simulium lampangense, Simulium malayense and Simulium triglobus) from 30 sites in Thailand, one in Malaysia and one in Vietnam were chromosomally analysed, and 69 specimens were molecularly analysed for cytochrome c oxidase I and II gene sequences. We recognize ten taxa, based largely on unique chromosome rearrangements. Simulium chainarongi, S. fenestratum and S. triglobus are homosequential species. Simulium malayense consists of three cytoforms (A, B and C). Identical chromosomes, minimal morphological differentiation and low COI+ COII differentiation (1.20%) suggest that S. lampangense is either a junior synonym or a homosequential cryptic species of S. chaliowae. High genetic diversity in the geographically widespread nominal species S. fenestratum suggests that it consists of at least two cryptic species. Chromosomal and molecular phylogenetic inference shows good agreement within the S. multistriatum species group, but not with regard to other species groups. chromosomes, COI gene, COII gene, cryptic species, phylogeny, systematics INTRODUCTION Morphological uniformity in many groups of the Simuliidae presents a challenge for the taxonomy and systematics of this medically important family. The extent of this challenge increased when cryptic diversity was discovered through studies of the banding patterns of the larval polytene chromosomes (Rothfels, 1979, 1988; Adler, Cheke & Post, 2010). These studies revealed a spectrum of differentiation that includes (1) morphologically and chromosomally unique species, (2) morphologically identical species with unique chromosomal banding sequences (cryptic species; Tangkawanit et al., 2009), (3) morphologically distinct species with identical chromosomal banding sequences (homosequential species; Adler, Currie & Wood, 2004), and (4) morphologically identical species with identical chromosomal sequences (homosequential cryptic species; Bedo, 1979; Henderson, 1986). Molecular approaches have offered a wealth of new characters for discovering and diagnosing biodiversity in the Simuliidae (Pramual & Wongpakam, 2011, 2013; Pramual & Kuvangkadilok, 2012). The Simulium multistriatum species group consists of 34 nominal species distributed primarily in the Oriental Region but also in the Palearctic Region (Adler & Crosskey, 2017). Nine species have been recorded from Thailand: Simulium bullatum Takaoka and Choochote, 2005; Simulium chainarongi Takaoka & Kuvangkadilok, 1999; Simulium chaliowae Takaoka & Boonkemtong, 1999; Simulium chanyae Takaoka & Choochote, 2007; Simulium fenestratum Edwards, 1934; Simulium lampangense Takaoka & Choochote, 2005; Simulium malayense Takaoka & Davies, 1995; Simulium takense Takaoka & Choochote, 2005; and Simulium triglobus Kuvangkadilok & Takaoka, 1999. All members of the group are morphologically similar in all life stages (Takaoka & Choochote, 2005a, b). Some frequently used mitochondrial (COI and COII) and nuclear genes (18S/ITS1) fail to separate all species of the group (Pramual & Nanork, 2012), whereas other genes (e.g. ECP1) provide better resolution (Thaijarern, Pramual & Adler, 2017). Morphological and molecular data, however, have been insufficient for fully resolving the taxonomy and systematics of this species group, and the chromosomes have not yet been investigated. Our primary objective was to use polytene chromosomes from the larval silk glands, supplemented with analyses of the COI and COII genes, to understand differentiation in the S. multistriatum species group. Our specific objectives were to test morphological hypotheses of species via chromosomal and molecular inference of reproductive isolation, inspect nominal species for hidden biodiversity and infer evolutionary relationships of the constituent members of the group in Thailand, and to other species groups in the subgenus Simulium, by using an integrated approach (Ilmonen et al., 2009; Adler & Huang, 2011; Pramual & Kuvangkadilok, 2012) based on morphology, cytology and gene sequences. We focused our study of this group in Thailand because the greatest number of species in Southeast Asia is found there (Adler & Crosskey, 2017) and because many of the group’s habitats in the country have been documented (Takaoka & Choochote, 2005a, b). MATERIAL AND METHODS Collection and identification Larvae and pupae of the S. multistriatum species group were collected from 30 stream sites in Thailand (Fig. 1), one in Malaysia and one in Vietnam (Table 1). They were fixed in three changes of Carnoy’s solution (1:3 glacial acetic acid:absolute ethanol) and stored at −20 °C until processing. Species were initially identified with the morphological keys and descriptions of Takaoka & Davies (1995), Takaoka & Kuvangkadilok (1999), Takaoka & Choochote (2004, 2005a, b) and Thaijarern et al. (2017). Representative larvae were deposited in the collection of the Department of Biology, Faculty of Science at Mahasarakham University in Thailand and in the Clemson University Arthropod Collection in Clemson, SC, USA. Table 1. Collection sites for larvae of the Simulium multistriatum group in Thailand, Malaysia and Vietnam Site Location Province Elevation (m) Latitude Longitude Date Larvae prepared (completely analysed) Taxon Thailand 270 Lert Pop waterfall Loei 1152 17°30′N 101°20′E 27 November 2013 17 (17) S. bullatum 279 Phu Suan Sai Loei 918 17°30′N 101°56′E 28 November 2013 9 (9) S. bullatum 396 Lert Pop waterfall Loei 1123 17°30′N101°20′E 20 September 2015 2 (2)17 (12) S. bullatumS. fenestratum 287 Na Ku Ha waterfall Phrae 556 18°07′N 100°17′E 6 December 2013 71 (59) S. chaliowae 294 Huai Ton Phueng waterfall Phayao 361 19°16′N 100°53′E 7 December 2013 89 (69) S. chaliowae 244 Pha Luang waterfall Ubon Ratchathani 166 15°36′N 105°22′E 2 December 2013 43 (28) S. chainarongi 253 Huai Sai Yai waterfall Ubon Ratchathani 218 14°55′N 105°30′E 3 December 2013 18 (7) S. chainarongi 254 Huai Luang waterfall Ubon Ratchathani 240 14°25′N 105°24′E 3 December 2013 51 (29) S. chainarongi 255 Kaeng Lam Duan waterfall Ubon Ratchathani 179 14°26′N 105°06′E 3 December 2013 71 (57) S. chainarongi 350 Wang Yai waterfall Si Saket 219 14°26′N 104°29′E 16 October 2014 16 (12) S. chainarongi 264 Piang Din waterfall Loei 627 17° 03′N 101°44′E 26 December 2013 16 (8*) S. fenestratum 268 Song Khon waterfall Loei 743 17°21′N 101°24′E 27 December 2013 14 (12*) S. fenestratum 275 Than Sawan waterfall Loei 506 17o 29′N 101°3.5′E 28 December 2013 14 (10*) S. fenestratum 276 Khring waterfall Loei 643 17°28′N 101°58′E 28 December 2013 16 (8*) S. fenestratum 280 Tad Huong waterfall Loei 560 17°33′N 100°59′E 28 December 2013 29 (19*) S. fenestratum 401 Huai Khamin Noy waterfall Loei 1208 16° 59′N 101°00′E 26 October 2015 7 (4*) S. fenestratum 363 Than Ngam waterfall Udon Thani 278 17° 09′N 102°44′E 7 December 2014 10 (3) S. fenestratum 364 Koi Nang waterfall Udon Thani 416 17° 07′N 102°43′E 7 December 2014 26 (16) S. fenestratum 297 Ban Pang Faen Chiang Mai 615 19° 00′N 99°18′E 7 November 2013 46 (29*) S. fenestratum 372 Huai Mae Om Long Chiang Mai 852 18°09′N 98°13′E 16 January 2015 23 (15) S. fenestratum 384 Mae Taeng District Chiang Mai 703 19°07′N 98°45′E 16 January 2015 11 (9) S. fenestratum 402 Phu Soi Dao waterfall Uttaradit 654 17°44′N 100°59′E 27 October 2015 23 (15) S. fenestratum 320 Mae Sot Tak 303 16°48′N 98°59′E 19 February 2014 7 (6*) S. fenestratum 342 Klong Na Rai waterfall Chan Tha Buri 43 12°34′N 102°10′E 5 March 2014 43 (30*) S. fenestratum 343 Pliw waterfall Chan Tha Buri 46 12°31′N 102°10′E 5 March 2014 66 (63*) S. fenestratum 345 Klong Gaew waterfall Trat 65 12°37′N 102°34′E 5 March 2014 7 (6) S. fenestratum 408 Wang Kaew waterfall Lampang 626 19°18′N 99°39′E 18 December 2015 26 (19) S. lampangense 409 Wang Thong waterfall Lampang 510 19°16′N 99°39′E 18 December 2015 27 (23) S. lampangense 298 Siribhumi waterfall Chiang Mai 1304 18°32′N98°30′E 8 December 2013 7 (5)1 (1) S. malayense AS. malayense B 9 (9) S. daoense 7 (7) S. fenestratum 13 October 2017† 14 (–)2 (2) S. fenestratumS. malayense A 291 Ton Tong waterfall Nan 1027 19°12′N 101°04′E 6 December 2013 43 (28) S. triglobus Malaysia MYC Chenderiang waterfall Perak 129 04°21′N 101°14′E 23 February 2015 11 (6) S. malayense C Vietnam Dao Sapa Lao Cai 1315 22°23′N 103°50′E 22 December 2014 3 (3) S. daoense Site Location Province Elevation (m) Latitude Longitude Date Larvae prepared (completely analysed) Taxon Thailand 270 Lert Pop waterfall Loei 1152 17°30′N 101°20′E 27 November 2013 17 (17) S. bullatum 279 Phu Suan Sai Loei 918 17°30′N 101°56′E 28 November 2013 9 (9) S. bullatum 396 Lert Pop waterfall Loei 1123 17°30′N101°20′E 20 September 2015 2 (2)17 (12) S. bullatumS. fenestratum 287 Na Ku Ha waterfall Phrae 556 18°07′N 100°17′E 6 December 2013 71 (59) S. chaliowae 294 Huai Ton Phueng waterfall Phayao 361 19°16′N 100°53′E 7 December 2013 89 (69) S. chaliowae 244 Pha Luang waterfall Ubon Ratchathani 166 15°36′N 105°22′E 2 December 2013 43 (28) S. chainarongi 253 Huai Sai Yai waterfall Ubon Ratchathani 218 14°55′N 105°30′E 3 December 2013 18 (7) S. chainarongi 254 Huai Luang waterfall Ubon Ratchathani 240 14°25′N 105°24′E 3 December 2013 51 (29) S. chainarongi 255 Kaeng Lam Duan waterfall Ubon Ratchathani 179 14°26′N 105°06′E 3 December 2013 71 (57) S. chainarongi 350 Wang Yai waterfall Si Saket 219 14°26′N 104°29′E 16 October 2014 16 (12) S. chainarongi 264 Piang Din waterfall Loei 627 17° 03′N 101°44′E 26 December 2013 16 (8*) S. fenestratum 268 Song Khon waterfall Loei 743 17°21′N 101°24′E 27 December 2013 14 (12*) S. fenestratum 275 Than Sawan waterfall Loei 506 17o 29′N 101°3.5′E 28 December 2013 14 (10*) S. fenestratum 276 Khring waterfall Loei 643 17°28′N 101°58′E 28 December 2013 16 (8*) S. fenestratum 280 Tad Huong waterfall Loei 560 17°33′N 100°59′E 28 December 2013 29 (19*) S. fenestratum 401 Huai Khamin Noy waterfall Loei 1208 16° 59′N 101°00′E 26 October 2015 7 (4*) S. fenestratum 363 Than Ngam waterfall Udon Thani 278 17° 09′N 102°44′E 7 December 2014 10 (3) S. fenestratum 364 Koi Nang waterfall Udon Thani 416 17° 07′N 102°43′E 7 December 2014 26 (16) S. fenestratum 297 Ban Pang Faen Chiang Mai 615 19° 00′N 99°18′E 7 November 2013 46 (29*) S. fenestratum 372 Huai Mae Om Long Chiang Mai 852 18°09′N 98°13′E 16 January 2015 23 (15) S. fenestratum 384 Mae Taeng District Chiang Mai 703 19°07′N 98°45′E 16 January 2015 11 (9) S. fenestratum 402 Phu Soi Dao waterfall Uttaradit 654 17°44′N 100°59′E 27 October 2015 23 (15) S. fenestratum 320 Mae Sot Tak 303 16°48′N 98°59′E 19 February 2014 7 (6*) S. fenestratum 342 Klong Na Rai waterfall Chan Tha Buri 43 12°34′N 102°10′E 5 March 2014 43 (30*) S. fenestratum 343 Pliw waterfall Chan Tha Buri 46 12°31′N 102°10′E 5 March 2014 66 (63*) S. fenestratum 345 Klong Gaew waterfall Trat 65 12°37′N 102°34′E 5 March 2014 7 (6) S. fenestratum 408 Wang Kaew waterfall Lampang 626 19°18′N 99°39′E 18 December 2015 26 (19) S. lampangense 409 Wang Thong waterfall Lampang 510 19°16′N 99°39′E 18 December 2015 27 (23) S. lampangense 298 Siribhumi waterfall Chiang Mai 1304 18°32′N98°30′E 8 December 2013 7 (5)1 (1) S. malayense AS. malayense B 9 (9) S. daoense 7 (7) S. fenestratum 13 October 2017† 14 (–)2 (2) S. fenestratumS. malayense A 291 Ton Tong waterfall Nan 1027 19°12′N 101°04′E 6 December 2013 43 (28) S. triglobus Malaysia MYC Chenderiang waterfall Perak 129 04°21′N 101°14′E 23 February 2015 11 (6) S. malayense C Vietnam Dao Sapa Lao Cai 1315 22°23′N 103°50′E 22 December 2014 3 (3) S. daoense *IIIL-2 was not analysed for all larvae; for each site, the number analysed is given in parentheses: site 264 (4), 268 (7), 275 (9), 276 (7), 280 (18), 401 (3), 297 (26), 320 (4), 342 (4) and 343 (30). †Of 16 larvae analysed from this collection, 14 were S. fenestratum and two were S. malayense A; larvae of S. daoense were not found on this date. These larvae of S. fenestratum were cytologically similar to those of 8 December 2013 but were not scored for polymorphisms and, therefore, were not included in any counts or analyses. View Large Table 1. Collection sites for larvae of the Simulium multistriatum group in Thailand, Malaysia and Vietnam Site Location Province Elevation (m) Latitude Longitude Date Larvae prepared (completely analysed) Taxon Thailand 270 Lert Pop waterfall Loei 1152 17°30′N 101°20′E 27 November 2013 17 (17) S. bullatum 279 Phu Suan Sai Loei 918 17°30′N 101°56′E 28 November 2013 9 (9) S. bullatum 396 Lert Pop waterfall Loei 1123 17°30′N101°20′E 20 September 2015 2 (2)17 (12) S. bullatumS. fenestratum 287 Na Ku Ha waterfall Phrae 556 18°07′N 100°17′E 6 December 2013 71 (59) S. chaliowae 294 Huai Ton Phueng waterfall Phayao 361 19°16′N 100°53′E 7 December 2013 89 (69) S. chaliowae 244 Pha Luang waterfall Ubon Ratchathani 166 15°36′N 105°22′E 2 December 2013 43 (28) S. chainarongi 253 Huai Sai Yai waterfall Ubon Ratchathani 218 14°55′N 105°30′E 3 December 2013 18 (7) S. chainarongi 254 Huai Luang waterfall Ubon Ratchathani 240 14°25′N 105°24′E 3 December 2013 51 (29) S. chainarongi 255 Kaeng Lam Duan waterfall Ubon Ratchathani 179 14°26′N 105°06′E 3 December 2013 71 (57) S. chainarongi 350 Wang Yai waterfall Si Saket 219 14°26′N 104°29′E 16 October 2014 16 (12) S. chainarongi 264 Piang Din waterfall Loei 627 17° 03′N 101°44′E 26 December 2013 16 (8*) S. fenestratum 268 Song Khon waterfall Loei 743 17°21′N 101°24′E 27 December 2013 14 (12*) S. fenestratum 275 Than Sawan waterfall Loei 506 17o 29′N 101°3.5′E 28 December 2013 14 (10*) S. fenestratum 276 Khring waterfall Loei 643 17°28′N 101°58′E 28 December 2013 16 (8*) S. fenestratum 280 Tad Huong waterfall Loei 560 17°33′N 100°59′E 28 December 2013 29 (19*) S. fenestratum 401 Huai Khamin Noy waterfall Loei 1208 16° 59′N 101°00′E 26 October 2015 7 (4*) S. fenestratum 363 Than Ngam waterfall Udon Thani 278 17° 09′N 102°44′E 7 December 2014 10 (3) S. fenestratum 364 Koi Nang waterfall Udon Thani 416 17° 07′N 102°43′E 7 December 2014 26 (16) S. fenestratum 297 Ban Pang Faen Chiang Mai 615 19° 00′N 99°18′E 7 November 2013 46 (29*) S. fenestratum 372 Huai Mae Om Long Chiang Mai 852 18°09′N 98°13′E 16 January 2015 23 (15) S. fenestratum 384 Mae Taeng District Chiang Mai 703 19°07′N 98°45′E 16 January 2015 11 (9) S. fenestratum 402 Phu Soi Dao waterfall Uttaradit 654 17°44′N 100°59′E 27 October 2015 23 (15) S. fenestratum 320 Mae Sot Tak 303 16°48′N 98°59′E 19 February 2014 7 (6*) S. fenestratum 342 Klong Na Rai waterfall Chan Tha Buri 43 12°34′N 102°10′E 5 March 2014 43 (30*) S. fenestratum 343 Pliw waterfall Chan Tha Buri 46 12°31′N 102°10′E 5 March 2014 66 (63*) S. fenestratum 345 Klong Gaew waterfall Trat 65 12°37′N 102°34′E 5 March 2014 7 (6) S. fenestratum 408 Wang Kaew waterfall Lampang 626 19°18′N 99°39′E 18 December 2015 26 (19) S. lampangense 409 Wang Thong waterfall Lampang 510 19°16′N 99°39′E 18 December 2015 27 (23) S. lampangense 298 Siribhumi waterfall Chiang Mai 1304 18°32′N98°30′E 8 December 2013 7 (5)1 (1) S. malayense AS. malayense B 9 (9) S. daoense 7 (7) S. fenestratum 13 October 2017† 14 (–)2 (2) S. fenestratumS. malayense A 291 Ton Tong waterfall Nan 1027 19°12′N 101°04′E 6 December 2013 43 (28) S. triglobus Malaysia MYC Chenderiang waterfall Perak 129 04°21′N 101°14′E 23 February 2015 11 (6) S. malayense C Vietnam Dao Sapa Lao Cai 1315 22°23′N 103°50′E 22 December 2014 3 (3) S. daoense Site Location Province Elevation (m) Latitude Longitude Date Larvae prepared (completely analysed) Taxon Thailand 270 Lert Pop waterfall Loei 1152 17°30′N 101°20′E 27 November 2013 17 (17) S. bullatum 279 Phu Suan Sai Loei 918 17°30′N 101°56′E 28 November 2013 9 (9) S. bullatum 396 Lert Pop waterfall Loei 1123 17°30′N101°20′E 20 September 2015 2 (2)17 (12) S. bullatumS. fenestratum 287 Na Ku Ha waterfall Phrae 556 18°07′N 100°17′E 6 December 2013 71 (59) S. chaliowae 294 Huai Ton Phueng waterfall Phayao 361 19°16′N 100°53′E 7 December 2013 89 (69) S. chaliowae 244 Pha Luang waterfall Ubon Ratchathani 166 15°36′N 105°22′E 2 December 2013 43 (28) S. chainarongi 253 Huai Sai Yai waterfall Ubon Ratchathani 218 14°55′N 105°30′E 3 December 2013 18 (7) S. chainarongi 254 Huai Luang waterfall Ubon Ratchathani 240 14°25′N 105°24′E 3 December 2013 51 (29) S. chainarongi 255 Kaeng Lam Duan waterfall Ubon Ratchathani 179 14°26′N 105°06′E 3 December 2013 71 (57) S. chainarongi 350 Wang Yai waterfall Si Saket 219 14°26′N 104°29′E 16 October 2014 16 (12) S. chainarongi 264 Piang Din waterfall Loei 627 17° 03′N 101°44′E 26 December 2013 16 (8*) S. fenestratum 268 Song Khon waterfall Loei 743 17°21′N 101°24′E 27 December 2013 14 (12*) S. fenestratum 275 Than Sawan waterfall Loei 506 17o 29′N 101°3.5′E 28 December 2013 14 (10*) S. fenestratum 276 Khring waterfall Loei 643 17°28′N 101°58′E 28 December 2013 16 (8*) S. fenestratum 280 Tad Huong waterfall Loei 560 17°33′N 100°59′E 28 December 2013 29 (19*) S. fenestratum 401 Huai Khamin Noy waterfall Loei 1208 16° 59′N 101°00′E 26 October 2015 7 (4*) S. fenestratum 363 Than Ngam waterfall Udon Thani 278 17° 09′N 102°44′E 7 December 2014 10 (3) S. fenestratum 364 Koi Nang waterfall Udon Thani 416 17° 07′N 102°43′E 7 December 2014 26 (16) S. fenestratum 297 Ban Pang Faen Chiang Mai 615 19° 00′N 99°18′E 7 November 2013 46 (29*) S. fenestratum 372 Huai Mae Om Long Chiang Mai 852 18°09′N 98°13′E 16 January 2015 23 (15) S. fenestratum 384 Mae Taeng District Chiang Mai 703 19°07′N 98°45′E 16 January 2015 11 (9) S. fenestratum 402 Phu Soi Dao waterfall Uttaradit 654 17°44′N 100°59′E 27 October 2015 23 (15) S. fenestratum 320 Mae Sot Tak 303 16°48′N 98°59′E 19 February 2014 7 (6*) S. fenestratum 342 Klong Na Rai waterfall Chan Tha Buri 43 12°34′N 102°10′E 5 March 2014 43 (30*) S. fenestratum 343 Pliw waterfall Chan Tha Buri 46 12°31′N 102°10′E 5 March 2014 66 (63*) S. fenestratum 345 Klong Gaew waterfall Trat 65 12°37′N 102°34′E 5 March 2014 7 (6) S. fenestratum 408 Wang Kaew waterfall Lampang 626 19°18′N 99°39′E 18 December 2015 26 (19) S. lampangense 409 Wang Thong waterfall Lampang 510 19°16′N 99°39′E 18 December 2015 27 (23) S. lampangense 298 Siribhumi waterfall Chiang Mai 1304 18°32′N98°30′E 8 December 2013 7 (5)1 (1) S. malayense AS. malayense B 9 (9) S. daoense 7 (7) S. fenestratum 13 October 2017† 14 (–)2 (2) S. fenestratumS. malayense A 291 Ton Tong waterfall Nan 1027 19°12′N 101°04′E 6 December 2013 43 (28) S. triglobus Malaysia MYC Chenderiang waterfall Perak 129 04°21′N 101°14′E 23 February 2015 11 (6) S. malayense C Vietnam Dao Sapa Lao Cai 1315 22°23′N 103°50′E 22 December 2014 3 (3) S. daoense *IIIL-2 was not analysed for all larvae; for each site, the number analysed is given in parentheses: site 264 (4), 268 (7), 275 (9), 276 (7), 280 (18), 401 (3), 297 (26), 320 (4), 342 (4) and 343 (30). †Of 16 larvae analysed from this collection, 14 were S. fenestratum and two were S. malayense A; larvae of S. daoense were not found on this date. These larvae of S. fenestratum were cytologically similar to those of 8 December 2013 but were not scored for polymorphisms and, therefore, were not included in any counts or analyses. View Large Figure 1. View largeDownload slide Sampling sites for eight nominal species of Simulium multistriatum species group in Thailand. Details of sampling sites are given in Table 1. Figure 1. View largeDownload slide Sampling sites for eight nominal species of Simulium multistriatum species group in Thailand. Details of sampling sites are given in Table 1. Chromosome preparation and analysis Middle to final instar larvae were prepared for analysis of the polytene chromosomes from their silk glands, using the Feulgen method (Adler et al., 2016a). Representative chromosome preparations were photographed under oil immersion on an Olympus BX40 microscope (Simulium daoense) or a BH-2 Olympus microscope with a Jenoptik ProgRes SpeedXT Core 5 digital camera (all other taxa). The images were imported into Adobe Photoshop Elements 8 or Photoshop CS6 to construct chromosomal maps. The standard map for the S. multistriatum species group was based on the most central sequence among the group members, relative to the standard sequence established by Rothfels, Feraday & Kaneps (1978) and Adler et al. (2016a) for the subgenus Simulium. Chromosomal terminology and section numbering for the three chromosomes (I, II and III) followed the practice of Rothfels et al. (1978) and Adler et al. (2016a). Fixed inversions are italicized in the text and underlined on the chromosome maps; polymorphic rearrangements are given in regular type. Additional rearrangements are coded in the text and on the maps as follows: heteroband (hb), diffuse centromere band (d), additional (insertion) band (i), flocculent band expression (fl), and band enhancement (+). The following centromere associations were recognized: (1) chromocentre, i.e. all centromeres of all chromosomes in each nucleus were attached to a central mass of heterochromatin; (2) partial chromocentre, i.e. the centromeres of any two chromosomes of each nucleus were attached to a heterochromatic mass; and (3) ectopic pairing (pseudochromocentre), i.e. two or more centromeres were associated with one another in at least some, but typically not all, nuclei, and additional heterochromatin was absent from the centromere associations (Adler et al., 2004). The maximal width of the terminal flare for each chromosome arm of S. bullatum, S. fenestratum and S. triglobus was measured against the width of a subterminal indicator band and grouped into size categories as follows: 0 (less than the width of the indicator band), 1 (equal to width of the indicator band to ≤ 1.5 times its width), 2 (> 1.5 to ≤ 2.0 times width of the indicator band) and 3 (> 2.0 times width of the indicator band). A Mann–Whitney U test was used to evaluate ranked characteristics. Polymorphic inversions were tested for Hardy–Weinberg equilibrium when samples were large enough to provide at least five larvae for each state (homozygous standard, heterozygous and homozygous inverted). Evolutionary relationships The following species groups in the subgenus Simulium with chromosome complements fully resolved relative to the subgeneric standard of Rothfels et al. (1978) and Adler et al. (2016a) were screened for rearrangements, primarily inversions, shared with the S. multistriatum group: S. bezzii, S. ornatum, S. petersoni, S. variegatum (P. Adler, unpublished), S. malyschevi-reptans (Adler et al., 2016a), S. nobile (Tangkawanit et al., 2011), S. noelleri (Adler & Kachvorian, 2001), S. tuberosum (Tangkawanit et al., 2009, Adler et al., 2016b) and S. venustum groups (Huang, Adler & Takaoka, 2011). To permit additional comparison, we also resolved the entire sequence of the S. striatum group, based on maps of Pramual (2006). Species groups with any rearrangement(s) uniquely shared with the S. multistriatum species group were selected as outgroups. Four of these groups share unique inversions with the S. multistriatum species group: S. malyschevi-reptans, S. nobile and S. striatum groups. Among the 13 remaining species groups in the subgenus Simulium, the S. jenningsi group, which is closely related to the S. malyschevi-reptans group, might share IIS-1, but its highly scrambled IIS sequence has not yet been resolved (Adler et al., 2016a). Additionally, the S. griseifrons species group might share rearrangements with the S. multistriatum species group, based on molecular relationships showing that the two groups are closely related (Thanwisai, Kuvangkadilok & Baimai, 2006); however, not a single species in the group has been studied chromosomally (Adler & Crosskey, 2015). Furthermore, the S. griseifrons group has recently been divided into six species groups (Takaoka, 2017). All rearrangements shared between two or more of the members of the S. multistriatum species group were included in the phylogenetic analysis, with one exception. We did not include the uniquely manifested ‘2 blocks’ marker of S. chaliowae and S. lampangense because we suspect that it reflects gene expression and is environmentally influenced. Thus, a character matrix of 14 rearrangements was used in our analysis. Two character states were recognized for the rearrangements: 0 = absent and 1 = fixed. The maximum parsimony (MP) tree for phylogenetic relationships was calculated in PAUP* v.4.10b (Swofford, 2002), using a heuristic search with 1000 random addition sequence replicates; bootstrap support was estimated for 1000 replicates. DNA extraction, polymerase chain reaction, sequencing and analysis DNA was extracted with the Vivantis GF-1 Nucleic Extraction kit. Polymerase chain reaction (PCR) of the cytochrome c oxidase subunit I gene (COI) used the primers following the description of Folmer et al. (1994). The PCR followed the methods described by Rivera & Currie (2009). The cytochrome c oxidase subunit II gene (COII) used the primers TL2-J-3034 5′-ATTATGGCAGATTAGTGCA-3′ and TK-N-3785 5′-GTTTAA GAGACCAGTACTTG-3′, and PCR followed the methods of Conflitti et al. (2010). The PCR products were checked with 1% agarose gel electrophoresis and purified using a HighYield Gel/PCR DNA Fragment Extraction kit (RBC BioScience, Taiwan). Sequencing was performed by the Macrogen DNA sequencing service (Seoul, Korea) and 1st BASE DNA Sequencing Services (Singapore Science Park, Singapore), using primers as for PCR. A total of 69 sequences from nine cytologically distinct taxa of the S. multistriatum species group was included in the analyses (GenBank accession numbers: MG733997–MG734134). Genetic distances were calculated using MEGA7 (Tamura et al., 2013), based on the Kimura 2-parameter (K2P). Phylogenetic analyses were conducted separately for the COI and COII sequences and for the combined dataset, and included neighbor-joining (NJ), maximum likelihood (ML), MP and Bayesian methods. The NJ method inferred phylogenetic trees in MEGA7 (Tamura et al., 2013), and MP was calculated separately for each gene in PAUP* v.4.10b (Swofford, 2002). Branch support for NJ and MP was calculated using the bootstrapping method with 1000 replicates. Maximum likelihood was performed with PhyML 3.0 (Guindon et al., 2010). Node support was determined using an approximate likelihood-ratio test (Anisimova & Gascuel, 2006; Guindon et al., 2010). Bayesian inference was performed with MrBayes 3.04b and run for 2000000 generations with a sampling frequency of 100 generations (Huelsenbeck & Ronquist, 2001). Simulium nodosum, a member of the S. nobile species group, was used as an outgroup in all molecular phylogenetic analyses. The efficiency of specimen identification was obtained using the best match method in TaxonDNA (Meier et al., 2006). RESULTS The chromosomes of 912 larvae of the S. multistriatum species group were prepared, and the banding patterns of 664 (72.8%) of the larvae were analysed completely (Table 1), with the exception of the IIIL-2 sequence of S. fenestratum, for which 184 (65.9%) of 279 otherwise fully evaluated individuals were analysed. All larvae of the S. multistriatum species group had three pairs of tightly synapsed homologues (2n = 6). The nucleolar organizer (NO) was in the base of IS, near the expanded centromere region. The sex chromosomes of all species were microscopically undifferentiated. Chromosomal relationships of Simulium multistriatum species group to Simulium subgeneric standard The standard banding sequence of the S. multistriatum species group differed from the Simulium subgeneric standard by having the nucleolar organizer in the base of IS (rather than in IIIL) and by seven fixed inversions, as follows: IS: The basic sequence of this arm (Fig. 2) was identical to the subgeneric standard sequence of Rothfels et al. (1978). IL: The standard sequence for the S. multistriatum species group (Fig. 3, cf. Fig. 4) was identical to the subgeneric standard of Rothfels et al. (1978). IIS: The IIS sequence for all members of the S. multistriatum species group differed from the subgeneric standard of Adler et al. (2016a) by three fixed inversions. These three inversions divided the arm into six fragments. The most parsimonious reassemblage of fragments to produce the subgeneric standard, one inversion at a time, is shown below, and the order of the fragments is represented by the letters a–j (Fig. 5), where slashes represent inversion breakpoints and square brackets represent the inversion in each sequence. Inversion 2: a / g f / b c [i h / e d] j (= S. multistriatum species group standard) Inversion 3: a / g f / [b c d e] h i j Inversion 1: a [g f e d c b] h i j a b c d e f g h i j (= Simulium subgeneric standard) IIL: All members of the S. multistriatum species group could be derived by one fixed inversion (IIL-1; Fig. 6) from the subgeneric standard sequence of Rothfels et al. (1978). IIIS: The basic sequence of this arm (Fig. 7) was identical with the subgeneric standard sequence of Rothfels et al. (1978). IIIL: The basic IIIL sequence for the S. multistriatum species group was removed from the standard sequence of Adler et al. (2016a) by three fixed inversions (IIIL-3, IIIL-5 and IIIL-6; Fig. 8B, C). Figure 2. View largeDownload slide IS arm of Simulium multistriatum species group. A, B, Simulium fenestratum (female larva), corresponding to the Simulium subgeneric standard of Rothfels et al. (1978). C, Simulium chaliowae (male larva). Breakpoints of floating inversion IS-1 and fixed inversion IS-2 are indicated. C, centromere; g, glazed; NO, nucleolar organizer. Figure 2. View largeDownload slide IS arm of Simulium multistriatum species group. A, B, Simulium fenestratum (female larva), corresponding to the Simulium subgeneric standard of Rothfels et al. (1978). C, Simulium chaliowae (male larva). Breakpoints of floating inversion IS-1 and fixed inversion IS-2 are indicated. C, centromere; g, glazed; NO, nucleolar organizer. Figure 3. View largeDownload slide IL arm of Simulium bullatum (female larva), corresponding to the Simulium subgeneric standard of Rothfels et al. (1978) and the standard IL sequence of the S. multistriatum species group. Limits of fixed inversion IL-9 are indicated with brackets. C, centromere; M, marker. Figure 3. View largeDownload slide IL arm of Simulium bullatum (female larva), corresponding to the Simulium subgeneric standard of Rothfels et al. (1978) and the standard IL sequence of the S. multistriatum species group. Limits of fixed inversion IL-9 are indicated with brackets. C, centromere; M, marker. Figure 4. View largeDownload slide IL arm of Simulium multistriatum species group. Simulium daoense (male larva, sections 20–32) and Simulium fenestratum (female, sections 33–41), showing the IL-9 sequence. Breakpoints of floating inversions of S. fenestratum are indicated by brackets. Arrows indicate location of a heterochromatic band insertion (i) in S. fenestratum. M, marker. Figure 4. View largeDownload slide IL arm of Simulium multistriatum species group. Simulium daoense (male larva, sections 20–32) and Simulium fenestratum (female, sections 33–41), showing the IL-9 sequence. Breakpoints of floating inversions of S. fenestratum are indicated by brackets. Arrows indicate location of a heterochromatic band insertion (i) in S. fenestratum. M, marker. Figure 5. View largeDownload slide IIS arm of Simulium daoense (male larva), showing the standard sequence for the Simulium multistriatum species group relative to the Simulium subgeneric standard of Adler et al. (2016a). Breakpoints of three fixed inversions are indicated by numbered arrows 1–3. The letters a–j, when alphabetized, produce the subgeneric standard sequence. C, centromere. Figure 5. View largeDownload slide IIS arm of Simulium daoense (male larva), showing the standard sequence for the Simulium multistriatum species group relative to the Simulium subgeneric standard of Adler et al. (2016a). Breakpoints of three fixed inversions are indicated by numbered arrows 1–3. The letters a–j, when alphabetized, produce the subgeneric standard sequence. C, centromere. Figure 6. View largeDownload slide IIL arm of Simulium daoense (male larva), showing the standard sequence for the Simulium multistriatum species group, including fixed inversion IIL-1. Breakpoints of floating inversion IIL-2 of S. fenestratum are indicated by a bracket. C, centromere; gB, grey band; J, jagged; Pb, parabalbiani; po, polar. Figure 6. View largeDownload slide IIL arm of Simulium daoense (male larva), showing the standard sequence for the Simulium multistriatum species group, including fixed inversion IIL-1. Breakpoints of floating inversion IIL-2 of S. fenestratum are indicated by a bracket. C, centromere; gB, grey band; J, jagged; Pb, parabalbiani; po, polar. Figure 7. View largeDownload slide IIIS arm of Simulium lampangense (male larva), representing the standard sequence for the Simulium multistriatum species group, which is identical to the Simulium subgeneric standard sequence of Rothfels et al. (1978). Breakpoints of floating inversion IIIS-1 of Simulium chainarongi are shown. Bl, blister; C, centromere; Ca, capsule. Figure 7. View largeDownload slide IIIS arm of Simulium lampangense (male larva), representing the standard sequence for the Simulium multistriatum species group, which is identical to the Simulium subgeneric standard sequence of Rothfels et al. (1978). Breakpoints of floating inversion IIIS-1 of Simulium chainarongi are shown. Bl, blister; C, centromere; Ca, capsule. Figure 8. View largeDownload slide IIIL arm of Simulium fenestratum. A, basal sections (female larva, site 402) showing the IIIL-2, IIIL-3, 6 sequence; breakpoints of floating inversion IIIL-7 are indicated by a bracket. B, C, entire arm (female larva, site 343), showing the IIIL-3, 4, 5, 6 sequence; ordering the letters a–h produces the Simulium subgeneric sequence of Adler et al. (2016a). Breakpoints of three floating inversions [IIIL-8, IIIL-9 and compound (cmpd) inversion] are indicated by brackets. C, centromere. Figure 8. View largeDownload slide IIIL arm of Simulium fenestratum. A, basal sections (female larva, site 402) showing the IIIL-2, IIIL-3, 6 sequence; breakpoints of floating inversion IIIL-7 are indicated by a bracket. B, C, entire arm (female larva, site 343), showing the IIIL-3, 4, 5, 6 sequence; ordering the letters a–h produces the Simulium subgeneric sequence of Adler et al. (2016a). Breakpoints of three floating inversions [IIIL-8, IIIL-9 and compound (cmpd) inversion] are indicated by brackets. C, centromere. Simulium bullatum Takaoka & Choochote Three populations of S. bullatum from Loei Province were studied, and all 28 chromosomally prepared larvae were analysed completely (Table 1). Simulium bullatum differed from the standard for the S. multistriatum species group by one fixed inversion (IIIL-10; Fig. 9B) and a true chromocentre characterized by a glassy or darkly staining heterochromatic mass (Fig. 10A). Flaring at the end of IIIS was 3.0 times greater than the width of the indicator band, whereas the ends of IS, IL, IIS, IIL and IIIL were ≤ 1.5 times the width of the respective indicator bands (cf. Fig. 11D). Autosomal polymorphisms were not found. Figure 9. View largeDownload slide IIIL arm of Simulium multistriatum species group. A, Simulium daoense (male larva) showing the IIIL-1 sequence on top of IIIL-3, 6. B, Simulium malayense cytoform C (female larva) showing the IIIL-3, 5, 6, 10 sequence; ordering the letters a–h produces the Simulium subgeneric sequence of Adler et al. (2016a). Figure 9. View largeDownload slide IIIL arm of Simulium multistriatum species group. A, Simulium daoense (male larva) showing the IIIL-1 sequence on top of IIIL-3, 6. B, Simulium malayense cytoform C (female larva) showing the IIIL-3, 5, 6, 10 sequence; ordering the letters a–h produces the Simulium subgeneric sequence of Adler et al. (2016a). Figure 10. View largeDownload slide Complete chromosomal complement. A, Simulium bullatum (male larva), showing all centromeres attached to a chromocentre. B, Simulium malayense cytoform C (female larva), showing partial chromocentre (CI + CII). NO, nucleolar organizer. Figure 10. View largeDownload slide Complete chromosomal complement. A, Simulium bullatum (male larva), showing all centromeres attached to a chromocentre. B, Simulium malayense cytoform C (female larva), showing partial chromocentre (CI + CII). NO, nucleolar organizer. Figure 11. View largeDownload slide A, B, basal rearrangements in chromosome II of Simulium chainarongi. CII, typical centromere band; CIId, diffuse centromere band; i, heterochromatic insert in the base of IIS; +, band enhancement in the base of IIL; fl, expanded and flocculent IIL centromere region. C, D, degree of terminal flaring of chromosome arms. C, IIL of Simulium fenestratum. D, IIIL of Simulium triglobus. Arrows show indicator bands, against which the width of the terminal flare was measured. Figure 11. View largeDownload slide A, B, basal rearrangements in chromosome II of Simulium chainarongi. CII, typical centromere band; CIId, diffuse centromere band; i, heterochromatic insert in the base of IIS; +, band enhancement in the base of IIL; fl, expanded and flocculent IIL centromere region. C, D, degree of terminal flaring of chromosome arms. C, IIL of Simulium fenestratum. D, IIIL of Simulium triglobus. Arrows show indicator bands, against which the width of the terminal flare was measured. Simulium chainarongi Takaoka & Kuvangkadilok Simulium chainarongi was found only in northeastern Thailand. A total of 133 (66.8%) larvae of 199 prepared larvae from five populations were analysed completely. Kaeng Lam Duan waterfall (site 255) was the type locality of this species. The banding pattern of S. chainarongi differed by two fixed inversions (IL-9 and IIIL-4; Fig. 4) from the group standard. Its banding pattern did not, however, differ from that of two other group members (S. fenestratum and S. triglobus), although its centromeric characteristics were unique. The centromere bands of all chromosomes were typically more darkly stained and well defined than in any other group members (Fig. 12A). One autosomal inversion (IIIS-1) was found heterozygously (Fig. 7) in one male. Four band polymorphisms were found heterozygously in the centromere region of chromosome II: a diffuse centromere band in one male; a band enhancement in the base of IIL (four males and one female); a heterochromatic insert in the base of IIS (eight males and two females); and an expanded and flocculent IIL base in one male (Table 2, Fig. 11). Table 2. Frequency of homologues with chromosome rearrangements in seven nominal species of the Simulium multistriatum group in Thailand and Malaysia Species S. chainarongi S. chaliowae S. lampangense S. bullatum S. triglobus S. malayense S. daoense A B C Site 244 253 254 255 350 287 294 408 409 270 279 396 291 298 298 MYC 298 (male:female) 15:13 2:5 13:16 29:28 5:7 31:28 31:38 13:6 14:9 15:2 7:2 1:1 20:8 2:7 1:0 2:4 4:5 CIId 0.02 IIL+ 0.02 0.02 0.03 IIS 54i 0.07 0.05 IIL 54fl 0.02 IS-1 0.01 IS-2 1.00 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIS-1 0.02 IIIL-1 1.00 IIIL-2 1.00 1.00 1.00 1.00 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-8 0.01 IIIL-9 0.02 IIIL-10 1.00 1.00 1.00 1.00 1.00 1.00 Chromocentre 1.00 1.00 1.00 Species S. chainarongi S. chaliowae S. lampangense S. bullatum S. triglobus S. malayense S. daoense A B C Site 244 253 254 255 350 287 294 408 409 270 279 396 291 298 298 MYC 298 (male:female) 15:13 2:5 13:16 29:28 5:7 31:28 31:38 13:6 14:9 15:2 7:2 1:1 20:8 2:7 1:0 2:4 4:5 CIId 0.02 IIL+ 0.02 0.02 0.03 IIS 54i 0.07 0.05 IIL 54fl 0.02 IS-1 0.01 IS-2 1.00 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIS-1 0.02 IIIL-1 1.00 IIIL-2 1.00 1.00 1.00 1.00 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-8 0.01 IIIL-9 0.02 IIIL-10 1.00 1.00 1.00 1.00 1.00 1.00 Chromocentre 1.00 1.00 1.00 CIId, diffuse centromere band of chromosome II; IIL+, band enhancement in the long arm of chromosome II; IIS 54i, additional (insertion) band in section 54; and IIL 54fl, flocculent band expression in section 54 of the long arm of chromosome II. View Large Table 2. Frequency of homologues with chromosome rearrangements in seven nominal species of the Simulium multistriatum group in Thailand and Malaysia Species S. chainarongi S. chaliowae S. lampangense S. bullatum S. triglobus S. malayense S. daoense A B C Site 244 253 254 255 350 287 294 408 409 270 279 396 291 298 298 MYC 298 (male:female) 15:13 2:5 13:16 29:28 5:7 31:28 31:38 13:6 14:9 15:2 7:2 1:1 20:8 2:7 1:0 2:4 4:5 CIId 0.02 IIL+ 0.02 0.02 0.03 IIS 54i 0.07 0.05 IIL 54fl 0.02 IS-1 0.01 IS-2 1.00 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIS-1 0.02 IIIL-1 1.00 IIIL-2 1.00 1.00 1.00 1.00 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-8 0.01 IIIL-9 0.02 IIIL-10 1.00 1.00 1.00 1.00 1.00 1.00 Chromocentre 1.00 1.00 1.00 Species S. chainarongi S. chaliowae S. lampangense S. bullatum S. triglobus S. malayense S. daoense A B C Site 244 253 254 255 350 287 294 408 409 270 279 396 291 298 298 MYC 298 (male:female) 15:13 2:5 13:16 29:28 5:7 31:28 31:38 13:6 14:9 15:2 7:2 1:1 20:8 2:7 1:0 2:4 4:5 CIId 0.02 IIL+ 0.02 0.02 0.03 IIS 54i 0.07 0.05 IIL 54fl 0.02 IS-1 0.01 IS-2 1.00 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIS-1 0.02 IIIL-1 1.00 IIIL-2 1.00 1.00 1.00 1.00 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-8 0.01 IIIL-9 0.02 IIIL-10 1.00 1.00 1.00 1.00 1.00 1.00 Chromocentre 1.00 1.00 1.00 CIId, diffuse centromere band of chromosome II; IIL+, band enhancement in the long arm of chromosome II; IIS 54i, additional (insertion) band in section 54; and IIL 54fl, flocculent band expression in section 54 of the long arm of chromosome II. View Large Figure 12. View largeDownload slide Characteristics of centromere regions. A, Simulium chainarongi. B, Simulium fenestratum. C, centromere; NO, nucleolar organizer. Figure 12. View largeDownload slide Characteristics of centromere regions. A, Simulium chainarongi. B, Simulium fenestratum. C, centromere; NO, nucleolar organizer. Simulium chaliowae Takaoka & Boonkemtong Simulium chaliowae was found only in limestone streams in northern Thailand. The chromosomes of 128 (80%) of 160 prepared larvae from two sites were analysed completely. Wang Kaew waterfall was the type locality of this species. Simulium chaliowae differed from the S. multistriatum species group standard by three fixed inversions (IL-9, IIIL-2 and IIIL-4). Two floating inversions were found: IS-1 (Fig. 2A) and IIIL-8 (Fig. 8C), each in a separate female (frequency = 0.01; Table 2). The ‘2 blocks’ marker in IS (section 4) was found in two configurations, either standard (Fig. 2B) or with the first heavy block divided by a gap (Fig. 2C). Based on ten larvae per location (ten nuclei per larva), an average of 58% of nuclei per larva from site 287 and 57% of nuclei per larva from site 294 had the first block divided by an unstained gap. Simulium daoense Takaoka & Adler All nine larvae of S. daoense from Siribhumi waterfall, Chiang Mai Province (site 298), were analysed completely. This species differed from the S. multistriatum species group standard by having fixed inversions IL-9 (Fig. 4), IIIL-1 (Fig. 9A) and IIIL-4 (Fig. 8C). Autosomal polymorphisms were not found. The identity of this species was confirmed by chromosomal analysis of three larvae collected from the type locality of S. daoense in Vietnam at the same time that the type series was collected by Takaoka et al. (2017). Simulium fenestratum Edwards This species was the most common and widely distributed member of the S. multistriatum species group in Thailand. The chromosomes of 279 (70.5%) of 396 prepared larvae of S. fenestratum were analysed completely, except for inversion IIIL-2, which was evaluated in 65.9% of the 279 fully analysed larvae (Tables 1, 3). The banding sequence differed from the group standard by two fixed inversions (IL-9 and IIIL-4). The centromere bands of all chromosomes were diffuse and weakly stained (Fig. 12B). Simulium fenestratum had 12 autosomal floating inversions (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IIL-2, IIIL-2, IIIL-7 and an unresolved compound (cmpd) inversion in IIIL), plus one band insert in the expanded centromere region of chromosome I (IL 20i) and a heteroband (IL 21hb) in one female (Table 3, Fig. 4). Table 3. Frequency of homologues with chromosome rearrangements in Simulium fenestratum at 18 sites in Thailand Site Simulium fenestratum 396 264 268 275 276 280 401 363 364 297 298* 372 384 402 320 342 343 345 (male: female)† 9:3 2:2 2:5 5:4 3:4 8:10 3:0 2:1 6:10 15:11 4:3 4:11 6:3 1:3 0:4 1:3 13:17 3:3 IL-1 0.02 0.02 IL 20i 0.02 IL 21hb 0.02 IL-2 0.08 IL-3 0.03 IL-4 0.02 IL-5 0.02 IL-6 0.17 0.02 IL-7 0.04 IL-8 0.04 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIL-2 0.04 IIIL-2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.13 0.43 0.07 0.55 1.00 0.50 0.66 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-7 0.06 IIIL-cmpd 0.08 Site Simulium fenestratum 396 264 268 275 276 280 401 363 364 297 298* 372 384 402 320 342 343 345 (male: female)† 9:3 2:2 2:5 5:4 3:4 8:10 3:0 2:1 6:10 15:11 4:3 4:11 6:3 1:3 0:4 1:3 13:17 3:3 IL-1 0.02 0.02 IL 20i 0.02 IL 21hb 0.02 IL-2 0.08 IL-3 0.03 IL-4 0.02 IL-5 0.02 IL-6 0.17 0.02 IL-7 0.04 IL-8 0.04 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIL-2 0.04 IIIL-2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.13 0.43 0.07 0.55 1.00 0.50 0.66 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-7 0.06 IIIL-cmpd 0.08 IL 20i, additional (insertion) band in section 20 of the long arm of chromosome I; IL 21hb, heteroband in section 21 of the long arm of chromosome I; IIIL-cmpd, compound inversion, i.e. two overlapping inversions with unresolved inner breakpoints between outer section limits 94–96 of the long arm of chromosome III. *8 December 2013 only. †For sites 264, 268, 275, 276, 280, 401, 297, 298, 320 and 342, the number of larvae analysed for IIIL-2 is given here; the frequency of all other rearrangements is based on numbers in Table 1. View Large Table 3. Frequency of homologues with chromosome rearrangements in Simulium fenestratum at 18 sites in Thailand Site Simulium fenestratum 396 264 268 275 276 280 401 363 364 297 298* 372 384 402 320 342 343 345 (male: female)† 9:3 2:2 2:5 5:4 3:4 8:10 3:0 2:1 6:10 15:11 4:3 4:11 6:3 1:3 0:4 1:3 13:17 3:3 IL-1 0.02 0.02 IL 20i 0.02 IL 21hb 0.02 IL-2 0.08 IL-3 0.03 IL-4 0.02 IL-5 0.02 IL-6 0.17 0.02 IL-7 0.04 IL-8 0.04 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIL-2 0.04 IIIL-2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.13 0.43 0.07 0.55 1.00 0.50 0.66 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-7 0.06 IIIL-cmpd 0.08 Site Simulium fenestratum 396 264 268 275 276 280 401 363 364 297 298* 372 384 402 320 342 343 345 (male: female)† 9:3 2:2 2:5 5:4 3:4 8:10 3:0 2:1 6:10 15:11 4:3 4:11 6:3 1:3 0:4 1:3 13:17 3:3 IL-1 0.02 0.02 IL 20i 0.02 IL 21hb 0.02 IL-2 0.08 IL-3 0.03 IL-4 0.02 IL-5 0.02 IL-6 0.17 0.02 IL-7 0.04 IL-8 0.04 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIL-2 0.04 IIIL-2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.13 0.43 0.07 0.55 1.00 0.50 0.66 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-7 0.06 IIIL-cmpd 0.08 IL 20i, additional (insertion) band in section 20 of the long arm of chromosome I; IL 21hb, heteroband in section 21 of the long arm of chromosome I; IIIL-cmpd, compound inversion, i.e. two overlapping inversions with unresolved inner breakpoints between outer section limits 94–96 of the long arm of chromosome III. *8 December 2013 only. †For sites 264, 268, 275, 276, 280, 401, 297, 298, 320 and 342, the number of larvae analysed for IIIL-2 is given here; the frequency of all other rearrangements is based on numbers in Table 1. View Large All floating inversions were in low frequency (typically < 0.10; 0.17 for IL-6 in one small sample of three larvae), except IIIL-2, which varied in frequency with location (Table 3, Fig. 13). IIIL-2 was found in all populations, except two in Chan Tha Buri Province (sites 342 and 343). All specimens from Loei Province and Udon Thani Province and one site (402) in Uttaradit Province were homozygous for IIIL-2 (Fig. 8A), whereas other populations varied in frequency from 0.07 to 0.66 (Fig. 13). One population (Chiang Mai Province, site 297) large enough for statistical evaluation was in Hardy–Weinberg equilibrium for IIIL-2 (χ2 = 0, d.f. = 2, P > 0.05). A small sample of six larvae from Trat Province (site 345) had a deficiency of heterozygotes: four homozygous inverted (three female:one male), one homozygous standard (male) and one heterozygote (male). Figure 13. View largeDownload slide Frequency of IIIL-2 inversion (black, inverted; grey, standard) in 17 populations of Simulium fenestratum in Thailand. Figure 13. View largeDownload slide Frequency of IIIL-2 inversion (black, inverted; grey, standard) in 17 populations of Simulium fenestratum in Thailand. Simulium lampangense Takaoka & Choochote Simulium lampangense was restricted to limestone streams in northern Thailand. Of 53 larvae from two populations, Wang Kaew waterfall (type locality) and Wang Thong waterfall in Lampang Province, 42 (79.2%) were analysed completely. The banding pattern of this species differed from that of the S. multistriatum species group standard by three fixed inversions (IL-9, IIIL-2 and IIIL-4). The banding pattern of S. lampangense differed from that of S. fenestratum only by expression of the ‘2 blocks’ marker and fixation of IIIL-2. Based on nine larvae per location (seven to 18 nuclei per larva), an average of 46.7% of nuclei in larvae from Wang Kaew waterfall (site 408) and 30.1% from Wang Thong waterfall (site 409) had the ‘2 blocks’ marker with an unstained gap in the first block; the same condition as in S. chaliowae. Presence of the gap was positively related to the degree of polytenization. Thus, larger larvae, which exhibited a greater degree of polytenization, had a higher proportion of the ‘2 blocks’ marker with an unstained gap (Fig. 2C). The chromosomal banding pattern of S. lampangense was, therefore, identical to that of S. chaliowae in all respects. Simulium malayense Takaoka & Davies A total of 14 specimens of S. malayense was analysed completely: eight (80.0%) of ten larvae from Thailand and six (54.5%) of 11 larvae from Malaysia. The banding pattern of S. malayense was distinguished from that of the S. multistriatum species group standard by one fixed inversion (IIIL-10; Fig. 9B). Three cytoforms were found in S. malayense. Cytoforms A (seven larvae) and B (one larva) were found sympatrically in Thailand, and cytoform C (six larvae) was collected in Malaysia. Cytoform A was uniquely characterized by IS-2 (Fig. 2B) and ectopic pairing of CI and CIII in 1–60% of nuclei per larva. Cytoform B, consisting of only one male larva, expressed ectopic pairing that involved all combinations of the three centromere bands. It was collected in the same sample as cytoform A but was homozygous standard for IS. Cytoform C was characterized by a partial chromocentre involving CI and CII. CIII was well defined and darkly staining but did not participate in the chromocentric association. Simulium triglobus Kuvangkadilok & Takaoka This species was found at only one site, the type locality in Nan Province. A total of 28 (65.1%) of 43 larvae was analysed completely. All larvae had two fixed inversions (IL-9 and IIIL-4), but otherwise had the standard banding sequence for the group. However, the degree of flaring of the ends of chromosome arms IL, IIL, IIIL and IIIS was significantly greater for S. triglobus (14 larvae, four to ten nuclei per larva) than for S. fenestratum (13 larvae, four to ten nuclei per larva), which represented the standard flaring condition for the group (Mann–Whitney U test, P < 0.01, d.f. = 25; Table 4, Fig. 11D). One floating inversion (IIIL-9; Fig. 8C) appeared heterozygously in one male larva. Table 4. Median (range) of size classes for degree of terminal flaring of chromosomes of Simulium triglobus and Simulium fenestratum Species (N; larvae, nuclei) Size of flared end IL IIL IIIS IIIL Simulium triglobus (14, 83) 1 (0–3) 1 (0–3) 3 (1–3) 2 (0–3) Simulium fenestratum (13, 83) 0 (0–2) 0 (0–2) 1 (0–3) 1 (0–3) Species (N; larvae, nuclei) Size of flared end IL IIL IIIS IIIL Simulium triglobus (14, 83) 1 (0–3) 1 (0–3) 3 (1–3) 2 (0–3) Simulium fenestratum (13, 83) 0 (0–2) 0 (0–2) 1 (0–3) 1 (0–3) All medians differed between species except for IS and IIS (Mann–Whitney U test, using ranks, P < 0.01, d.f. = 165). View Large Table 4. Median (range) of size classes for degree of terminal flaring of chromosomes of Simulium triglobus and Simulium fenestratum Species (N; larvae, nuclei) Size of flared end IL IIL IIIS IIIL Simulium triglobus (14, 83) 1 (0–3) 1 (0–3) 3 (1–3) 2 (0–3) Simulium fenestratum (13, 83) 0 (0–2) 0 (0–2) 1 (0–3) 1 (0–3) Species (N; larvae, nuclei) Size of flared end IL IIL IIIS IIIL Simulium triglobus (14, 83) 1 (0–3) 1 (0–3) 3 (1–3) 2 (0–3) Simulium fenestratum (13, 83) 0 (0–2) 0 (0–2) 1 (0–3) 1 (0–3) All medians differed between species except for IS and IIS (Mann–Whitney U test, using ranks, P < 0.01, d.f. = 165). View Large Chromosomal relationships Our outgroup analysis indicated that the basic IS and IIIS sequences of the S. multistriatum species group, which are standard for the subgenus Simulium, carry no shared inversions with other analysed species groups of the subgenus. Although the S. striatum species group has multiple inversions in the base of IL, as do a number of other species groups in the subgenus Simulium (Adler et al., 2016a), none of the breakpoints is shared with the S. multistriatum species group. Thus, all rearrangements in IL are unique to the S. multistriatum species group. IIS-1 is shared with the S. malyschevi-reptans and S. striatum species groups, and IIS-3 is shared with the S. striatum species group (Table 5). The S. striatum and S. multistriatum species groups each have one additional, but distinctly different, fixed inversion in IIS. IIL-1 of the S. multistriatum species group is shared with the S. striatum species group. IIIL-5 of the S. multistriatum species group is shared with the S. nobile species group, in which it is referred to by Tangkawanit et al. (2011) as IIIL-b, and with the S. striatum species group. All other fixed IIIL inversions are unique to the S. multistriatum and S. striatum species groups. The position of the nucleolar organizer in the base of IS is shared with the S. striatum species group. Table 5. Matrix of 14 chromosomal rearrangements of the Simulium striatum, Simulium malyschevi-reptans and Simulium nobile groups (outgroups) and ten taxa in the Simulium multistriatum group in Thailand and Malaysia Rearrangements IL-9 IIS -1 IIS-2 IIS-3 IIL-1 IIIL-3 IIIL-4 IIIL-5 IIIL-6 IIIL-2 IIIL-10 Chromocentric Centromere association Position of nucleolar organizer in IS S. striatum group 0 1 0 1 1 0 0 1 0 0 0 0 0 1 S. malyschevi-reptans group 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S. nobile group 0 0 0 0 0 0 0 1 0 0 0 0 0 0 S. bullatum 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. chaliowae 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. chainarongi 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. daoense 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. fenestratum 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. lampangense 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. malayense A 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense B 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense C 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. triglobus 1 1 1 1 1 1 1 1 1 0 0 0 0 1 Rearrangements IL-9 IIS -1 IIS-2 IIS-3 IIL-1 IIIL-3 IIIL-4 IIIL-5 IIIL-6 IIIL-2 IIIL-10 Chromocentric Centromere association Position of nucleolar organizer in IS S. striatum group 0 1 0 1 1 0 0 1 0 0 0 0 0 1 S. malyschevi-reptans group 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S. nobile group 0 0 0 0 0 0 0 1 0 0 0 0 0 0 S. bullatum 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. chaliowae 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. chainarongi 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. daoense 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. fenestratum 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. lampangense 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. malayense A 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense B 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense C 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. triglobus 1 1 1 1 1 1 1 1 1 0 0 0 0 1 View Large Table 5. Matrix of 14 chromosomal rearrangements of the Simulium striatum, Simulium malyschevi-reptans and Simulium nobile groups (outgroups) and ten taxa in the Simulium multistriatum group in Thailand and Malaysia Rearrangements IL-9 IIS -1 IIS-2 IIS-3 IIL-1 IIIL-3 IIIL-4 IIIL-5 IIIL-6 IIIL-2 IIIL-10 Chromocentric Centromere association Position of nucleolar organizer in IS S. striatum group 0 1 0 1 1 0 0 1 0 0 0 0 0 1 S. malyschevi-reptans group 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S. nobile group 0 0 0 0 0 0 0 1 0 0 0 0 0 0 S. bullatum 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. chaliowae 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. chainarongi 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. daoense 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. fenestratum 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. lampangense 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. malayense A 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense B 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense C 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. triglobus 1 1 1 1 1 1 1 1 1 0 0 0 0 1 Rearrangements IL-9 IIS -1 IIS-2 IIS-3 IIL-1 IIIL-3 IIIL-4 IIIL-5 IIIL-6 IIIL-2 IIIL-10 Chromocentric Centromere association Position of nucleolar organizer in IS S. striatum group 0 1 0 1 1 0 0 1 0 0 0 0 0 1 S. malyschevi-reptans group 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S. nobile group 0 0 0 0 0 0 0 1 0 0 0 0 0 0 S. bullatum 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. chaliowae 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. chainarongi 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. daoense 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. fenestratum 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. lampangense 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. malayense A 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense B 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense C 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. triglobus 1 1 1 1 1 1 1 1 1 0 0 0 0 1 View Large The evolutionary relationships in the S. multistriatum species group, based on shared rearrangements, show two main lineages (Fig. 14). The first lineage, consisting of six formally described species, was defined by IL-9 and IIIL-4. Within this lineage, a group of three nominal species (S. chainarongi, S. fenestratum and S. triglobus) is defined by IIIL-2, which was fixed in S. chaliowae and S. lampangense but polymorphic in S. fenestratum. Simulium bullatum and the three cytoforms of S. malayense formed the second primary lineage, defined by IIIL-10. All chromosomal features for the S. multistriatum group are summarized in idiograms (Fig. 15). Figure 14. View largeDownload slide Cytophylogeny of ten taxa in the Simulium multistriatum species group in Thailand (and Malaysia). Characters in brackets indicate possible environmental influence. C, centromere. Bootstrap values for maximum parsimony are shown above or near the branches. Figure 14. View largeDownload slide Cytophylogeny of ten taxa in the Simulium multistriatum species group in Thailand (and Malaysia). Characters in brackets indicate possible environmental influence. C, centromere. Bootstrap values for maximum parsimony are shown above or near the branches. Figure 15. View largeDownload slide Idiograms of ten cytoforms in the Simulium multistriatum species group, summarizing all chromosomal features relative to the Simulium subgeneric standard, including all fixed inversions (underlined and bracketed on the left side of the chromosomes) and autosomal polymorphisms (bracketed on the right side). C, centromere; Ch, chromocentre; M, marker; NO, nucleolar organizer; Pb, parabalbiani. Figure 15. View largeDownload slide Idiograms of ten cytoforms in the Simulium multistriatum species group, summarizing all chromosomal features relative to the Simulium subgeneric standard, including all fixed inversions (underlined and bracketed on the left side of the chromosomes) and autosomal polymorphisms (bracketed on the right side). C, centromere; Ch, chromocentre; M, marker; NO, nucleolar organizer; Pb, parabalbiani. Mitochondrial DNA sequence variation The COI and COII gene sequences were analysed for 69 specimens from nine taxa of the S. multistriatum species group in Thailand. The sequence length of the COI gene was 581 bp. There were 168 variable sites, of which 143 were parsimony informative. The maximum intraspecific genetic divergence based on the COI sequence (Table 6) was in S. daoense (5.60%), and the minimum values were in S. bullatum, S. chainarongi and S. chaliowae (0.10%). The minimum interspecific genetic divergence was between S. chaliowae and S. lampangense (1.30%). The maximum interspecific genetic divergence was between S. daoense and S. malayense cytoform C from Thailand (14.70%). Table 6. Range (mean) of intraspecific and interspecific genetic distances for mitochondrial cytochrome c oxidase I (COI) sequences of nine taxa in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam, based on Kimura 2-parameter Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.004 (0.001) S. chainarongi (CN) 0.088–0.090 (0.088) 0.00–0.002(0.001) S. chaliowae (CW) 0.092–0.097 (0.094) 0.036–0.042 (0.038) 0.000–0.004 (0.001) S. daoense (DA) 0.128–0.166 (0.145) 0.077–0.158 (0.112) 0.066–0.170 (0.112) 0.000–0.101 (0.056) S. fenestratum (FN) 0.083–0.095 (0.090) 0.021–0.042 (0.033) 0.006–0.032 (0.016) 0.058–0.202 (0.110) 0.004–0.026 (0.015) S. lampangense (LP) 0.086–0.095(0.092) 0.028–0.038(0.033) 0.004–0.019 (0.013) 0.056–0.170 (0.106) 0.002–0.026 (0.013) 0.000–0.016 (0.009) S. malayense cytoform A (MYA) 0.063–0.073 (0.068) 0.101–0.110 (0.105) 0.103–0.115 (0.109) 0.125–0.161 (0.144) 0.101–0.147 (0.110) 0.103–0.117 (0.108) 0.006–0.014 (0.013) S. malayense cytoform C(MYC) 0.021–0.050(0.026) 0.083–0.110(0.097) 0.088–0.114 (0.096) 0.123–0.187 (0.147) 0.077–0.112(0.091) 0.081–0.112(0.092) 0.058–0.097 (0.071) 0.004–0.031(0.014) S. triglobus (TB) 0.092–0.094(0.093) 0.093–0.102(0.097) 0.093–0.102(0.097) 0.093–0.172 (0.124) 0.088–0.106(0.097) 0.091–0.102(0.096) 0.110–0.115 (0.113) 0.090–0.118 (0.104) 0.002–0.012(0.007) Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.004 (0.001) S. chainarongi (CN) 0.088–0.090 (0.088) 0.00–0.002(0.001) S. chaliowae (CW) 0.092–0.097 (0.094) 0.036–0.042 (0.038) 0.000–0.004 (0.001) S. daoense (DA) 0.128–0.166 (0.145) 0.077–0.158 (0.112) 0.066–0.170 (0.112) 0.000–0.101 (0.056) S. fenestratum (FN) 0.083–0.095 (0.090) 0.021–0.042 (0.033) 0.006–0.032 (0.016) 0.058–0.202 (0.110) 0.004–0.026 (0.015) S. lampangense (LP) 0.086–0.095(0.092) 0.028–0.038(0.033) 0.004–0.019 (0.013) 0.056–0.170 (0.106) 0.002–0.026 (0.013) 0.000–0.016 (0.009) S. malayense cytoform A (MYA) 0.063–0.073 (0.068) 0.101–0.110 (0.105) 0.103–0.115 (0.109) 0.125–0.161 (0.144) 0.101–0.147 (0.110) 0.103–0.117 (0.108) 0.006–0.014 (0.013) S. malayense cytoform C(MYC) 0.021–0.050(0.026) 0.083–0.110(0.097) 0.088–0.114 (0.096) 0.123–0.187 (0.147) 0.077–0.112(0.091) 0.081–0.112(0.092) 0.058–0.097 (0.071) 0.004–0.031(0.014) S. triglobus (TB) 0.092–0.094(0.093) 0.093–0.102(0.097) 0.093–0.102(0.097) 0.093–0.172 (0.124) 0.088–0.106(0.097) 0.091–0.102(0.096) 0.110–0.115 (0.113) 0.090–0.118 (0.104) 0.002–0.012(0.007) View Large Table 6. Range (mean) of intraspecific and interspecific genetic distances for mitochondrial cytochrome c oxidase I (COI) sequences of nine taxa in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam, based on Kimura 2-parameter Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.004 (0.001) S. chainarongi (CN) 0.088–0.090 (0.088) 0.00–0.002(0.001) S. chaliowae (CW) 0.092–0.097 (0.094) 0.036–0.042 (0.038) 0.000–0.004 (0.001) S. daoense (DA) 0.128–0.166 (0.145) 0.077–0.158 (0.112) 0.066–0.170 (0.112) 0.000–0.101 (0.056) S. fenestratum (FN) 0.083–0.095 (0.090) 0.021–0.042 (0.033) 0.006–0.032 (0.016) 0.058–0.202 (0.110) 0.004–0.026 (0.015) S. lampangense (LP) 0.086–0.095(0.092) 0.028–0.038(0.033) 0.004–0.019 (0.013) 0.056–0.170 (0.106) 0.002–0.026 (0.013) 0.000–0.016 (0.009) S. malayense cytoform A (MYA) 0.063–0.073 (0.068) 0.101–0.110 (0.105) 0.103–0.115 (0.109) 0.125–0.161 (0.144) 0.101–0.147 (0.110) 0.103–0.117 (0.108) 0.006–0.014 (0.013) S. malayense cytoform C(MYC) 0.021–0.050(0.026) 0.083–0.110(0.097) 0.088–0.114 (0.096) 0.123–0.187 (0.147) 0.077–0.112(0.091) 0.081–0.112(0.092) 0.058–0.097 (0.071) 0.004–0.031(0.014) S. triglobus (TB) 0.092–0.094(0.093) 0.093–0.102(0.097) 0.093–0.102(0.097) 0.093–0.172 (0.124) 0.088–0.106(0.097) 0.091–0.102(0.096) 0.110–0.115 (0.113) 0.090–0.118 (0.104) 0.002–0.012(0.007) Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.004 (0.001) S. chainarongi (CN) 0.088–0.090 (0.088) 0.00–0.002(0.001) S. chaliowae (CW) 0.092–0.097 (0.094) 0.036–0.042 (0.038) 0.000–0.004 (0.001) S. daoense (DA) 0.128–0.166 (0.145) 0.077–0.158 (0.112) 0.066–0.170 (0.112) 0.000–0.101 (0.056) S. fenestratum (FN) 0.083–0.095 (0.090) 0.021–0.042 (0.033) 0.006–0.032 (0.016) 0.058–0.202 (0.110) 0.004–0.026 (0.015) S. lampangense (LP) 0.086–0.095(0.092) 0.028–0.038(0.033) 0.004–0.019 (0.013) 0.056–0.170 (0.106) 0.002–0.026 (0.013) 0.000–0.016 (0.009) S. malayense cytoform A (MYA) 0.063–0.073 (0.068) 0.101–0.110 (0.105) 0.103–0.115 (0.109) 0.125–0.161 (0.144) 0.101–0.147 (0.110) 0.103–0.117 (0.108) 0.006–0.014 (0.013) S. malayense cytoform C(MYC) 0.021–0.050(0.026) 0.083–0.110(0.097) 0.088–0.114 (0.096) 0.123–0.187 (0.147) 0.077–0.112(0.091) 0.081–0.112(0.092) 0.058–0.097 (0.071) 0.004–0.031(0.014) S. triglobus (TB) 0.092–0.094(0.093) 0.093–0.102(0.097) 0.093–0.102(0.097) 0.093–0.172 (0.124) 0.088–0.106(0.097) 0.091–0.102(0.096) 0.110–0.115 (0.113) 0.090–0.118 (0.104) 0.002–0.012(0.007) View Large The sequence length of the COII gene was 697 bp, with 235 variable sites, of which 184 were parsimony informative. Maximum intraspecific genetic divergence based on the COII sequence (Table 7) was in S. daoense (3.30%), and the lowest intraspecific genetic divergence was in S. chaliowae (0.40%). The maximum interspecific genetic divergence was between S. lampangense and S. malayense cytoform A, and S. malayense cytoform A and S. malayense cytoform C (16.00%), and the minimum interspecific genetic divergence was between S. chaliowae and S. lampangense (1.20%). Table 7. Range (mean) of intraspecific and interspecific genetic distances for mitochondrial cytochrome c oxidase II (COII) sequences of nine taxa in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam, based on Kimura 2-parameter Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.012 (0.007) S. chainarongi (CN) 0.092–0.137 (0.105) 0.002–0.038 (0.019) S. chaliowae (CW) 0.074–0.087 (0.081) 0.034–0.074 (0.048) 0.000–0.006 (0.004) S. daoense (DA) 0.121–0.147 (0.134) 0.054–0.090 (0.067) 0.082–0.135 (0.107) 0.029–0.060 (0.033) S. fenestratum (FN) 0.081–0.119 (0.095) 0.016–0.063 (0.033) 0.010–0.053 (0.029) 0.044–0.118 (0.072) 0.004–0.040 (0.019) S. lampangense (LP) 0.084–0.103 (0.095) 0.018–0.067 (0.038) 0.003–0.020 (0.012) 0.046–0.083 (0.061) 0.014–0.044 (0.025) 0.000–0.028 (0.010) S. malayense cytoform A (MYA) 0.094–0.115 (0.105) 0.114–0.134 (0.123) 0.148–0.167 (0.158) 0.145–0.170 (0.158) 0.116–0.168 (0.138) 0.154–0.167 (0.160) 0.006–0.017 (0.012) S. malayense cytoform C (MYC) 0.032–0.055 (0.046) 0.096–0.137 (0.110) 0.098–0.119 (0.109) 0.072–0.089 (0.081) 0.094–0.123 (0.106) 0.092–0.116 (0.105) 0.154–0.167 (0.160) 0.010–0.034 (0.020) S. triglobus (TB) 0.114–0.152(0.127) 0.072–0.105 (0.090) 0.098–0.132 (0.111) 0.072–0.098 (0.083) 0.078–0.123 (0.098) 0.087–0.130 (0.106) 0.132–0.161 (0.144) 0.114–0.146 (0.127) 0.002–0.026 (0.015) Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.012 (0.007) S. chainarongi (CN) 0.092–0.137 (0.105) 0.002–0.038 (0.019) S. chaliowae (CW) 0.074–0.087 (0.081) 0.034–0.074 (0.048) 0.000–0.006 (0.004) S. daoense (DA) 0.121–0.147 (0.134) 0.054–0.090 (0.067) 0.082–0.135 (0.107) 0.029–0.060 (0.033) S. fenestratum (FN) 0.081–0.119 (0.095) 0.016–0.063 (0.033) 0.010–0.053 (0.029) 0.044–0.118 (0.072) 0.004–0.040 (0.019) S. lampangense (LP) 0.084–0.103 (0.095) 0.018–0.067 (0.038) 0.003–0.020 (0.012) 0.046–0.083 (0.061) 0.014–0.044 (0.025) 0.000–0.028 (0.010) S. malayense cytoform A (MYA) 0.094–0.115 (0.105) 0.114–0.134 (0.123) 0.148–0.167 (0.158) 0.145–0.170 (0.158) 0.116–0.168 (0.138) 0.154–0.167 (0.160) 0.006–0.017 (0.012) S. malayense cytoform C (MYC) 0.032–0.055 (0.046) 0.096–0.137 (0.110) 0.098–0.119 (0.109) 0.072–0.089 (0.081) 0.094–0.123 (0.106) 0.092–0.116 (0.105) 0.154–0.167 (0.160) 0.010–0.034 (0.020) S. triglobus (TB) 0.114–0.152(0.127) 0.072–0.105 (0.090) 0.098–0.132 (0.111) 0.072–0.098 (0.083) 0.078–0.123 (0.098) 0.087–0.130 (0.106) 0.132–0.161 (0.144) 0.114–0.146 (0.127) 0.002–0.026 (0.015) View Large Table 7. Range (mean) of intraspecific and interspecific genetic distances for mitochondrial cytochrome c oxidase II (COII) sequences of nine taxa in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam, based on Kimura 2-parameter Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.012 (0.007) S. chainarongi (CN) 0.092–0.137 (0.105) 0.002–0.038 (0.019) S. chaliowae (CW) 0.074–0.087 (0.081) 0.034–0.074 (0.048) 0.000–0.006 (0.004) S. daoense (DA) 0.121–0.147 (0.134) 0.054–0.090 (0.067) 0.082–0.135 (0.107) 0.029–0.060 (0.033) S. fenestratum (FN) 0.081–0.119 (0.095) 0.016–0.063 (0.033) 0.010–0.053 (0.029) 0.044–0.118 (0.072) 0.004–0.040 (0.019) S. lampangense (LP) 0.084–0.103 (0.095) 0.018–0.067 (0.038) 0.003–0.020 (0.012) 0.046–0.083 (0.061) 0.014–0.044 (0.025) 0.000–0.028 (0.010) S. malayense cytoform A (MYA) 0.094–0.115 (0.105) 0.114–0.134 (0.123) 0.148–0.167 (0.158) 0.145–0.170 (0.158) 0.116–0.168 (0.138) 0.154–0.167 (0.160) 0.006–0.017 (0.012) S. malayense cytoform C (MYC) 0.032–0.055 (0.046) 0.096–0.137 (0.110) 0.098–0.119 (0.109) 0.072–0.089 (0.081) 0.094–0.123 (0.106) 0.092–0.116 (0.105) 0.154–0.167 (0.160) 0.010–0.034 (0.020) S. triglobus (TB) 0.114–0.152(0.127) 0.072–0.105 (0.090) 0.098–0.132 (0.111) 0.072–0.098 (0.083) 0.078–0.123 (0.098) 0.087–0.130 (0.106) 0.132–0.161 (0.144) 0.114–0.146 (0.127) 0.002–0.026 (0.015) Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.012 (0.007) S. chainarongi (CN) 0.092–0.137 (0.105) 0.002–0.038 (0.019) S. chaliowae (CW) 0.074–0.087 (0.081) 0.034–0.074 (0.048) 0.000–0.006 (0.004) S. daoense (DA) 0.121–0.147 (0.134) 0.054–0.090 (0.067) 0.082–0.135 (0.107) 0.029–0.060 (0.033) S. fenestratum (FN) 0.081–0.119 (0.095) 0.016–0.063 (0.033) 0.010–0.053 (0.029) 0.044–0.118 (0.072) 0.004–0.040 (0.019) S. lampangense (LP) 0.084–0.103 (0.095) 0.018–0.067 (0.038) 0.003–0.020 (0.012) 0.046–0.083 (0.061) 0.014–0.044 (0.025) 0.000–0.028 (0.010) S. malayense cytoform A (MYA) 0.094–0.115 (0.105) 0.114–0.134 (0.123) 0.148–0.167 (0.158) 0.145–0.170 (0.158) 0.116–0.168 (0.138) 0.154–0.167 (0.160) 0.006–0.017 (0.012) S. malayense cytoform C (MYC) 0.032–0.055 (0.046) 0.096–0.137 (0.110) 0.098–0.119 (0.109) 0.072–0.089 (0.081) 0.094–0.123 (0.106) 0.092–0.116 (0.105) 0.154–0.167 (0.160) 0.010–0.034 (0.020) S. triglobus (TB) 0.114–0.152(0.127) 0.072–0.105 (0.090) 0.098–0.132 (0.111) 0.072–0.098 (0.083) 0.078–0.123 (0.098) 0.087–0.130 (0.106) 0.132–0.161 (0.144) 0.114–0.146 (0.127) 0.002–0.026 (0.015) View Large COII was more effective than COI for differentiating members of the S. multistriatum species group (Table 8). Identifications based on best match for COI were 73.9% correct (51 of 69 sequences), with 14.5% (ten of 69 sequences) misidentifications and 11.6% (eight of 69 sequences) ambiguous identifications. Identifications based on COII were 97.1% (67 of 69 sequences) correct, with 2.9% (two of 69 sequences) misidentifications and no ambiguous identifications. Table 8. Nucleotide sequence statistics based on COI and COII of nine taxa of black flies in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam Gene Number of taxa Number of sequences Variable sites (V) Parsimony-informative sites (Pi) Length of sequences (bp) Percentage of correct identifications (N) Percentage of misidentifications (N) Percentage ambiguous (N) COI 9 69 168 143 581 73.9% (51) 14.5% (10) 11.6% (8) COII 9 69 235 184 697 97.1% (67) 2.9% (2) 0 Gene Number of taxa Number of sequences Variable sites (V) Parsimony-informative sites (Pi) Length of sequences (bp) Percentage of correct identifications (N) Percentage of misidentifications (N) Percentage ambiguous (N) COI 9 69 168 143 581 73.9% (51) 14.5% (10) 11.6% (8) COII 9 69 235 184 697 97.1% (67) 2.9% (2) 0 Percentage of correct identification is based on best match method in TaxonDNA (Meier et al. 2006). View Large Table 8. Nucleotide sequence statistics based on COI and COII of nine taxa of black flies in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam Gene Number of taxa Number of sequences Variable sites (V) Parsimony-informative sites (Pi) Length of sequences (bp) Percentage of correct identifications (N) Percentage of misidentifications (N) Percentage ambiguous (N) COI 9 69 168 143 581 73.9% (51) 14.5% (10) 11.6% (8) COII 9 69 235 184 697 97.1% (67) 2.9% (2) 0 Gene Number of taxa Number of sequences Variable sites (V) Parsimony-informative sites (Pi) Length of sequences (bp) Percentage of correct identifications (N) Percentage of misidentifications (N) Percentage ambiguous (N) COI 9 69 168 143 581 73.9% (51) 14.5% (10) 11.6% (8) COII 9 69 235 184 697 97.1% (67) 2.9% (2) 0 Percentage of correct identification is based on best match method in TaxonDNA (Meier et al. 2006). View Large Molecular phylogenetic relationships Phylogenetic analyses were conducted for COI, COII and the combined dataset (COI + COII). All phylogenetic analysis methods (NJ, MP, ML and Bayesian) revealed similar tree topologies; thus, only NJ trees are shown. The NJ tree based on COI sequences revealed two major clades (Fig. 16). Simulium chainarongi, S. chaliowae, S. daoense, S. fenestratum, S. lampangense and S. triglobus formed clade I. Simulium fenestratum and S. lampangense were paraphyletic, whereas S. chainarongi and S. chaliowae were monophyletic, although they nested within S. fenestratum. All members of S. daoense from Thailand and Vietnam were clustered in the same clade with moderate bootstrap support (> 71%). Clade II comprised S. bullatum, S. malayense cytoform A from Thailand and S. malayense cytoform C from Malaysia. Simulium bullatum and S. malayense cytoforms A and C in clade II were monophyletic with strong support. Simulium malayense cytoform B was not available for molecular analysis. Figure 16. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on COI sequences. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. Figure 16. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on COI sequences. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. The phylogenetic analysis based on the COII gene (Fig. 17) provided better resolution than did the COI gene. The COII sequences revealed two main clades. Clade I comprised six species, namely S. chainarongi, S. chaliowae, S. daoense, S. fenestratum, S. lampangense and S. triglobus. All species in this clade were monophyletic, except S. chaliowae and S. lampangense. Simulium chainarongi nested within S. fenestratum. All specimens of S. lampangense formed a single well-supported clade with S. chaliowae. Clade II was composed of S. bullatum, S. malayense cytoform A from Thailand and cytoform C from Malaysia. Figure 17. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on COII sequences. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. Figure 17. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on COII sequences. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. The phylogenetic tree based on the combined data (Fig. 18) showed two main clades similar to the trees derived from the COI and COII sequences. Simulium chainarongi, S. chaliowae, S. daoense, S. fenestratum, S. lampangense and S. triglobus each formed a monophyletic cluster within clade I. Simulium lampangense formed a single well-supported subclade with S. chaliowae in clade I. Simulium chainarongi, however, nested within S. fenestratum. Simulium bullatum and S. malayense cytoforms A and C formed a monophyletic cluster within clade II. Figure 18. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on combined (COI + COII) data set. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. Figure 18. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on combined (COI + COII) data set. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. Chromosomal identification key for members of the Simulium multistriatum species group in Thailand and Malaysia 1. Centromere bands associated in > 50% of nuclei (e.g. Fig. 10). IL-9 absent (Fig. 3), IIIL-10 present (Fig. 9B) 2 Centromere bands associated in < 10% of nuclei. IL-9 present, IIIL-10 absent 5 2. True chromocentre with extra heterochromatin present (Fig. 10A) Simulium bullatum Partial chromocentre (Fig. 10B) or ectopic pairing present 3 3. Ectopic pairing of all combinations of centromere bands (CI, CII and CIII) Simulium malayense cytoform B (Thailand) Partial chromocentre or ectopic pairing of only two centromere bands 4 4. IS-2 present (Fig. 2B). Ectopic pairing involving centromeres I and III in 1–60% of nuclei. …………………… Simulium malayense cytoform A (Thailand) IS-2 absent. Partial chromocentre (Fig. 10B) present, with extra heterochromatin, involving centromeres CI and CII. .Simulium malayense cytoform C (Malaysia) 5. Centromere bands well defined, darkly stained (Fig. 12A) Simulium chainarongi Centromere bands weakly defined, lightly stained (Fig. 12B) 6 6. Flaring of ends of IL, IIL, IIIL and IIIS > 1.5 times wider than respective indicator bands (Fig. 11D) Simulium triglobus Flaring of ends of all chromosome arms < 1.5 times wider than respective indicator bands (Fig. 11C) 7 7. ‘2 blocks’ marker appearing as 3 blocks in at least some nuclei (Fig. 2C). IIIL-2 fixed (Fig. 8A) Simulium chaliowae, Simulium lampangense ‘2 blocks’ marker standard in all nuclei (Fig. 2B). IIIL-2 polymorphic 8 8. IIIL-1 present (Fig. 9A) Simulium daoense IIIL-1 absent Simulium fenestratum DISCUSSION Taxonomic status of chromosomal entities Our chromosomal study recognizes ten distinct taxa among eight nominal morphospecies in the S. multistriatum species group. Chromosomal analyses confirm reproductive isolation (species status), via absence of hybrids, of eight species, reveal two undescribed taxa (S. malayense cytoforms A and B), and suggest possible conspecificity of at least two nominal species (S. chaliowae and S. lampangense) and possible cryptic species in another (S. fenestratum). The species status of S. bullatum separate from all other studied species is supported chromosomally by the presence of a large chromocentre. Morphologically, the large unpigmented organ of its pupal gill is unique (Takaoka & Choochote, 2005b). Molecular analyses also support its distinction from other species, with K2P genetic distances ranging from 2.60 to 14.50% for COI and from 4.60 to 15.60% for COII. Molecular phylogenetic analysis shows that all specimens of S. bullatum form a monophyletic group, in agreement with a previous molecular study based on the COI and ECP1 genes (Thaijarern et al., 2017). Species status is tentatively suggested for the three cytoforms of S. malayense. The presence of IS-2 in homozygous and standard arrangements (with no heterozygotes), coupled with unique centromeric associations, in sympatry suggests that cytoforms A and B are distinct species. This suggestion, however, is tempered by an inadequate sample size. Molecular analysis corroborates chromosomal data that S. malayense cytoform C is genetically distinct from A. However, the allopatric distribution of A relative to C does not permit a strict evaluation of species status; A and B are ~1605 km distant from C. The collection site of cytoform C is < 100 km from the type locality of S. malayense and, therefore, is the best candidate for typical S. malayense. Simulium chainarongi is homosequential with S. fenestratum and S. triglobus but has unique centromeric expression and polymorphisms. Differential expression of centromere bands is not uncommon in closely related species of black flies (Bedo, 1975). Although S. chainarongi is difficult to separate morphologically from some species in the S. multistriatum species group (Takaoka & Kuvangkadilok, 1999), its unique centromeric expression, coupled with molecular and ecological data (i.e. low-elevation habitats, < 250 m), support its species status. Gene sequences indicate that S. chainarongi is monophyletic, with interspecific genetic distances ranging from 3.30 to 11.20% for COI and from 3.30 to 12.30% for COII. Its status as a species distinct from S. fenestratum, however, is questionable based on COI and COII sequences, which depict it nesting within S. fenestratum. Given the geographical separation of our samples of the two taxa (~330 km), additional study is needed to test their current status as distinct species. Simulium daoense differs by only one fixed inversion (IIIL-1) from S. chainarongi, S. fenestratum and S. triglobus. Our chromosomal analysis shows the presence of S. daoense in Thailand (Chiang Mai Province) for the first time. The Thai population is chromosomally identical with S. daoense from the type locality in Vietnam ~700 km distant. The type locality of S. daoense is at high elevation (1315 m), similar to the location in Thailand (1304 m). The stream width at the type locality is small (0.5 m) and the water temperature cold (7.0 °C), whereas in Thailand, the stream is wider (5 m) and the water temperature warmer (15.7 °C). The COI and COII sequences place S. daoense from Thailand and Vietnam together, with maximum intraspecific genetic divergence of 5.60% for COI and 3.30% for COII. The chromosomes suggest that the two populations are a single species, but environmental effects and geographical distance might be driving the molecular differentiation between the two populations. Geographical distance is the main factor limiting gene flow between populations of many black fly species (Pramual et al., 2005), particularly those inhabiting high-elevation areas (Finn & Adler, 2006; Finn et al., 2006; Pramual & Wongpakam, 2013). The banding pattern of S. triglobus is homosequential with that of S. chainarongi and S. fenestratum, but differs from that of S. chainarongi by standard centromere band expression and from both species by enhanced terminal flaring of the chromosome arms. Typically, the chromosomal ends would be heterochromatinized via duplication processes related to repeat DNA elements and perhaps heterochromatic genes. Thus, when the ends flare, some form of expression is probably occurring with regard to these elements or genes. Gene expression is necessary to produce the appropriate proteins for development in particular environments (Gottlieb, 1998). The enhanced flaring of S. triglobus might be associated with conditions in calcareous streams, such as high calcium carbonate, pH and conductivity. Although the effect of calcium carbonate on genomic expression has not been investigated for black flies, some evidence suggests an indirect effect on the structure of polytene chromosomes in larval chironomids via surface adsorption of heavy metals on marl surfaces (Jabłońska-Barna, Szarek-Gwiazda & Michailova, 2013). If terminal flaring of chromosomes is environmentally influenced, no chromosomal evidence is available to support species status of S. triglobus. Morphological support for the species status of S. triglobus separate from S. chainarongi and S. fenestratum is based primarily on the more branched thoracic trichomes and corbicular cocoon of the pupa, lack of dorsolateral protuberances on the larval abdomen, and three (vs. one) spermathecae in females of S. triglobus (Takaoka & Kuvangkadilok, 1999). Molecular analysis based on COI and COII support the unique, albeit weak chromosomal feature (flaring); all specimens of S. triglobus from the type locality form a monophyletic group with strong support (100%), agreeing with a previous DNA barcode tree by Pramual & Wongpakam (2014). This species also shows a high level of interspecific molecular genetic differentiation (K2P genetic distance: 9.30–12.40% for COI and 8.30–14.40% for COII). The banding patterns and chromosomal characteristics of S. chaliowae and S. lampangense are identical, and both species are known only from limestone streams. The unique expression of the ‘2 blocks’ marker is positively related to the degree of polytenization and might represent gene expression related to environmental influence from the calcareous streams they inhabit, as discussed for S. triglobus. The COII sequence and the combined COI + COII data set show a close relationship between S. chaliowae and S. lampangense, with low interspecific genetic divergence (1.20% for COII). The results agree with those based on the ECP1 gene, which found that S. chaliowae and S. lampangense are closely related but fall into separate clusters (Thaijarern et al., 2017). The molecular difference might be a site effect, rather than a species effect. Known locations of S. chaliowae are ~130–150 km from those of S. lampangense. If these taxa are restricted to calcareous streams, which have patchy distributions (Pramual & Pangjanda, 2015), the implication is that females tend to return to their natal streams to oviposit, enhancing the build-up of location-specific genetic differences. Morphologically, the larvae differ only by the presence of dorsal protuberances on abdominal segments two to six in S. chaliowae vs. three to seven in S. lampangense (Thaijarern et al., 2017); however, this character varies intraspecifically in some members of the S. multistriatum species group (Takaoka & Kuvangkadilok, 1999; Takaoka & Choochote, 2005a). For example, some populations of S. fenestratum have dorsal protuberances, whereas others do not (J. Thaijarern and P. Pramual, unpublished data). The cocoon of S. lampangense is fenestrated and either slipper or shoe shaped, whereas that of S. chaliowae is unfenestrated and shoe shaped (Takaoka & Kuvangkadilok, 1999; Takaoka & Choochote, 2005a). However, the presence of windows in the cocoon can vary intraspecifically in other simuliid species (Adler et al., 2004). Differences in the adults are based on minor characteristics, such as the length-to-width ratio of the anal lobe (Takaoka & Kuvangkadilok, 1999; Takaoka & Choochote, 2005a). Morphological differences might also be location specific. Thus, the evidence that S. chaliowae and S. lampangense are distinct species is weak, suggesting that S. lampangense might be a junior synonym of S. chaliowae. Until evidence to the contrary can be adduced, we continue to recognize S. chaliowae and S. lampangense as separate species, based on recovery of distinct clusters in molecular phylogenies using the ECP1 (Thaijarern et al., 2017) and COII genes, and minor morphological differences. Thus, they would be nearly homosequential cryptic species. The chromosomes of S. chaliowae and S. lampangense are fundamentally the same as those in populations of S. fenestratum homozygous for the IIIL-2 inversion. Larvae and pupae of S. chaliowae and S. lampangense have conventionally been distinguished from S. fenestratum by body colour, dorsal protuberances on the abdomen, and shape of the cocoon (Takaoka & Kuvangkadilok, 1999). These characters, however, are subject to intraspecific variation. The male of S. chaliowae is similar to that of S. fenestratum but is distinguished by the horn-like basal protuberance of the gonostylus with many teeth along the anterior margin (Takaoka & Kuvangkadilok, 1999), and S. lampangense can be distinguished from S. fenestratum by having a bare radial vein in females and several spines on the basal protuberance of the gonostylus (Takaoka & Choochote, 2005a); S. fenestratum has one apical spine on the basal protuberance (Takaoka, 1977). Although the ECP1 gene separates S. fenestratum from S. chaliowae and S. lampangense with strong support, the COI gene groups some populations of S. fenestratum in a clade with S. chaliowae (Pramual & Wongpakam, 2014; Thaijarern et al., 2017). Our chromosomal results provide some indication that S. fenestratum consists of cryptic species. A previous report of cryptic species (i.e. chromocentric individuals) in S. fenestratum (Pramual & Nanork, 2012) in fact pertains to S. bullatum. Our evidence for cryptic species of S. fenestratum involves the IIIL-2 inversion. The existence of populations homozygous standard, homozygous inverted and polymorphic for this inversion presents four hypotheses: (1) a single polymorphic species, with IIIL-2 perhaps reflecting local adaptation; (2) two species, one fixed for IIIL-2 and one polymorphic for IIIL-2; (3) two species, one homozygous standard and one polymorphic for IIIL-2; and (4) three species, one fixed for IIIL-2, one fixed for standard and one polymorphic. This same scenario has been found in the S. tani complex in Thailand, with all four possibilities (although involving other inversions), representing different cytoforms (Tangkawanit et al., 2009). A population in Trat Province where individuals homozygous for IIIL-2 and for standard were found, with a dearth of heterozygotes, might provide a test for cryptic species if larger samples are available to test the inversion for Hardy–Weinberg equilibrium or to conduct a molecular evaluation. The biogeographical pattern for IIIL-2 in S. fenestratum is similar to that for inversions in Simulium aureohirtum and Simulium tani s.l., which increase in frequency with latitude (Pramual et al., 2005; Pramual, Wongpakam & Kuvangkadilok, 2008). Phylogenetic relationships Monophyly of the S. multistriatum species group, based on species from Thailand, is demonstrated on the basis of four shared chromosomal rearrangements (IIS-2, IIIL-3, IIIL-5 and IIIL-6), providing a framework for further testing of the other 25 known species (Adler & Crosskey, 2017) in the group. The chromosomal characters should prove especially useful for evaluating group membership of species such as S. takense, which does not cluster with the S. multistriatum species group on the basis of molecular evidence (COI, COII and 18S/ITS; Pramaul & Nanork, 2012; Pramual & Adler, 2014). Chromosomal characters suggest that the S. multistriatum group is most closely related to the S. striatum group and that these two groups, in turn, are related to the S. malyschevi-reptans and S. nobile groups. This cluster of species groups finds morphological support based on the presence of fenestrated cocoons. Although the fenestra can be lost in some species (Moulton & Adler, 1995), their presence is strongly associated with these groups. If this structural character has phylogenetic value, we would expect the S. griseifrons species group s.l. (including its recent divisions; Takaoka, 2017) also to be a member of this clade; to date, no chromosomal information is available for S. griseifrons and its relatives. The chromosomal phylogeny showing that species of the S. multistriatum species group in Thailand fall into two distinct groups based on three fixed chromosome inversions (IL-9, IIIL-4 and IIIL-10) and centromere associations agrees with phylogenetic analysis based on COI and COII gene sequences: S. bullatum and S. malayense cytoforms A, B and C form one clade, and S. chainarongi, S. chaliowae, S. daoense, S. fenestratum, S. lampangense and S. triglobus form another clade. These results agree with a previous DNA-barcode tree (Pramual & Wongpakam, 2014). A paucity of shared structural characters does not permit inference of a morphological phylogeny. The chromosomal evidence that the S. multistriatum group is most closely related to the S. striatum species group agrees with some molecular analyses (Thanwisai et al., 2006) but not others (Otsuka et al., 2003; Phayuhasena et al., 2010; Pramual & Adler, 2014). Speciation in the S. multistriatum species group Limited morphological differentiation is found among the species in the S. multistriatum group, consistent with the low levels of cytogenetic differentiation. Only 30 rearrangements, other than those common to the basic sequence, have been found in this species group in Thailand. This number is low compared with other Southeast Asian species groups, such as the S. tuberosum species group in Vietnam, with 88 rearrangements (Adler et al., 2016a). Speciation in the Simuliidae is typically associated with chromosomal phenomena, particularly coadaptation of sex chromosomes, cooption of individual rearrangements for different roles in different lineages and, more rarely, larger genomic restructuring events (Rothfels, 1989; Adler et al., 2016c). Speciation in the S. multistriatum group, however, is largely not associated with the typical chromosomal phenomena in the Simuliidae. The sex chromosomes, for example, are undifferentiated in all taxa in our study. Only one inversion, IIIL-2, functions in multiple roles: fixed in S. chaliowae and S. lampangense and polymorphic in S. fenestratum. Sharing of the IIIL-2 inversion suggests that S. fenestratum, S. chaliowae and S. lampangense are derived from a common ancestor. Accordingly, this inversion would have been polymorphic in the ancestor, remained so in S. fenestratum, and become fixed in S. chaliowae and S. lampangense. The latter two species occur only in a specific habitat, calcareous streams; thus, fixation of IIIL-2 might be associated with habitat specialization. Chromosomal and molecular study of another calcareous stream specialist, S. weji, in Thailand suggests that females return to their natal sites to oviposit (Pramual & Pangjanda, 2015). If this scenario also applies to S. chaliowae and S. lampangense, inbreeding could occur and facilitate the fixation of IIIL-2 in the populations. The low molecular diversity in the COI and COII genes of S. chaliowae and S. lampangense supports this possibility. Rather than the typical chromosomal rearrangements associated with simuliid speciation (e.g. inversions), other chromosomal phenomena occur in the S. multistriatum species group, including centromere associations with and without heterochromatinization (S. bullatum and S. malayense cytoforms A, B and C). Centromere associations occur throughout the Simuliidae in > 12% of all species (Adler et al., 2010). Heterochromatinization within the genome can serve as a driving force in speciation (Ferree & Barbasha, 2009). The chromosomal and molecular phylogenetic clustering of the three types of centromeric associations (i.e. ectopic pairing, partial chromocentre and chromocentre) suggests a common origin. The simplest form of centric association, ectopic pairing, is a frequent phenomenon in diverse species, and is often restricted to certain populations of a species, although it is not necessarily expressed in all individuals or even in all nuclei of an individual (Rothfels & Freeman, 1977). The origin of heterochromatinization is inferred to be a result of over-replication of repetitive elements (Thapa et al., 2014). An occasional tendency for centromeres to associate ectopically might represent the first step in acquisition of a permanent, species-specific chromocentre or partial chromocentre, with the next step being addition of heterochromatin. A similar progression from non-chromocentric to fully chromocentric, via ectopic pairing (pseudochromocentre), has also been proposed for some members of the Simulium vernum species group, of which at least one species exhibits chromocentre polymorphism (Brockhouse, Bass & Straus, 1989). The partial chromocentric state, which is typically a species-specific trait, might result from the loss of repetitive DNA sequences responsible for the chromocentre from only one of the three chromosomes (Brockhouse et al., 1989). ACKNOWLEDGEMENTS This work was funded by the Thailand Research Fund (TRF) special programme for the Royal Golden Jubilee (RGJ) Ph.D. Program and Mahasarakham University. We thank C. E. Beard of the Clemson University Cherry Farm Insectary, where J.T. worked in the laboratory of P.H.A. for 11 months in 2016–2017. We are grateful to Zubaidah Ya’cob (University of Malaya) for providing specimens of S. malayense C from Malaysia and to Professor Hiroyuki Takaoka for graciously providing larvae of S. daoense from Vietnam. REFERENCES Adler PH , Cheke RA , Post RJ . 2010 . Evolution, epidemiology, and population genetics of black flies (Diptera: Simuliidae) . Infection, Genetics and Evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 10 : 846 – 865 . Google Scholar CrossRef Search ADS Adler PH , Crosskey RW . 2015 . Cytotaxonomy of the Simuliidae (Diptera): a systematic and bibliographic conspectus . Zootaxa 3975 : 1 – 139 . Google Scholar CrossRef Search ADS Adler PH , Crosskey RW . 2017 . World blackflies (Diptera: Simuliidae): a comprehensive revision of the taxonomic and geographical inventory [2017] . Available at: https://biomia.sites.clemson.edu/pdfs/blackflyinventory.pdf Adler PH , Currie DC , Wood DM . 2004 . The black flies (Simuliidae) of North America . New York : Cornell University Press . Adler PH , Huang YT . 2011 . Integrated systematics of the Simuliidae (Diptera): evolutionary relationships of the little-known Palearctic black fly Simulium acrotrichum . Canadian Entomologist 143 : 612 – 628 . Google Scholar CrossRef Search ADS Adler PH , Kachvorian EA . 2001 . Cytogenetics of the Holarctic black fly Simulium noelleri (Diptera: Simuliidae) . Canadian Journal of Zoology 79 : 1972 – 1979 . Google Scholar CrossRef Search ADS Adler PH , Kúdelová T , Kúdela M , Seitz G , Ignjatović-Ćupina A . 2016a . Cryptic biodiversity and the origins of pest status revealed in the macrogenome of Simulium colombaschense (Diptera: Simuliidae), history’s most destructive black fly . PLoS ONE 11 : e0147673 . Google Scholar CrossRef Search ADS Adler PH , Takaoka H , Sofian-Azirun M , Low VL , Ya’cob Z , Chen CD , Lau KW , Pham XD . 2016b . Vietnam, a hotspot for chromosomal diversity and cryptic species in black flies (Diptera: Simuliidae) . PLoS ONE 11 : e0163881 . Google Scholar CrossRef Search ADS Adler PH , Yadamsuren O , Procunier WS . 2016c . Chromosomal translocations in black flies (Diptera: Simuliidae)—facilitators of adaptive radiation ? PLoS ONE 11 : e0158272 . Google Scholar CrossRef Search ADS Anisimova M , Gascuel O . 2006 . Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative . Systematic Biology 55 : 539 – 552 . Google Scholar CrossRef Search ADS Bedo DG . 1975 . Polytene chromosomes of three species of blackflies in the Simulium pictipes group (Diptera:Simuliidae) . Canadian Journal of Zoology 53 : 1147 – 1164 . Google Scholar CrossRef Search ADS Bedo DG . 1979 . Cytogenetics and evolution of Simulium ornatipes Skuse (Diptera: Simuliidae). II. Temporal variation in chromosomal polymorphisms and homosequential sibling species . Evolution; international journal of organic evolution 33 : 296 – 308 . Google Scholar CrossRef Search ADS Brockhouse C , Bass JAB , Straus NA . 1989 . Chromocentre polymorphism in polytene chromosomes of Simulium costatum (Diptera: Simuliidae) . Genome 32 : 510 – 515 . Google Scholar CrossRef Search ADS Conflitti IM , Kratochvil MJ , Spironello M , Shields GF , Currie DC . 2010 . Good species behaving badly: non-monophyly of black fly sibling species in the Simulium arcticum complex (Diptera: Simuliidae) . Molecular Phylogenetics and Evolution 57 : 245 – 257 . Google Scholar CrossRef Search ADS Ferree PM , Barbash DA . 2009 . Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila . PLoS Biology 7 : e1000234 . Google Scholar CrossRef Search ADS Finn DS , Adler PH . 2006 . Population genetic structure of a rare high‐elevation black fly, Metacnephia coloradensis, occupying Colorado lake outlet streams . Freshwater Biology 51 : 2240 – 2251 . Google Scholar CrossRef Search ADS Finn DS , Theobald DM , Black WC 4th , Poff NL . 2006 . Spatial population genetic structure and limited dispersal in a Rocky Mountain alpine stream insect . Molecular Ecology 15 : 3553 – 3566 . Google Scholar CrossRef Search ADS 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 . Molecular Marine Biology and Biotechnology 3 : 294 – 299 . Gottlieb G . 1998 . Normally occurring environmental and behavioral influences on gene activity: from central dogma to probabilistic epigenesis . Psychological Review 105 : 792 – 802 . Google Scholar CrossRef Search ADS Guindon S , Dufayard JF , Lefort V , Anisimova M , Hordijk W , Gascuel O . 2010 . New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0 . Systematic Biology 59 : 307 – 321 . Google Scholar CrossRef Search ADS Henderson CAP . 1986 . Homosequential species 2a and 2b within the Prosimulium onychodactylum complex (Diptera): temporal heterogeneity, linkage disequilibrium, and Wahlund effect . Canadian Journal of Zoology 64 : 859 – 866 . Google Scholar CrossRef Search ADS Huang YT , Adler PH , Takaoka H . 2011 . Polytene chromosomes of Simulium arakawae, a pest species in the Simulium venustum group (Diptera: Simuliidae) from Japan . Tropical Biomedicine 28 : 376 – 381 . Huelsenbeck JP , Ronquist F , Nielsen R , Bollback JP . 2001 . Bayesian inference of phylogeny and its impact on evolutionary biology . Science (New York, N.Y.) 294 : 2310 – 2314 . Google Scholar CrossRef Search ADS Ilmonen J , Adler PH , Malmqvist B , Cywinska A . 2009 . The Simulium vernum group (Diptera: Simuliidae) in Europe: multiple character sets for assessing species status . Zoological Journal of the Linnean Society 156 : 847 – 863 . Google Scholar CrossRef Search ADS Jabłońska-Barna I , Szarek-Gwiazda E , Michailova P . 2013 . Environmental agents in Lake Łuknajno (Poland) affecting the genome of Chironomus melanotus Keyl, 1961 (Diptera, Chironomidae)—a new species of Polish fauna . Oceanological and Hydrobiological Studies 42 : 164 – 172 . Google Scholar CrossRef Search ADS Meier R , Shiyang K , Vaidya G , Ng PK . 2006 . DNA barcoding and taxonomy in Diptera: a tale of high intraspecific variability and low identification success . Systematic Biology 55 : 715 – 728 . Google Scholar CrossRef Search ADS Moulton JK , Adler PH . 1995 . Revision of the Simulium jenningsi species-group (Diptera: Simuliidae) . Transactions of the American Entomological Society 121 : 1 – 57 . Otsuka Y , Takaoka H , Aoki C , Choochote W . 2003 . Phylogenetic analysis of the subgenus Himalayum within the genus Simulium sl (Diptera: Simuliidae) using mitochondrial 16S rRNA gene sequences . Medical Entomology and Zoology 54 : 113 – 120 . Google Scholar CrossRef Search ADS Phayuhasena S , Colgan DJ , Kuvangkadilok C , Pramual P , Baimai V . 2010 . Phylogenetic relationships among the black fly species (Diptera: Simuliidae) of Thailand based on multiple gene sequences . Genetica 138 : 633 – 648 . Google Scholar CrossRef Search ADS Pramual P . 2006 . Population genetic structure of black flies (Diptera: Simuliidae) from Thailand inferred from mitochondrial DNA sequences . Ph.D. Thesis, Mahidol University , Bangkok, Thailand . Pramual P , Adler PH . 2014 . DNA barcoding of tropical black flies (Diptera: Simuliidae) of Thailand . Molecular Ecology Resources 14 : 262 – 271 . Google Scholar CrossRef Search ADS Pramual P , Kuvangkadilok C . 2012 . Integrated cytogenetic, ecological, and DNA barcode study reveals cryptic diversity in Simulium (Gomphostilbia) angulistylum (Diptera: Simuliidae) . Genome 55 : 447 – 458 . Google Scholar CrossRef Search ADS Pramual P , Kuvangkadilok C , Baimai V , Walton C . 2005 . Phylogeography of the black fly Simulium tani (Diptera: Simuliidae) from Thailand as inferred from mtDNA sequences . Molecular Ecology 14 : 3989 – 4001 . Google Scholar CrossRef Search ADS Pramual P , Nanork P . 2012 . Phylogenetic analysis based on multiple gene sequences revealing cryptic biodiversity in Simulium multistriatum group (Diptera: Simuliidae) in Thailand . Entomological Science 15 : 202 – 213 . Google Scholar CrossRef Search ADS Pramual P , Pangjanda S . 2015 . Effects of habitat specialization on population genetic structure of black fly Simulium weji Takaoka (Diptera: Simuliidae) . Journal of Asia-Pacific Entomology 18 : 33 – 37 . Google Scholar CrossRef Search ADS Pramual P , Wongpakam K . 2011 . Cytogenetics of Simulium siamense Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand . Aquatic Insects 33 : 171 – 184 . Google Scholar CrossRef Search ADS Pramual P , Wongpakam K . 2013 . Population genetics of the high elevation black fly Simulium (Nevermannia) feuerborni Edwards in Thailand . Entomological Science 16 : 298 – 308 . Google Scholar CrossRef Search ADS Pramual P , Wongpakam K . 2014 . Association of black fly (Diptera: Simuliidae) life stages using DNA barcode . Journal of Asia-Pacific Entomology 17 : 549 – 554 . Google Scholar CrossRef Search ADS Pramual P , Wongpakam K , Kuvangkadilok C . 2008 . Cytogenetics of the black fly Simulium aureohirtum Brunetti from Thailand . Cytologia 73 : 293 – 304 . Google Scholar CrossRef Search ADS Rivera J , Currie DC . 2009 . Identification of Nearctic black flies using DNA barcodes (Diptera: Simuliidae) . Molecular Ecology Resources 9 ( Suppl s1 ): 224 – 236 . Google Scholar CrossRef Search ADS Rothfels KH . 1979 . Cytotaxonomy of black flies (Simuliidae) . Annual Review of Entomology 24 : 507 – 539 . Google Scholar CrossRef Search ADS Rothfels KH . 1988 . Cytological approaches to black fly taxonomy . In: Kim KC , Merritt RW , eds. Black flies: ecology, population management, and annotated world list . University Park, PA : Pennsylvania State University Press , 39 – 52 . Rothfels K . 1989 . Speciation in black flies . Genome 32 : 500 – 509 . Google Scholar CrossRef Search ADS Rothfels K , Feraday R , Kaneps A . 1978 . A cytological description of sibling species of Simulium venustum and S. verecundum with standard maps for the subgenus Simulium Davies (Diptera) . Canadian Journal of Zoology 56 : 1110 – 1128 . Google Scholar CrossRef Search ADS Rothfels KH , Freeman DM . 1977 . The salivary gland chromosomes of seven species of Prosimulium (Diptera, Simuliidae) in the mixtum (IIIL-1) group . Canadian Journal of Zoology 55 : 482 – 507 . Google Scholar CrossRef Search ADS Swofford DL . 2002 . PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4.b.10 . Sunderland : Sinauer . Takaoka H . 1977 . Studies on black flies of the Nansei Islands, Japan (Simuliidae; Diptera): III. On six species of the subgenus Simulium Latreille . Medical Entomology and Zoology 28 : 193 – 217 . Google Scholar CrossRef Search ADS Takaoka H . 2017 . Morphotaxonomic revision of species-groups of Simulium (Simulium) (Diptera: Simuliidae) in the Oriental Region . Zootaxa 4353 : 425 – 446 . Google Scholar CrossRef Search ADS Takaoka H , Choochote W . 2004 . A list of and keys to black flies (Diptera: Simuliidae) in Thailand . Tropical Medicine and Health 32 : 189 – 197 . Google Scholar CrossRef Search ADS Takaoka H , Choochote W . 2005a . Two new species of black flies (Diptera: Simuliidae) from northern Thailand . Medical Entomology and Zoology 56 : 319 – 333 . Google Scholar CrossRef Search ADS Takaoka H , Choochote W . 2005b . Two new species of Simulium Latreille (Diptera: Simuliidae) from northwestern Thailand . Medical Entomology and Zoology 56 : 123 – 133 . Google Scholar CrossRef Search ADS Takaoka H , Choochote W . 2007 . A new species of the multistriatum species group of Simulium (Simulium) (Diptera: Simuliidae) from Northern Thailand . Tropical Medicine and Health 35 : 19 – 22 . Google Scholar CrossRef Search ADS Takaoka H , Davies DM . 1995 . The black flies (Diptera: Simuliidae) of West Malaysia . Fukuoka, Japan : Kyushu University Press . Takaoka H , Kuvangkadilok C . 1999 . Four new species of black flies (Diptera: Simuliidae) from Thailand . Japanese Journal of Tropical Medicine and Hygiene 27 : 497 – 509 . Google Scholar CrossRef Search ADS Tamura K , Stecher G , Peterson D , Filipski A , Kumar S . 2013 . MEGA6: molecular evolutionary genetics analysis version 6.0 . Molecular Biology and Evolution 30 : 2725 – 2729 . Google Scholar CrossRef Search ADS Tangkawanit U , Kuvangkadilok C , Baimai V , Adler PH . 2009 . Cytosystematics of the Simulium tuberosum group (Diptera: Simuliidae) in Thailand . Zoological Journal of the Linnean Society 155 : 289 – 315 . Google Scholar CrossRef Search ADS Tangkawanit U , Kuvangkadilok C , Trinachartvanit W , Baimai V . 2011 . Cytotaxonomy, morphology and ecology of the Simulium nobile species group (Diptera: Simuliidae) in Thailand . Cytogenetic and Genome Research 134 : 308 – 318 . Google Scholar CrossRef Search ADS Thaijarern J , Pramual P , Adler PH . 2017 . Life-stage association of black flies, using a fast-evolving nuclear gene sequence, and description of the larva of Simulium lampangense Takaoka & Choochote (Diptera: Simuliidae) from Thailand . Zootaxa 4299 : 263 – 270 . Google Scholar CrossRef Search ADS Thanwisai A , Kuvangkadilok C , Baimai V . 2006 . Molecular phylogeny of black flies (Diptera: Simuliidae) from Thailand, using ITS2 rDNA . Genetica 128 : 177 – 204 . Google Scholar CrossRef Search ADS Thapa S , Procunier W , Henry W , Chhetri S . 2014 . Heterochromatin and sibling species of Simulium praelargum s.l. (Diptera: Simuliidae) . Genome 57 : 223 – 232 . Google Scholar CrossRef Search ADS © 2018 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Zoological Journal of the Linnean Society Oxford University Press

Limited differentiation among black flies in the Simulium multistriatum species group (Diptera: Simuliidae) in Thailand: cryptic species, homosequential species and homosequential cryptic species

Loading next page...
 
/lp/ou_press/limited-differentiation-among-black-flies-in-the-simulium-WnwLVCMHyO
Publisher
Oxford University Press
Copyright
© 2018 The Linnean Society of London, Zoological Journal of the Linnean Society
ISSN
0024-4082
eISSN
1096-3642
D.O.I.
10.1093/zoolinnean/zly023
Publisher site
See Article on Publisher Site

Abstract

Abstract The high degree of morphological homogeneity in the Simuliidae often necessitates an integrated approach to the systematics of the family. We used cytological and molecular approaches to examine species delimitation and evolutionary relationships of black flies in the Simulium multistriatum species group in Thailand. A total of 664 larvae of eight nominal morphospecies (Simulium bullatum, Simulium chainarongi, Simulium chaliowae, Simulium daoense, Simulium fenestratum, Simulium lampangense, Simulium malayense and Simulium triglobus) from 30 sites in Thailand, one in Malaysia and one in Vietnam were chromosomally analysed, and 69 specimens were molecularly analysed for cytochrome c oxidase I and II gene sequences. We recognize ten taxa, based largely on unique chromosome rearrangements. Simulium chainarongi, S. fenestratum and S. triglobus are homosequential species. Simulium malayense consists of three cytoforms (A, B and C). Identical chromosomes, minimal morphological differentiation and low COI+ COII differentiation (1.20%) suggest that S. lampangense is either a junior synonym or a homosequential cryptic species of S. chaliowae. High genetic diversity in the geographically widespread nominal species S. fenestratum suggests that it consists of at least two cryptic species. Chromosomal and molecular phylogenetic inference shows good agreement within the S. multistriatum species group, but not with regard to other species groups. chromosomes, COI gene, COII gene, cryptic species, phylogeny, systematics INTRODUCTION Morphological uniformity in many groups of the Simuliidae presents a challenge for the taxonomy and systematics of this medically important family. The extent of this challenge increased when cryptic diversity was discovered through studies of the banding patterns of the larval polytene chromosomes (Rothfels, 1979, 1988; Adler, Cheke & Post, 2010). These studies revealed a spectrum of differentiation that includes (1) morphologically and chromosomally unique species, (2) morphologically identical species with unique chromosomal banding sequences (cryptic species; Tangkawanit et al., 2009), (3) morphologically distinct species with identical chromosomal banding sequences (homosequential species; Adler, Currie & Wood, 2004), and (4) morphologically identical species with identical chromosomal sequences (homosequential cryptic species; Bedo, 1979; Henderson, 1986). Molecular approaches have offered a wealth of new characters for discovering and diagnosing biodiversity in the Simuliidae (Pramual & Wongpakam, 2011, 2013; Pramual & Kuvangkadilok, 2012). The Simulium multistriatum species group consists of 34 nominal species distributed primarily in the Oriental Region but also in the Palearctic Region (Adler & Crosskey, 2017). Nine species have been recorded from Thailand: Simulium bullatum Takaoka and Choochote, 2005; Simulium chainarongi Takaoka & Kuvangkadilok, 1999; Simulium chaliowae Takaoka & Boonkemtong, 1999; Simulium chanyae Takaoka & Choochote, 2007; Simulium fenestratum Edwards, 1934; Simulium lampangense Takaoka & Choochote, 2005; Simulium malayense Takaoka & Davies, 1995; Simulium takense Takaoka & Choochote, 2005; and Simulium triglobus Kuvangkadilok & Takaoka, 1999. All members of the group are morphologically similar in all life stages (Takaoka & Choochote, 2005a, b). Some frequently used mitochondrial (COI and COII) and nuclear genes (18S/ITS1) fail to separate all species of the group (Pramual & Nanork, 2012), whereas other genes (e.g. ECP1) provide better resolution (Thaijarern, Pramual & Adler, 2017). Morphological and molecular data, however, have been insufficient for fully resolving the taxonomy and systematics of this species group, and the chromosomes have not yet been investigated. Our primary objective was to use polytene chromosomes from the larval silk glands, supplemented with analyses of the COI and COII genes, to understand differentiation in the S. multistriatum species group. Our specific objectives were to test morphological hypotheses of species via chromosomal and molecular inference of reproductive isolation, inspect nominal species for hidden biodiversity and infer evolutionary relationships of the constituent members of the group in Thailand, and to other species groups in the subgenus Simulium, by using an integrated approach (Ilmonen et al., 2009; Adler & Huang, 2011; Pramual & Kuvangkadilok, 2012) based on morphology, cytology and gene sequences. We focused our study of this group in Thailand because the greatest number of species in Southeast Asia is found there (Adler & Crosskey, 2017) and because many of the group’s habitats in the country have been documented (Takaoka & Choochote, 2005a, b). MATERIAL AND METHODS Collection and identification Larvae and pupae of the S. multistriatum species group were collected from 30 stream sites in Thailand (Fig. 1), one in Malaysia and one in Vietnam (Table 1). They were fixed in three changes of Carnoy’s solution (1:3 glacial acetic acid:absolute ethanol) and stored at −20 °C until processing. Species were initially identified with the morphological keys and descriptions of Takaoka & Davies (1995), Takaoka & Kuvangkadilok (1999), Takaoka & Choochote (2004, 2005a, b) and Thaijarern et al. (2017). Representative larvae were deposited in the collection of the Department of Biology, Faculty of Science at Mahasarakham University in Thailand and in the Clemson University Arthropod Collection in Clemson, SC, USA. Table 1. Collection sites for larvae of the Simulium multistriatum group in Thailand, Malaysia and Vietnam Site Location Province Elevation (m) Latitude Longitude Date Larvae prepared (completely analysed) Taxon Thailand 270 Lert Pop waterfall Loei 1152 17°30′N 101°20′E 27 November 2013 17 (17) S. bullatum 279 Phu Suan Sai Loei 918 17°30′N 101°56′E 28 November 2013 9 (9) S. bullatum 396 Lert Pop waterfall Loei 1123 17°30′N101°20′E 20 September 2015 2 (2)17 (12) S. bullatumS. fenestratum 287 Na Ku Ha waterfall Phrae 556 18°07′N 100°17′E 6 December 2013 71 (59) S. chaliowae 294 Huai Ton Phueng waterfall Phayao 361 19°16′N 100°53′E 7 December 2013 89 (69) S. chaliowae 244 Pha Luang waterfall Ubon Ratchathani 166 15°36′N 105°22′E 2 December 2013 43 (28) S. chainarongi 253 Huai Sai Yai waterfall Ubon Ratchathani 218 14°55′N 105°30′E 3 December 2013 18 (7) S. chainarongi 254 Huai Luang waterfall Ubon Ratchathani 240 14°25′N 105°24′E 3 December 2013 51 (29) S. chainarongi 255 Kaeng Lam Duan waterfall Ubon Ratchathani 179 14°26′N 105°06′E 3 December 2013 71 (57) S. chainarongi 350 Wang Yai waterfall Si Saket 219 14°26′N 104°29′E 16 October 2014 16 (12) S. chainarongi 264 Piang Din waterfall Loei 627 17° 03′N 101°44′E 26 December 2013 16 (8*) S. fenestratum 268 Song Khon waterfall Loei 743 17°21′N 101°24′E 27 December 2013 14 (12*) S. fenestratum 275 Than Sawan waterfall Loei 506 17o 29′N 101°3.5′E 28 December 2013 14 (10*) S. fenestratum 276 Khring waterfall Loei 643 17°28′N 101°58′E 28 December 2013 16 (8*) S. fenestratum 280 Tad Huong waterfall Loei 560 17°33′N 100°59′E 28 December 2013 29 (19*) S. fenestratum 401 Huai Khamin Noy waterfall Loei 1208 16° 59′N 101°00′E 26 October 2015 7 (4*) S. fenestratum 363 Than Ngam waterfall Udon Thani 278 17° 09′N 102°44′E 7 December 2014 10 (3) S. fenestratum 364 Koi Nang waterfall Udon Thani 416 17° 07′N 102°43′E 7 December 2014 26 (16) S. fenestratum 297 Ban Pang Faen Chiang Mai 615 19° 00′N 99°18′E 7 November 2013 46 (29*) S. fenestratum 372 Huai Mae Om Long Chiang Mai 852 18°09′N 98°13′E 16 January 2015 23 (15) S. fenestratum 384 Mae Taeng District Chiang Mai 703 19°07′N 98°45′E 16 January 2015 11 (9) S. fenestratum 402 Phu Soi Dao waterfall Uttaradit 654 17°44′N 100°59′E 27 October 2015 23 (15) S. fenestratum 320 Mae Sot Tak 303 16°48′N 98°59′E 19 February 2014 7 (6*) S. fenestratum 342 Klong Na Rai waterfall Chan Tha Buri 43 12°34′N 102°10′E 5 March 2014 43 (30*) S. fenestratum 343 Pliw waterfall Chan Tha Buri 46 12°31′N 102°10′E 5 March 2014 66 (63*) S. fenestratum 345 Klong Gaew waterfall Trat 65 12°37′N 102°34′E 5 March 2014 7 (6) S. fenestratum 408 Wang Kaew waterfall Lampang 626 19°18′N 99°39′E 18 December 2015 26 (19) S. lampangense 409 Wang Thong waterfall Lampang 510 19°16′N 99°39′E 18 December 2015 27 (23) S. lampangense 298 Siribhumi waterfall Chiang Mai 1304 18°32′N98°30′E 8 December 2013 7 (5)1 (1) S. malayense AS. malayense B 9 (9) S. daoense 7 (7) S. fenestratum 13 October 2017† 14 (–)2 (2) S. fenestratumS. malayense A 291 Ton Tong waterfall Nan 1027 19°12′N 101°04′E 6 December 2013 43 (28) S. triglobus Malaysia MYC Chenderiang waterfall Perak 129 04°21′N 101°14′E 23 February 2015 11 (6) S. malayense C Vietnam Dao Sapa Lao Cai 1315 22°23′N 103°50′E 22 December 2014 3 (3) S. daoense Site Location Province Elevation (m) Latitude Longitude Date Larvae prepared (completely analysed) Taxon Thailand 270 Lert Pop waterfall Loei 1152 17°30′N 101°20′E 27 November 2013 17 (17) S. bullatum 279 Phu Suan Sai Loei 918 17°30′N 101°56′E 28 November 2013 9 (9) S. bullatum 396 Lert Pop waterfall Loei 1123 17°30′N101°20′E 20 September 2015 2 (2)17 (12) S. bullatumS. fenestratum 287 Na Ku Ha waterfall Phrae 556 18°07′N 100°17′E 6 December 2013 71 (59) S. chaliowae 294 Huai Ton Phueng waterfall Phayao 361 19°16′N 100°53′E 7 December 2013 89 (69) S. chaliowae 244 Pha Luang waterfall Ubon Ratchathani 166 15°36′N 105°22′E 2 December 2013 43 (28) S. chainarongi 253 Huai Sai Yai waterfall Ubon Ratchathani 218 14°55′N 105°30′E 3 December 2013 18 (7) S. chainarongi 254 Huai Luang waterfall Ubon Ratchathani 240 14°25′N 105°24′E 3 December 2013 51 (29) S. chainarongi 255 Kaeng Lam Duan waterfall Ubon Ratchathani 179 14°26′N 105°06′E 3 December 2013 71 (57) S. chainarongi 350 Wang Yai waterfall Si Saket 219 14°26′N 104°29′E 16 October 2014 16 (12) S. chainarongi 264 Piang Din waterfall Loei 627 17° 03′N 101°44′E 26 December 2013 16 (8*) S. fenestratum 268 Song Khon waterfall Loei 743 17°21′N 101°24′E 27 December 2013 14 (12*) S. fenestratum 275 Than Sawan waterfall Loei 506 17o 29′N 101°3.5′E 28 December 2013 14 (10*) S. fenestratum 276 Khring waterfall Loei 643 17°28′N 101°58′E 28 December 2013 16 (8*) S. fenestratum 280 Tad Huong waterfall Loei 560 17°33′N 100°59′E 28 December 2013 29 (19*) S. fenestratum 401 Huai Khamin Noy waterfall Loei 1208 16° 59′N 101°00′E 26 October 2015 7 (4*) S. fenestratum 363 Than Ngam waterfall Udon Thani 278 17° 09′N 102°44′E 7 December 2014 10 (3) S. fenestratum 364 Koi Nang waterfall Udon Thani 416 17° 07′N 102°43′E 7 December 2014 26 (16) S. fenestratum 297 Ban Pang Faen Chiang Mai 615 19° 00′N 99°18′E 7 November 2013 46 (29*) S. fenestratum 372 Huai Mae Om Long Chiang Mai 852 18°09′N 98°13′E 16 January 2015 23 (15) S. fenestratum 384 Mae Taeng District Chiang Mai 703 19°07′N 98°45′E 16 January 2015 11 (9) S. fenestratum 402 Phu Soi Dao waterfall Uttaradit 654 17°44′N 100°59′E 27 October 2015 23 (15) S. fenestratum 320 Mae Sot Tak 303 16°48′N 98°59′E 19 February 2014 7 (6*) S. fenestratum 342 Klong Na Rai waterfall Chan Tha Buri 43 12°34′N 102°10′E 5 March 2014 43 (30*) S. fenestratum 343 Pliw waterfall Chan Tha Buri 46 12°31′N 102°10′E 5 March 2014 66 (63*) S. fenestratum 345 Klong Gaew waterfall Trat 65 12°37′N 102°34′E 5 March 2014 7 (6) S. fenestratum 408 Wang Kaew waterfall Lampang 626 19°18′N 99°39′E 18 December 2015 26 (19) S. lampangense 409 Wang Thong waterfall Lampang 510 19°16′N 99°39′E 18 December 2015 27 (23) S. lampangense 298 Siribhumi waterfall Chiang Mai 1304 18°32′N98°30′E 8 December 2013 7 (5)1 (1) S. malayense AS. malayense B 9 (9) S. daoense 7 (7) S. fenestratum 13 October 2017† 14 (–)2 (2) S. fenestratumS. malayense A 291 Ton Tong waterfall Nan 1027 19°12′N 101°04′E 6 December 2013 43 (28) S. triglobus Malaysia MYC Chenderiang waterfall Perak 129 04°21′N 101°14′E 23 February 2015 11 (6) S. malayense C Vietnam Dao Sapa Lao Cai 1315 22°23′N 103°50′E 22 December 2014 3 (3) S. daoense *IIIL-2 was not analysed for all larvae; for each site, the number analysed is given in parentheses: site 264 (4), 268 (7), 275 (9), 276 (7), 280 (18), 401 (3), 297 (26), 320 (4), 342 (4) and 343 (30). †Of 16 larvae analysed from this collection, 14 were S. fenestratum and two were S. malayense A; larvae of S. daoense were not found on this date. These larvae of S. fenestratum were cytologically similar to those of 8 December 2013 but were not scored for polymorphisms and, therefore, were not included in any counts or analyses. View Large Table 1. Collection sites for larvae of the Simulium multistriatum group in Thailand, Malaysia and Vietnam Site Location Province Elevation (m) Latitude Longitude Date Larvae prepared (completely analysed) Taxon Thailand 270 Lert Pop waterfall Loei 1152 17°30′N 101°20′E 27 November 2013 17 (17) S. bullatum 279 Phu Suan Sai Loei 918 17°30′N 101°56′E 28 November 2013 9 (9) S. bullatum 396 Lert Pop waterfall Loei 1123 17°30′N101°20′E 20 September 2015 2 (2)17 (12) S. bullatumS. fenestratum 287 Na Ku Ha waterfall Phrae 556 18°07′N 100°17′E 6 December 2013 71 (59) S. chaliowae 294 Huai Ton Phueng waterfall Phayao 361 19°16′N 100°53′E 7 December 2013 89 (69) S. chaliowae 244 Pha Luang waterfall Ubon Ratchathani 166 15°36′N 105°22′E 2 December 2013 43 (28) S. chainarongi 253 Huai Sai Yai waterfall Ubon Ratchathani 218 14°55′N 105°30′E 3 December 2013 18 (7) S. chainarongi 254 Huai Luang waterfall Ubon Ratchathani 240 14°25′N 105°24′E 3 December 2013 51 (29) S. chainarongi 255 Kaeng Lam Duan waterfall Ubon Ratchathani 179 14°26′N 105°06′E 3 December 2013 71 (57) S. chainarongi 350 Wang Yai waterfall Si Saket 219 14°26′N 104°29′E 16 October 2014 16 (12) S. chainarongi 264 Piang Din waterfall Loei 627 17° 03′N 101°44′E 26 December 2013 16 (8*) S. fenestratum 268 Song Khon waterfall Loei 743 17°21′N 101°24′E 27 December 2013 14 (12*) S. fenestratum 275 Than Sawan waterfall Loei 506 17o 29′N 101°3.5′E 28 December 2013 14 (10*) S. fenestratum 276 Khring waterfall Loei 643 17°28′N 101°58′E 28 December 2013 16 (8*) S. fenestratum 280 Tad Huong waterfall Loei 560 17°33′N 100°59′E 28 December 2013 29 (19*) S. fenestratum 401 Huai Khamin Noy waterfall Loei 1208 16° 59′N 101°00′E 26 October 2015 7 (4*) S. fenestratum 363 Than Ngam waterfall Udon Thani 278 17° 09′N 102°44′E 7 December 2014 10 (3) S. fenestratum 364 Koi Nang waterfall Udon Thani 416 17° 07′N 102°43′E 7 December 2014 26 (16) S. fenestratum 297 Ban Pang Faen Chiang Mai 615 19° 00′N 99°18′E 7 November 2013 46 (29*) S. fenestratum 372 Huai Mae Om Long Chiang Mai 852 18°09′N 98°13′E 16 January 2015 23 (15) S. fenestratum 384 Mae Taeng District Chiang Mai 703 19°07′N 98°45′E 16 January 2015 11 (9) S. fenestratum 402 Phu Soi Dao waterfall Uttaradit 654 17°44′N 100°59′E 27 October 2015 23 (15) S. fenestratum 320 Mae Sot Tak 303 16°48′N 98°59′E 19 February 2014 7 (6*) S. fenestratum 342 Klong Na Rai waterfall Chan Tha Buri 43 12°34′N 102°10′E 5 March 2014 43 (30*) S. fenestratum 343 Pliw waterfall Chan Tha Buri 46 12°31′N 102°10′E 5 March 2014 66 (63*) S. fenestratum 345 Klong Gaew waterfall Trat 65 12°37′N 102°34′E 5 March 2014 7 (6) S. fenestratum 408 Wang Kaew waterfall Lampang 626 19°18′N 99°39′E 18 December 2015 26 (19) S. lampangense 409 Wang Thong waterfall Lampang 510 19°16′N 99°39′E 18 December 2015 27 (23) S. lampangense 298 Siribhumi waterfall Chiang Mai 1304 18°32′N98°30′E 8 December 2013 7 (5)1 (1) S. malayense AS. malayense B 9 (9) S. daoense 7 (7) S. fenestratum 13 October 2017† 14 (–)2 (2) S. fenestratumS. malayense A 291 Ton Tong waterfall Nan 1027 19°12′N 101°04′E 6 December 2013 43 (28) S. triglobus Malaysia MYC Chenderiang waterfall Perak 129 04°21′N 101°14′E 23 February 2015 11 (6) S. malayense C Vietnam Dao Sapa Lao Cai 1315 22°23′N 103°50′E 22 December 2014 3 (3) S. daoense Site Location Province Elevation (m) Latitude Longitude Date Larvae prepared (completely analysed) Taxon Thailand 270 Lert Pop waterfall Loei 1152 17°30′N 101°20′E 27 November 2013 17 (17) S. bullatum 279 Phu Suan Sai Loei 918 17°30′N 101°56′E 28 November 2013 9 (9) S. bullatum 396 Lert Pop waterfall Loei 1123 17°30′N101°20′E 20 September 2015 2 (2)17 (12) S. bullatumS. fenestratum 287 Na Ku Ha waterfall Phrae 556 18°07′N 100°17′E 6 December 2013 71 (59) S. chaliowae 294 Huai Ton Phueng waterfall Phayao 361 19°16′N 100°53′E 7 December 2013 89 (69) S. chaliowae 244 Pha Luang waterfall Ubon Ratchathani 166 15°36′N 105°22′E 2 December 2013 43 (28) S. chainarongi 253 Huai Sai Yai waterfall Ubon Ratchathani 218 14°55′N 105°30′E 3 December 2013 18 (7) S. chainarongi 254 Huai Luang waterfall Ubon Ratchathani 240 14°25′N 105°24′E 3 December 2013 51 (29) S. chainarongi 255 Kaeng Lam Duan waterfall Ubon Ratchathani 179 14°26′N 105°06′E 3 December 2013 71 (57) S. chainarongi 350 Wang Yai waterfall Si Saket 219 14°26′N 104°29′E 16 October 2014 16 (12) S. chainarongi 264 Piang Din waterfall Loei 627 17° 03′N 101°44′E 26 December 2013 16 (8*) S. fenestratum 268 Song Khon waterfall Loei 743 17°21′N 101°24′E 27 December 2013 14 (12*) S. fenestratum 275 Than Sawan waterfall Loei 506 17o 29′N 101°3.5′E 28 December 2013 14 (10*) S. fenestratum 276 Khring waterfall Loei 643 17°28′N 101°58′E 28 December 2013 16 (8*) S. fenestratum 280 Tad Huong waterfall Loei 560 17°33′N 100°59′E 28 December 2013 29 (19*) S. fenestratum 401 Huai Khamin Noy waterfall Loei 1208 16° 59′N 101°00′E 26 October 2015 7 (4*) S. fenestratum 363 Than Ngam waterfall Udon Thani 278 17° 09′N 102°44′E 7 December 2014 10 (3) S. fenestratum 364 Koi Nang waterfall Udon Thani 416 17° 07′N 102°43′E 7 December 2014 26 (16) S. fenestratum 297 Ban Pang Faen Chiang Mai 615 19° 00′N 99°18′E 7 November 2013 46 (29*) S. fenestratum 372 Huai Mae Om Long Chiang Mai 852 18°09′N 98°13′E 16 January 2015 23 (15) S. fenestratum 384 Mae Taeng District Chiang Mai 703 19°07′N 98°45′E 16 January 2015 11 (9) S. fenestratum 402 Phu Soi Dao waterfall Uttaradit 654 17°44′N 100°59′E 27 October 2015 23 (15) S. fenestratum 320 Mae Sot Tak 303 16°48′N 98°59′E 19 February 2014 7 (6*) S. fenestratum 342 Klong Na Rai waterfall Chan Tha Buri 43 12°34′N 102°10′E 5 March 2014 43 (30*) S. fenestratum 343 Pliw waterfall Chan Tha Buri 46 12°31′N 102°10′E 5 March 2014 66 (63*) S. fenestratum 345 Klong Gaew waterfall Trat 65 12°37′N 102°34′E 5 March 2014 7 (6) S. fenestratum 408 Wang Kaew waterfall Lampang 626 19°18′N 99°39′E 18 December 2015 26 (19) S. lampangense 409 Wang Thong waterfall Lampang 510 19°16′N 99°39′E 18 December 2015 27 (23) S. lampangense 298 Siribhumi waterfall Chiang Mai 1304 18°32′N98°30′E 8 December 2013 7 (5)1 (1) S. malayense AS. malayense B 9 (9) S. daoense 7 (7) S. fenestratum 13 October 2017† 14 (–)2 (2) S. fenestratumS. malayense A 291 Ton Tong waterfall Nan 1027 19°12′N 101°04′E 6 December 2013 43 (28) S. triglobus Malaysia MYC Chenderiang waterfall Perak 129 04°21′N 101°14′E 23 February 2015 11 (6) S. malayense C Vietnam Dao Sapa Lao Cai 1315 22°23′N 103°50′E 22 December 2014 3 (3) S. daoense *IIIL-2 was not analysed for all larvae; for each site, the number analysed is given in parentheses: site 264 (4), 268 (7), 275 (9), 276 (7), 280 (18), 401 (3), 297 (26), 320 (4), 342 (4) and 343 (30). †Of 16 larvae analysed from this collection, 14 were S. fenestratum and two were S. malayense A; larvae of S. daoense were not found on this date. These larvae of S. fenestratum were cytologically similar to those of 8 December 2013 but were not scored for polymorphisms and, therefore, were not included in any counts or analyses. View Large Figure 1. View largeDownload slide Sampling sites for eight nominal species of Simulium multistriatum species group in Thailand. Details of sampling sites are given in Table 1. Figure 1. View largeDownload slide Sampling sites for eight nominal species of Simulium multistriatum species group in Thailand. Details of sampling sites are given in Table 1. Chromosome preparation and analysis Middle to final instar larvae were prepared for analysis of the polytene chromosomes from their silk glands, using the Feulgen method (Adler et al., 2016a). Representative chromosome preparations were photographed under oil immersion on an Olympus BX40 microscope (Simulium daoense) or a BH-2 Olympus microscope with a Jenoptik ProgRes SpeedXT Core 5 digital camera (all other taxa). The images were imported into Adobe Photoshop Elements 8 or Photoshop CS6 to construct chromosomal maps. The standard map for the S. multistriatum species group was based on the most central sequence among the group members, relative to the standard sequence established by Rothfels, Feraday & Kaneps (1978) and Adler et al. (2016a) for the subgenus Simulium. Chromosomal terminology and section numbering for the three chromosomes (I, II and III) followed the practice of Rothfels et al. (1978) and Adler et al. (2016a). Fixed inversions are italicized in the text and underlined on the chromosome maps; polymorphic rearrangements are given in regular type. Additional rearrangements are coded in the text and on the maps as follows: heteroband (hb), diffuse centromere band (d), additional (insertion) band (i), flocculent band expression (fl), and band enhancement (+). The following centromere associations were recognized: (1) chromocentre, i.e. all centromeres of all chromosomes in each nucleus were attached to a central mass of heterochromatin; (2) partial chromocentre, i.e. the centromeres of any two chromosomes of each nucleus were attached to a heterochromatic mass; and (3) ectopic pairing (pseudochromocentre), i.e. two or more centromeres were associated with one another in at least some, but typically not all, nuclei, and additional heterochromatin was absent from the centromere associations (Adler et al., 2004). The maximal width of the terminal flare for each chromosome arm of S. bullatum, S. fenestratum and S. triglobus was measured against the width of a subterminal indicator band and grouped into size categories as follows: 0 (less than the width of the indicator band), 1 (equal to width of the indicator band to ≤ 1.5 times its width), 2 (> 1.5 to ≤ 2.0 times width of the indicator band) and 3 (> 2.0 times width of the indicator band). A Mann–Whitney U test was used to evaluate ranked characteristics. Polymorphic inversions were tested for Hardy–Weinberg equilibrium when samples were large enough to provide at least five larvae for each state (homozygous standard, heterozygous and homozygous inverted). Evolutionary relationships The following species groups in the subgenus Simulium with chromosome complements fully resolved relative to the subgeneric standard of Rothfels et al. (1978) and Adler et al. (2016a) were screened for rearrangements, primarily inversions, shared with the S. multistriatum group: S. bezzii, S. ornatum, S. petersoni, S. variegatum (P. Adler, unpublished), S. malyschevi-reptans (Adler et al., 2016a), S. nobile (Tangkawanit et al., 2011), S. noelleri (Adler & Kachvorian, 2001), S. tuberosum (Tangkawanit et al., 2009, Adler et al., 2016b) and S. venustum groups (Huang, Adler & Takaoka, 2011). To permit additional comparison, we also resolved the entire sequence of the S. striatum group, based on maps of Pramual (2006). Species groups with any rearrangement(s) uniquely shared with the S. multistriatum species group were selected as outgroups. Four of these groups share unique inversions with the S. multistriatum species group: S. malyschevi-reptans, S. nobile and S. striatum groups. Among the 13 remaining species groups in the subgenus Simulium, the S. jenningsi group, which is closely related to the S. malyschevi-reptans group, might share IIS-1, but its highly scrambled IIS sequence has not yet been resolved (Adler et al., 2016a). Additionally, the S. griseifrons species group might share rearrangements with the S. multistriatum species group, based on molecular relationships showing that the two groups are closely related (Thanwisai, Kuvangkadilok & Baimai, 2006); however, not a single species in the group has been studied chromosomally (Adler & Crosskey, 2015). Furthermore, the S. griseifrons group has recently been divided into six species groups (Takaoka, 2017). All rearrangements shared between two or more of the members of the S. multistriatum species group were included in the phylogenetic analysis, with one exception. We did not include the uniquely manifested ‘2 blocks’ marker of S. chaliowae and S. lampangense because we suspect that it reflects gene expression and is environmentally influenced. Thus, a character matrix of 14 rearrangements was used in our analysis. Two character states were recognized for the rearrangements: 0 = absent and 1 = fixed. The maximum parsimony (MP) tree for phylogenetic relationships was calculated in PAUP* v.4.10b (Swofford, 2002), using a heuristic search with 1000 random addition sequence replicates; bootstrap support was estimated for 1000 replicates. DNA extraction, polymerase chain reaction, sequencing and analysis DNA was extracted with the Vivantis GF-1 Nucleic Extraction kit. Polymerase chain reaction (PCR) of the cytochrome c oxidase subunit I gene (COI) used the primers following the description of Folmer et al. (1994). The PCR followed the methods described by Rivera & Currie (2009). The cytochrome c oxidase subunit II gene (COII) used the primers TL2-J-3034 5′-ATTATGGCAGATTAGTGCA-3′ and TK-N-3785 5′-GTTTAA GAGACCAGTACTTG-3′, and PCR followed the methods of Conflitti et al. (2010). The PCR products were checked with 1% agarose gel electrophoresis and purified using a HighYield Gel/PCR DNA Fragment Extraction kit (RBC BioScience, Taiwan). Sequencing was performed by the Macrogen DNA sequencing service (Seoul, Korea) and 1st BASE DNA Sequencing Services (Singapore Science Park, Singapore), using primers as for PCR. A total of 69 sequences from nine cytologically distinct taxa of the S. multistriatum species group was included in the analyses (GenBank accession numbers: MG733997–MG734134). Genetic distances were calculated using MEGA7 (Tamura et al., 2013), based on the Kimura 2-parameter (K2P). Phylogenetic analyses were conducted separately for the COI and COII sequences and for the combined dataset, and included neighbor-joining (NJ), maximum likelihood (ML), MP and Bayesian methods. The NJ method inferred phylogenetic trees in MEGA7 (Tamura et al., 2013), and MP was calculated separately for each gene in PAUP* v.4.10b (Swofford, 2002). Branch support for NJ and MP was calculated using the bootstrapping method with 1000 replicates. Maximum likelihood was performed with PhyML 3.0 (Guindon et al., 2010). Node support was determined using an approximate likelihood-ratio test (Anisimova & Gascuel, 2006; Guindon et al., 2010). Bayesian inference was performed with MrBayes 3.04b and run for 2000000 generations with a sampling frequency of 100 generations (Huelsenbeck & Ronquist, 2001). Simulium nodosum, a member of the S. nobile species group, was used as an outgroup in all molecular phylogenetic analyses. The efficiency of specimen identification was obtained using the best match method in TaxonDNA (Meier et al., 2006). RESULTS The chromosomes of 912 larvae of the S. multistriatum species group were prepared, and the banding patterns of 664 (72.8%) of the larvae were analysed completely (Table 1), with the exception of the IIIL-2 sequence of S. fenestratum, for which 184 (65.9%) of 279 otherwise fully evaluated individuals were analysed. All larvae of the S. multistriatum species group had three pairs of tightly synapsed homologues (2n = 6). The nucleolar organizer (NO) was in the base of IS, near the expanded centromere region. The sex chromosomes of all species were microscopically undifferentiated. Chromosomal relationships of Simulium multistriatum species group to Simulium subgeneric standard The standard banding sequence of the S. multistriatum species group differed from the Simulium subgeneric standard by having the nucleolar organizer in the base of IS (rather than in IIIL) and by seven fixed inversions, as follows: IS: The basic sequence of this arm (Fig. 2) was identical to the subgeneric standard sequence of Rothfels et al. (1978). IL: The standard sequence for the S. multistriatum species group (Fig. 3, cf. Fig. 4) was identical to the subgeneric standard of Rothfels et al. (1978). IIS: The IIS sequence for all members of the S. multistriatum species group differed from the subgeneric standard of Adler et al. (2016a) by three fixed inversions. These three inversions divided the arm into six fragments. The most parsimonious reassemblage of fragments to produce the subgeneric standard, one inversion at a time, is shown below, and the order of the fragments is represented by the letters a–j (Fig. 5), where slashes represent inversion breakpoints and square brackets represent the inversion in each sequence. Inversion 2: a / g f / b c [i h / e d] j (= S. multistriatum species group standard) Inversion 3: a / g f / [b c d e] h i j Inversion 1: a [g f e d c b] h i j a b c d e f g h i j (= Simulium subgeneric standard) IIL: All members of the S. multistriatum species group could be derived by one fixed inversion (IIL-1; Fig. 6) from the subgeneric standard sequence of Rothfels et al. (1978). IIIS: The basic sequence of this arm (Fig. 7) was identical with the subgeneric standard sequence of Rothfels et al. (1978). IIIL: The basic IIIL sequence for the S. multistriatum species group was removed from the standard sequence of Adler et al. (2016a) by three fixed inversions (IIIL-3, IIIL-5 and IIIL-6; Fig. 8B, C). Figure 2. View largeDownload slide IS arm of Simulium multistriatum species group. A, B, Simulium fenestratum (female larva), corresponding to the Simulium subgeneric standard of Rothfels et al. (1978). C, Simulium chaliowae (male larva). Breakpoints of floating inversion IS-1 and fixed inversion IS-2 are indicated. C, centromere; g, glazed; NO, nucleolar organizer. Figure 2. View largeDownload slide IS arm of Simulium multistriatum species group. A, B, Simulium fenestratum (female larva), corresponding to the Simulium subgeneric standard of Rothfels et al. (1978). C, Simulium chaliowae (male larva). Breakpoints of floating inversion IS-1 and fixed inversion IS-2 are indicated. C, centromere; g, glazed; NO, nucleolar organizer. Figure 3. View largeDownload slide IL arm of Simulium bullatum (female larva), corresponding to the Simulium subgeneric standard of Rothfels et al. (1978) and the standard IL sequence of the S. multistriatum species group. Limits of fixed inversion IL-9 are indicated with brackets. C, centromere; M, marker. Figure 3. View largeDownload slide IL arm of Simulium bullatum (female larva), corresponding to the Simulium subgeneric standard of Rothfels et al. (1978) and the standard IL sequence of the S. multistriatum species group. Limits of fixed inversion IL-9 are indicated with brackets. C, centromere; M, marker. Figure 4. View largeDownload slide IL arm of Simulium multistriatum species group. Simulium daoense (male larva, sections 20–32) and Simulium fenestratum (female, sections 33–41), showing the IL-9 sequence. Breakpoints of floating inversions of S. fenestratum are indicated by brackets. Arrows indicate location of a heterochromatic band insertion (i) in S. fenestratum. M, marker. Figure 4. View largeDownload slide IL arm of Simulium multistriatum species group. Simulium daoense (male larva, sections 20–32) and Simulium fenestratum (female, sections 33–41), showing the IL-9 sequence. Breakpoints of floating inversions of S. fenestratum are indicated by brackets. Arrows indicate location of a heterochromatic band insertion (i) in S. fenestratum. M, marker. Figure 5. View largeDownload slide IIS arm of Simulium daoense (male larva), showing the standard sequence for the Simulium multistriatum species group relative to the Simulium subgeneric standard of Adler et al. (2016a). Breakpoints of three fixed inversions are indicated by numbered arrows 1–3. The letters a–j, when alphabetized, produce the subgeneric standard sequence. C, centromere. Figure 5. View largeDownload slide IIS arm of Simulium daoense (male larva), showing the standard sequence for the Simulium multistriatum species group relative to the Simulium subgeneric standard of Adler et al. (2016a). Breakpoints of three fixed inversions are indicated by numbered arrows 1–3. The letters a–j, when alphabetized, produce the subgeneric standard sequence. C, centromere. Figure 6. View largeDownload slide IIL arm of Simulium daoense (male larva), showing the standard sequence for the Simulium multistriatum species group, including fixed inversion IIL-1. Breakpoints of floating inversion IIL-2 of S. fenestratum are indicated by a bracket. C, centromere; gB, grey band; J, jagged; Pb, parabalbiani; po, polar. Figure 6. View largeDownload slide IIL arm of Simulium daoense (male larva), showing the standard sequence for the Simulium multistriatum species group, including fixed inversion IIL-1. Breakpoints of floating inversion IIL-2 of S. fenestratum are indicated by a bracket. C, centromere; gB, grey band; J, jagged; Pb, parabalbiani; po, polar. Figure 7. View largeDownload slide IIIS arm of Simulium lampangense (male larva), representing the standard sequence for the Simulium multistriatum species group, which is identical to the Simulium subgeneric standard sequence of Rothfels et al. (1978). Breakpoints of floating inversion IIIS-1 of Simulium chainarongi are shown. Bl, blister; C, centromere; Ca, capsule. Figure 7. View largeDownload slide IIIS arm of Simulium lampangense (male larva), representing the standard sequence for the Simulium multistriatum species group, which is identical to the Simulium subgeneric standard sequence of Rothfels et al. (1978). Breakpoints of floating inversion IIIS-1 of Simulium chainarongi are shown. Bl, blister; C, centromere; Ca, capsule. Figure 8. View largeDownload slide IIIL arm of Simulium fenestratum. A, basal sections (female larva, site 402) showing the IIIL-2, IIIL-3, 6 sequence; breakpoints of floating inversion IIIL-7 are indicated by a bracket. B, C, entire arm (female larva, site 343), showing the IIIL-3, 4, 5, 6 sequence; ordering the letters a–h produces the Simulium subgeneric sequence of Adler et al. (2016a). Breakpoints of three floating inversions [IIIL-8, IIIL-9 and compound (cmpd) inversion] are indicated by brackets. C, centromere. Figure 8. View largeDownload slide IIIL arm of Simulium fenestratum. A, basal sections (female larva, site 402) showing the IIIL-2, IIIL-3, 6 sequence; breakpoints of floating inversion IIIL-7 are indicated by a bracket. B, C, entire arm (female larva, site 343), showing the IIIL-3, 4, 5, 6 sequence; ordering the letters a–h produces the Simulium subgeneric sequence of Adler et al. (2016a). Breakpoints of three floating inversions [IIIL-8, IIIL-9 and compound (cmpd) inversion] are indicated by brackets. C, centromere. Simulium bullatum Takaoka & Choochote Three populations of S. bullatum from Loei Province were studied, and all 28 chromosomally prepared larvae were analysed completely (Table 1). Simulium bullatum differed from the standard for the S. multistriatum species group by one fixed inversion (IIIL-10; Fig. 9B) and a true chromocentre characterized by a glassy or darkly staining heterochromatic mass (Fig. 10A). Flaring at the end of IIIS was 3.0 times greater than the width of the indicator band, whereas the ends of IS, IL, IIS, IIL and IIIL were ≤ 1.5 times the width of the respective indicator bands (cf. Fig. 11D). Autosomal polymorphisms were not found. Figure 9. View largeDownload slide IIIL arm of Simulium multistriatum species group. A, Simulium daoense (male larva) showing the IIIL-1 sequence on top of IIIL-3, 6. B, Simulium malayense cytoform C (female larva) showing the IIIL-3, 5, 6, 10 sequence; ordering the letters a–h produces the Simulium subgeneric sequence of Adler et al. (2016a). Figure 9. View largeDownload slide IIIL arm of Simulium multistriatum species group. A, Simulium daoense (male larva) showing the IIIL-1 sequence on top of IIIL-3, 6. B, Simulium malayense cytoform C (female larva) showing the IIIL-3, 5, 6, 10 sequence; ordering the letters a–h produces the Simulium subgeneric sequence of Adler et al. (2016a). Figure 10. View largeDownload slide Complete chromosomal complement. A, Simulium bullatum (male larva), showing all centromeres attached to a chromocentre. B, Simulium malayense cytoform C (female larva), showing partial chromocentre (CI + CII). NO, nucleolar organizer. Figure 10. View largeDownload slide Complete chromosomal complement. A, Simulium bullatum (male larva), showing all centromeres attached to a chromocentre. B, Simulium malayense cytoform C (female larva), showing partial chromocentre (CI + CII). NO, nucleolar organizer. Figure 11. View largeDownload slide A, B, basal rearrangements in chromosome II of Simulium chainarongi. CII, typical centromere band; CIId, diffuse centromere band; i, heterochromatic insert in the base of IIS; +, band enhancement in the base of IIL; fl, expanded and flocculent IIL centromere region. C, D, degree of terminal flaring of chromosome arms. C, IIL of Simulium fenestratum. D, IIIL of Simulium triglobus. Arrows show indicator bands, against which the width of the terminal flare was measured. Figure 11. View largeDownload slide A, B, basal rearrangements in chromosome II of Simulium chainarongi. CII, typical centromere band; CIId, diffuse centromere band; i, heterochromatic insert in the base of IIS; +, band enhancement in the base of IIL; fl, expanded and flocculent IIL centromere region. C, D, degree of terminal flaring of chromosome arms. C, IIL of Simulium fenestratum. D, IIIL of Simulium triglobus. Arrows show indicator bands, against which the width of the terminal flare was measured. Simulium chainarongi Takaoka & Kuvangkadilok Simulium chainarongi was found only in northeastern Thailand. A total of 133 (66.8%) larvae of 199 prepared larvae from five populations were analysed completely. Kaeng Lam Duan waterfall (site 255) was the type locality of this species. The banding pattern of S. chainarongi differed by two fixed inversions (IL-9 and IIIL-4; Fig. 4) from the group standard. Its banding pattern did not, however, differ from that of two other group members (S. fenestratum and S. triglobus), although its centromeric characteristics were unique. The centromere bands of all chromosomes were typically more darkly stained and well defined than in any other group members (Fig. 12A). One autosomal inversion (IIIS-1) was found heterozygously (Fig. 7) in one male. Four band polymorphisms were found heterozygously in the centromere region of chromosome II: a diffuse centromere band in one male; a band enhancement in the base of IIL (four males and one female); a heterochromatic insert in the base of IIS (eight males and two females); and an expanded and flocculent IIL base in one male (Table 2, Fig. 11). Table 2. Frequency of homologues with chromosome rearrangements in seven nominal species of the Simulium multistriatum group in Thailand and Malaysia Species S. chainarongi S. chaliowae S. lampangense S. bullatum S. triglobus S. malayense S. daoense A B C Site 244 253 254 255 350 287 294 408 409 270 279 396 291 298 298 MYC 298 (male:female) 15:13 2:5 13:16 29:28 5:7 31:28 31:38 13:6 14:9 15:2 7:2 1:1 20:8 2:7 1:0 2:4 4:5 CIId 0.02 IIL+ 0.02 0.02 0.03 IIS 54i 0.07 0.05 IIL 54fl 0.02 IS-1 0.01 IS-2 1.00 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIS-1 0.02 IIIL-1 1.00 IIIL-2 1.00 1.00 1.00 1.00 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-8 0.01 IIIL-9 0.02 IIIL-10 1.00 1.00 1.00 1.00 1.00 1.00 Chromocentre 1.00 1.00 1.00 Species S. chainarongi S. chaliowae S. lampangense S. bullatum S. triglobus S. malayense S. daoense A B C Site 244 253 254 255 350 287 294 408 409 270 279 396 291 298 298 MYC 298 (male:female) 15:13 2:5 13:16 29:28 5:7 31:28 31:38 13:6 14:9 15:2 7:2 1:1 20:8 2:7 1:0 2:4 4:5 CIId 0.02 IIL+ 0.02 0.02 0.03 IIS 54i 0.07 0.05 IIL 54fl 0.02 IS-1 0.01 IS-2 1.00 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIS-1 0.02 IIIL-1 1.00 IIIL-2 1.00 1.00 1.00 1.00 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-8 0.01 IIIL-9 0.02 IIIL-10 1.00 1.00 1.00 1.00 1.00 1.00 Chromocentre 1.00 1.00 1.00 CIId, diffuse centromere band of chromosome II; IIL+, band enhancement in the long arm of chromosome II; IIS 54i, additional (insertion) band in section 54; and IIL 54fl, flocculent band expression in section 54 of the long arm of chromosome II. View Large Table 2. Frequency of homologues with chromosome rearrangements in seven nominal species of the Simulium multistriatum group in Thailand and Malaysia Species S. chainarongi S. chaliowae S. lampangense S. bullatum S. triglobus S. malayense S. daoense A B C Site 244 253 254 255 350 287 294 408 409 270 279 396 291 298 298 MYC 298 (male:female) 15:13 2:5 13:16 29:28 5:7 31:28 31:38 13:6 14:9 15:2 7:2 1:1 20:8 2:7 1:0 2:4 4:5 CIId 0.02 IIL+ 0.02 0.02 0.03 IIS 54i 0.07 0.05 IIL 54fl 0.02 IS-1 0.01 IS-2 1.00 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIS-1 0.02 IIIL-1 1.00 IIIL-2 1.00 1.00 1.00 1.00 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-8 0.01 IIIL-9 0.02 IIIL-10 1.00 1.00 1.00 1.00 1.00 1.00 Chromocentre 1.00 1.00 1.00 Species S. chainarongi S. chaliowae S. lampangense S. bullatum S. triglobus S. malayense S. daoense A B C Site 244 253 254 255 350 287 294 408 409 270 279 396 291 298 298 MYC 298 (male:female) 15:13 2:5 13:16 29:28 5:7 31:28 31:38 13:6 14:9 15:2 7:2 1:1 20:8 2:7 1:0 2:4 4:5 CIId 0.02 IIL+ 0.02 0.02 0.03 IIS 54i 0.07 0.05 IIL 54fl 0.02 IS-1 0.01 IS-2 1.00 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIS-1 0.02 IIIL-1 1.00 IIIL-2 1.00 1.00 1.00 1.00 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-8 0.01 IIIL-9 0.02 IIIL-10 1.00 1.00 1.00 1.00 1.00 1.00 Chromocentre 1.00 1.00 1.00 CIId, diffuse centromere band of chromosome II; IIL+, band enhancement in the long arm of chromosome II; IIS 54i, additional (insertion) band in section 54; and IIL 54fl, flocculent band expression in section 54 of the long arm of chromosome II. View Large Figure 12. View largeDownload slide Characteristics of centromere regions. A, Simulium chainarongi. B, Simulium fenestratum. C, centromere; NO, nucleolar organizer. Figure 12. View largeDownload slide Characteristics of centromere regions. A, Simulium chainarongi. B, Simulium fenestratum. C, centromere; NO, nucleolar organizer. Simulium chaliowae Takaoka & Boonkemtong Simulium chaliowae was found only in limestone streams in northern Thailand. The chromosomes of 128 (80%) of 160 prepared larvae from two sites were analysed completely. Wang Kaew waterfall was the type locality of this species. Simulium chaliowae differed from the S. multistriatum species group standard by three fixed inversions (IL-9, IIIL-2 and IIIL-4). Two floating inversions were found: IS-1 (Fig. 2A) and IIIL-8 (Fig. 8C), each in a separate female (frequency = 0.01; Table 2). The ‘2 blocks’ marker in IS (section 4) was found in two configurations, either standard (Fig. 2B) or with the first heavy block divided by a gap (Fig. 2C). Based on ten larvae per location (ten nuclei per larva), an average of 58% of nuclei per larva from site 287 and 57% of nuclei per larva from site 294 had the first block divided by an unstained gap. Simulium daoense Takaoka & Adler All nine larvae of S. daoense from Siribhumi waterfall, Chiang Mai Province (site 298), were analysed completely. This species differed from the S. multistriatum species group standard by having fixed inversions IL-9 (Fig. 4), IIIL-1 (Fig. 9A) and IIIL-4 (Fig. 8C). Autosomal polymorphisms were not found. The identity of this species was confirmed by chromosomal analysis of three larvae collected from the type locality of S. daoense in Vietnam at the same time that the type series was collected by Takaoka et al. (2017). Simulium fenestratum Edwards This species was the most common and widely distributed member of the S. multistriatum species group in Thailand. The chromosomes of 279 (70.5%) of 396 prepared larvae of S. fenestratum were analysed completely, except for inversion IIIL-2, which was evaluated in 65.9% of the 279 fully analysed larvae (Tables 1, 3). The banding sequence differed from the group standard by two fixed inversions (IL-9 and IIIL-4). The centromere bands of all chromosomes were diffuse and weakly stained (Fig. 12B). Simulium fenestratum had 12 autosomal floating inversions (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IIL-2, IIIL-2, IIIL-7 and an unresolved compound (cmpd) inversion in IIIL), plus one band insert in the expanded centromere region of chromosome I (IL 20i) and a heteroband (IL 21hb) in one female (Table 3, Fig. 4). Table 3. Frequency of homologues with chromosome rearrangements in Simulium fenestratum at 18 sites in Thailand Site Simulium fenestratum 396 264 268 275 276 280 401 363 364 297 298* 372 384 402 320 342 343 345 (male: female)† 9:3 2:2 2:5 5:4 3:4 8:10 3:0 2:1 6:10 15:11 4:3 4:11 6:3 1:3 0:4 1:3 13:17 3:3 IL-1 0.02 0.02 IL 20i 0.02 IL 21hb 0.02 IL-2 0.08 IL-3 0.03 IL-4 0.02 IL-5 0.02 IL-6 0.17 0.02 IL-7 0.04 IL-8 0.04 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIL-2 0.04 IIIL-2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.13 0.43 0.07 0.55 1.00 0.50 0.66 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-7 0.06 IIIL-cmpd 0.08 Site Simulium fenestratum 396 264 268 275 276 280 401 363 364 297 298* 372 384 402 320 342 343 345 (male: female)† 9:3 2:2 2:5 5:4 3:4 8:10 3:0 2:1 6:10 15:11 4:3 4:11 6:3 1:3 0:4 1:3 13:17 3:3 IL-1 0.02 0.02 IL 20i 0.02 IL 21hb 0.02 IL-2 0.08 IL-3 0.03 IL-4 0.02 IL-5 0.02 IL-6 0.17 0.02 IL-7 0.04 IL-8 0.04 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIL-2 0.04 IIIL-2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.13 0.43 0.07 0.55 1.00 0.50 0.66 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-7 0.06 IIIL-cmpd 0.08 IL 20i, additional (insertion) band in section 20 of the long arm of chromosome I; IL 21hb, heteroband in section 21 of the long arm of chromosome I; IIIL-cmpd, compound inversion, i.e. two overlapping inversions with unresolved inner breakpoints between outer section limits 94–96 of the long arm of chromosome III. *8 December 2013 only. †For sites 264, 268, 275, 276, 280, 401, 297, 298, 320 and 342, the number of larvae analysed for IIIL-2 is given here; the frequency of all other rearrangements is based on numbers in Table 1. View Large Table 3. Frequency of homologues with chromosome rearrangements in Simulium fenestratum at 18 sites in Thailand Site Simulium fenestratum 396 264 268 275 276 280 401 363 364 297 298* 372 384 402 320 342 343 345 (male: female)† 9:3 2:2 2:5 5:4 3:4 8:10 3:0 2:1 6:10 15:11 4:3 4:11 6:3 1:3 0:4 1:3 13:17 3:3 IL-1 0.02 0.02 IL 20i 0.02 IL 21hb 0.02 IL-2 0.08 IL-3 0.03 IL-4 0.02 IL-5 0.02 IL-6 0.17 0.02 IL-7 0.04 IL-8 0.04 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIL-2 0.04 IIIL-2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.13 0.43 0.07 0.55 1.00 0.50 0.66 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-7 0.06 IIIL-cmpd 0.08 Site Simulium fenestratum 396 264 268 275 276 280 401 363 364 297 298* 372 384 402 320 342 343 345 (male: female)† 9:3 2:2 2:5 5:4 3:4 8:10 3:0 2:1 6:10 15:11 4:3 4:11 6:3 1:3 0:4 1:3 13:17 3:3 IL-1 0.02 0.02 IL 20i 0.02 IL 21hb 0.02 IL-2 0.08 IL-3 0.03 IL-4 0.02 IL-5 0.02 IL-6 0.17 0.02 IL-7 0.04 IL-8 0.04 IL-9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIL-2 0.04 IIIL-2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.13 0.43 0.07 0.55 1.00 0.50 0.66 IIIL-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 IIIL-7 0.06 IIIL-cmpd 0.08 IL 20i, additional (insertion) band in section 20 of the long arm of chromosome I; IL 21hb, heteroband in section 21 of the long arm of chromosome I; IIIL-cmpd, compound inversion, i.e. two overlapping inversions with unresolved inner breakpoints between outer section limits 94–96 of the long arm of chromosome III. *8 December 2013 only. †For sites 264, 268, 275, 276, 280, 401, 297, 298, 320 and 342, the number of larvae analysed for IIIL-2 is given here; the frequency of all other rearrangements is based on numbers in Table 1. View Large All floating inversions were in low frequency (typically < 0.10; 0.17 for IL-6 in one small sample of three larvae), except IIIL-2, which varied in frequency with location (Table 3, Fig. 13). IIIL-2 was found in all populations, except two in Chan Tha Buri Province (sites 342 and 343). All specimens from Loei Province and Udon Thani Province and one site (402) in Uttaradit Province were homozygous for IIIL-2 (Fig. 8A), whereas other populations varied in frequency from 0.07 to 0.66 (Fig. 13). One population (Chiang Mai Province, site 297) large enough for statistical evaluation was in Hardy–Weinberg equilibrium for IIIL-2 (χ2 = 0, d.f. = 2, P > 0.05). A small sample of six larvae from Trat Province (site 345) had a deficiency of heterozygotes: four homozygous inverted (three female:one male), one homozygous standard (male) and one heterozygote (male). Figure 13. View largeDownload slide Frequency of IIIL-2 inversion (black, inverted; grey, standard) in 17 populations of Simulium fenestratum in Thailand. Figure 13. View largeDownload slide Frequency of IIIL-2 inversion (black, inverted; grey, standard) in 17 populations of Simulium fenestratum in Thailand. Simulium lampangense Takaoka & Choochote Simulium lampangense was restricted to limestone streams in northern Thailand. Of 53 larvae from two populations, Wang Kaew waterfall (type locality) and Wang Thong waterfall in Lampang Province, 42 (79.2%) were analysed completely. The banding pattern of this species differed from that of the S. multistriatum species group standard by three fixed inversions (IL-9, IIIL-2 and IIIL-4). The banding pattern of S. lampangense differed from that of S. fenestratum only by expression of the ‘2 blocks’ marker and fixation of IIIL-2. Based on nine larvae per location (seven to 18 nuclei per larva), an average of 46.7% of nuclei in larvae from Wang Kaew waterfall (site 408) and 30.1% from Wang Thong waterfall (site 409) had the ‘2 blocks’ marker with an unstained gap in the first block; the same condition as in S. chaliowae. Presence of the gap was positively related to the degree of polytenization. Thus, larger larvae, which exhibited a greater degree of polytenization, had a higher proportion of the ‘2 blocks’ marker with an unstained gap (Fig. 2C). The chromosomal banding pattern of S. lampangense was, therefore, identical to that of S. chaliowae in all respects. Simulium malayense Takaoka & Davies A total of 14 specimens of S. malayense was analysed completely: eight (80.0%) of ten larvae from Thailand and six (54.5%) of 11 larvae from Malaysia. The banding pattern of S. malayense was distinguished from that of the S. multistriatum species group standard by one fixed inversion (IIIL-10; Fig. 9B). Three cytoforms were found in S. malayense. Cytoforms A (seven larvae) and B (one larva) were found sympatrically in Thailand, and cytoform C (six larvae) was collected in Malaysia. Cytoform A was uniquely characterized by IS-2 (Fig. 2B) and ectopic pairing of CI and CIII in 1–60% of nuclei per larva. Cytoform B, consisting of only one male larva, expressed ectopic pairing that involved all combinations of the three centromere bands. It was collected in the same sample as cytoform A but was homozygous standard for IS. Cytoform C was characterized by a partial chromocentre involving CI and CII. CIII was well defined and darkly staining but did not participate in the chromocentric association. Simulium triglobus Kuvangkadilok & Takaoka This species was found at only one site, the type locality in Nan Province. A total of 28 (65.1%) of 43 larvae was analysed completely. All larvae had two fixed inversions (IL-9 and IIIL-4), but otherwise had the standard banding sequence for the group. However, the degree of flaring of the ends of chromosome arms IL, IIL, IIIL and IIIS was significantly greater for S. triglobus (14 larvae, four to ten nuclei per larva) than for S. fenestratum (13 larvae, four to ten nuclei per larva), which represented the standard flaring condition for the group (Mann–Whitney U test, P < 0.01, d.f. = 25; Table 4, Fig. 11D). One floating inversion (IIIL-9; Fig. 8C) appeared heterozygously in one male larva. Table 4. Median (range) of size classes for degree of terminal flaring of chromosomes of Simulium triglobus and Simulium fenestratum Species (N; larvae, nuclei) Size of flared end IL IIL IIIS IIIL Simulium triglobus (14, 83) 1 (0–3) 1 (0–3) 3 (1–3) 2 (0–3) Simulium fenestratum (13, 83) 0 (0–2) 0 (0–2) 1 (0–3) 1 (0–3) Species (N; larvae, nuclei) Size of flared end IL IIL IIIS IIIL Simulium triglobus (14, 83) 1 (0–3) 1 (0–3) 3 (1–3) 2 (0–3) Simulium fenestratum (13, 83) 0 (0–2) 0 (0–2) 1 (0–3) 1 (0–3) All medians differed between species except for IS and IIS (Mann–Whitney U test, using ranks, P < 0.01, d.f. = 165). View Large Table 4. Median (range) of size classes for degree of terminal flaring of chromosomes of Simulium triglobus and Simulium fenestratum Species (N; larvae, nuclei) Size of flared end IL IIL IIIS IIIL Simulium triglobus (14, 83) 1 (0–3) 1 (0–3) 3 (1–3) 2 (0–3) Simulium fenestratum (13, 83) 0 (0–2) 0 (0–2) 1 (0–3) 1 (0–3) Species (N; larvae, nuclei) Size of flared end IL IIL IIIS IIIL Simulium triglobus (14, 83) 1 (0–3) 1 (0–3) 3 (1–3) 2 (0–3) Simulium fenestratum (13, 83) 0 (0–2) 0 (0–2) 1 (0–3) 1 (0–3) All medians differed between species except for IS and IIS (Mann–Whitney U test, using ranks, P < 0.01, d.f. = 165). View Large Chromosomal relationships Our outgroup analysis indicated that the basic IS and IIIS sequences of the S. multistriatum species group, which are standard for the subgenus Simulium, carry no shared inversions with other analysed species groups of the subgenus. Although the S. striatum species group has multiple inversions in the base of IL, as do a number of other species groups in the subgenus Simulium (Adler et al., 2016a), none of the breakpoints is shared with the S. multistriatum species group. Thus, all rearrangements in IL are unique to the S. multistriatum species group. IIS-1 is shared with the S. malyschevi-reptans and S. striatum species groups, and IIS-3 is shared with the S. striatum species group (Table 5). The S. striatum and S. multistriatum species groups each have one additional, but distinctly different, fixed inversion in IIS. IIL-1 of the S. multistriatum species group is shared with the S. striatum species group. IIIL-5 of the S. multistriatum species group is shared with the S. nobile species group, in which it is referred to by Tangkawanit et al. (2011) as IIIL-b, and with the S. striatum species group. All other fixed IIIL inversions are unique to the S. multistriatum and S. striatum species groups. The position of the nucleolar organizer in the base of IS is shared with the S. striatum species group. Table 5. Matrix of 14 chromosomal rearrangements of the Simulium striatum, Simulium malyschevi-reptans and Simulium nobile groups (outgroups) and ten taxa in the Simulium multistriatum group in Thailand and Malaysia Rearrangements IL-9 IIS -1 IIS-2 IIS-3 IIL-1 IIIL-3 IIIL-4 IIIL-5 IIIL-6 IIIL-2 IIIL-10 Chromocentric Centromere association Position of nucleolar organizer in IS S. striatum group 0 1 0 1 1 0 0 1 0 0 0 0 0 1 S. malyschevi-reptans group 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S. nobile group 0 0 0 0 0 0 0 1 0 0 0 0 0 0 S. bullatum 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. chaliowae 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. chainarongi 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. daoense 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. fenestratum 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. lampangense 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. malayense A 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense B 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense C 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. triglobus 1 1 1 1 1 1 1 1 1 0 0 0 0 1 Rearrangements IL-9 IIS -1 IIS-2 IIS-3 IIL-1 IIIL-3 IIIL-4 IIIL-5 IIIL-6 IIIL-2 IIIL-10 Chromocentric Centromere association Position of nucleolar organizer in IS S. striatum group 0 1 0 1 1 0 0 1 0 0 0 0 0 1 S. malyschevi-reptans group 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S. nobile group 0 0 0 0 0 0 0 1 0 0 0 0 0 0 S. bullatum 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. chaliowae 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. chainarongi 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. daoense 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. fenestratum 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. lampangense 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. malayense A 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense B 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense C 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. triglobus 1 1 1 1 1 1 1 1 1 0 0 0 0 1 View Large Table 5. Matrix of 14 chromosomal rearrangements of the Simulium striatum, Simulium malyschevi-reptans and Simulium nobile groups (outgroups) and ten taxa in the Simulium multistriatum group in Thailand and Malaysia Rearrangements IL-9 IIS -1 IIS-2 IIS-3 IIL-1 IIIL-3 IIIL-4 IIIL-5 IIIL-6 IIIL-2 IIIL-10 Chromocentric Centromere association Position of nucleolar organizer in IS S. striatum group 0 1 0 1 1 0 0 1 0 0 0 0 0 1 S. malyschevi-reptans group 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S. nobile group 0 0 0 0 0 0 0 1 0 0 0 0 0 0 S. bullatum 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. chaliowae 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. chainarongi 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. daoense 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. fenestratum 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. lampangense 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. malayense A 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense B 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense C 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. triglobus 1 1 1 1 1 1 1 1 1 0 0 0 0 1 Rearrangements IL-9 IIS -1 IIS-2 IIS-3 IIL-1 IIIL-3 IIIL-4 IIIL-5 IIIL-6 IIIL-2 IIIL-10 Chromocentric Centromere association Position of nucleolar organizer in IS S. striatum group 0 1 0 1 1 0 0 1 0 0 0 0 0 1 S. malyschevi-reptans group 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S. nobile group 0 0 0 0 0 0 0 1 0 0 0 0 0 0 S. bullatum 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. chaliowae 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. chainarongi 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. daoense 1 1 1 1 1 1 1 1 1 0 0 0 0 1 S. fenestratum 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. lampangense 1 1 1 1 1 1 1 1 1 1 0 0 0 1 S. malayense A 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense B 0 1 1 1 1 1 0 1 1 0 1 0 1 1 S. malayense C 0 1 1 1 1 1 0 1 1 0 1 1 1 1 S. triglobus 1 1 1 1 1 1 1 1 1 0 0 0 0 1 View Large The evolutionary relationships in the S. multistriatum species group, based on shared rearrangements, show two main lineages (Fig. 14). The first lineage, consisting of six formally described species, was defined by IL-9 and IIIL-4. Within this lineage, a group of three nominal species (S. chainarongi, S. fenestratum and S. triglobus) is defined by IIIL-2, which was fixed in S. chaliowae and S. lampangense but polymorphic in S. fenestratum. Simulium bullatum and the three cytoforms of S. malayense formed the second primary lineage, defined by IIIL-10. All chromosomal features for the S. multistriatum group are summarized in idiograms (Fig. 15). Figure 14. View largeDownload slide Cytophylogeny of ten taxa in the Simulium multistriatum species group in Thailand (and Malaysia). Characters in brackets indicate possible environmental influence. C, centromere. Bootstrap values for maximum parsimony are shown above or near the branches. Figure 14. View largeDownload slide Cytophylogeny of ten taxa in the Simulium multistriatum species group in Thailand (and Malaysia). Characters in brackets indicate possible environmental influence. C, centromere. Bootstrap values for maximum parsimony are shown above or near the branches. Figure 15. View largeDownload slide Idiograms of ten cytoforms in the Simulium multistriatum species group, summarizing all chromosomal features relative to the Simulium subgeneric standard, including all fixed inversions (underlined and bracketed on the left side of the chromosomes) and autosomal polymorphisms (bracketed on the right side). C, centromere; Ch, chromocentre; M, marker; NO, nucleolar organizer; Pb, parabalbiani. Figure 15. View largeDownload slide Idiograms of ten cytoforms in the Simulium multistriatum species group, summarizing all chromosomal features relative to the Simulium subgeneric standard, including all fixed inversions (underlined and bracketed on the left side of the chromosomes) and autosomal polymorphisms (bracketed on the right side). C, centromere; Ch, chromocentre; M, marker; NO, nucleolar organizer; Pb, parabalbiani. Mitochondrial DNA sequence variation The COI and COII gene sequences were analysed for 69 specimens from nine taxa of the S. multistriatum species group in Thailand. The sequence length of the COI gene was 581 bp. There were 168 variable sites, of which 143 were parsimony informative. The maximum intraspecific genetic divergence based on the COI sequence (Table 6) was in S. daoense (5.60%), and the minimum values were in S. bullatum, S. chainarongi and S. chaliowae (0.10%). The minimum interspecific genetic divergence was between S. chaliowae and S. lampangense (1.30%). The maximum interspecific genetic divergence was between S. daoense and S. malayense cytoform C from Thailand (14.70%). Table 6. Range (mean) of intraspecific and interspecific genetic distances for mitochondrial cytochrome c oxidase I (COI) sequences of nine taxa in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam, based on Kimura 2-parameter Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.004 (0.001) S. chainarongi (CN) 0.088–0.090 (0.088) 0.00–0.002(0.001) S. chaliowae (CW) 0.092–0.097 (0.094) 0.036–0.042 (0.038) 0.000–0.004 (0.001) S. daoense (DA) 0.128–0.166 (0.145) 0.077–0.158 (0.112) 0.066–0.170 (0.112) 0.000–0.101 (0.056) S. fenestratum (FN) 0.083–0.095 (0.090) 0.021–0.042 (0.033) 0.006–0.032 (0.016) 0.058–0.202 (0.110) 0.004–0.026 (0.015) S. lampangense (LP) 0.086–0.095(0.092) 0.028–0.038(0.033) 0.004–0.019 (0.013) 0.056–0.170 (0.106) 0.002–0.026 (0.013) 0.000–0.016 (0.009) S. malayense cytoform A (MYA) 0.063–0.073 (0.068) 0.101–0.110 (0.105) 0.103–0.115 (0.109) 0.125–0.161 (0.144) 0.101–0.147 (0.110) 0.103–0.117 (0.108) 0.006–0.014 (0.013) S. malayense cytoform C(MYC) 0.021–0.050(0.026) 0.083–0.110(0.097) 0.088–0.114 (0.096) 0.123–0.187 (0.147) 0.077–0.112(0.091) 0.081–0.112(0.092) 0.058–0.097 (0.071) 0.004–0.031(0.014) S. triglobus (TB) 0.092–0.094(0.093) 0.093–0.102(0.097) 0.093–0.102(0.097) 0.093–0.172 (0.124) 0.088–0.106(0.097) 0.091–0.102(0.096) 0.110–0.115 (0.113) 0.090–0.118 (0.104) 0.002–0.012(0.007) Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.004 (0.001) S. chainarongi (CN) 0.088–0.090 (0.088) 0.00–0.002(0.001) S. chaliowae (CW) 0.092–0.097 (0.094) 0.036–0.042 (0.038) 0.000–0.004 (0.001) S. daoense (DA) 0.128–0.166 (0.145) 0.077–0.158 (0.112) 0.066–0.170 (0.112) 0.000–0.101 (0.056) S. fenestratum (FN) 0.083–0.095 (0.090) 0.021–0.042 (0.033) 0.006–0.032 (0.016) 0.058–0.202 (0.110) 0.004–0.026 (0.015) S. lampangense (LP) 0.086–0.095(0.092) 0.028–0.038(0.033) 0.004–0.019 (0.013) 0.056–0.170 (0.106) 0.002–0.026 (0.013) 0.000–0.016 (0.009) S. malayense cytoform A (MYA) 0.063–0.073 (0.068) 0.101–0.110 (0.105) 0.103–0.115 (0.109) 0.125–0.161 (0.144) 0.101–0.147 (0.110) 0.103–0.117 (0.108) 0.006–0.014 (0.013) S. malayense cytoform C(MYC) 0.021–0.050(0.026) 0.083–0.110(0.097) 0.088–0.114 (0.096) 0.123–0.187 (0.147) 0.077–0.112(0.091) 0.081–0.112(0.092) 0.058–0.097 (0.071) 0.004–0.031(0.014) S. triglobus (TB) 0.092–0.094(0.093) 0.093–0.102(0.097) 0.093–0.102(0.097) 0.093–0.172 (0.124) 0.088–0.106(0.097) 0.091–0.102(0.096) 0.110–0.115 (0.113) 0.090–0.118 (0.104) 0.002–0.012(0.007) View Large Table 6. Range (mean) of intraspecific and interspecific genetic distances for mitochondrial cytochrome c oxidase I (COI) sequences of nine taxa in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam, based on Kimura 2-parameter Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.004 (0.001) S. chainarongi (CN) 0.088–0.090 (0.088) 0.00–0.002(0.001) S. chaliowae (CW) 0.092–0.097 (0.094) 0.036–0.042 (0.038) 0.000–0.004 (0.001) S. daoense (DA) 0.128–0.166 (0.145) 0.077–0.158 (0.112) 0.066–0.170 (0.112) 0.000–0.101 (0.056) S. fenestratum (FN) 0.083–0.095 (0.090) 0.021–0.042 (0.033) 0.006–0.032 (0.016) 0.058–0.202 (0.110) 0.004–0.026 (0.015) S. lampangense (LP) 0.086–0.095(0.092) 0.028–0.038(0.033) 0.004–0.019 (0.013) 0.056–0.170 (0.106) 0.002–0.026 (0.013) 0.000–0.016 (0.009) S. malayense cytoform A (MYA) 0.063–0.073 (0.068) 0.101–0.110 (0.105) 0.103–0.115 (0.109) 0.125–0.161 (0.144) 0.101–0.147 (0.110) 0.103–0.117 (0.108) 0.006–0.014 (0.013) S. malayense cytoform C(MYC) 0.021–0.050(0.026) 0.083–0.110(0.097) 0.088–0.114 (0.096) 0.123–0.187 (0.147) 0.077–0.112(0.091) 0.081–0.112(0.092) 0.058–0.097 (0.071) 0.004–0.031(0.014) S. triglobus (TB) 0.092–0.094(0.093) 0.093–0.102(0.097) 0.093–0.102(0.097) 0.093–0.172 (0.124) 0.088–0.106(0.097) 0.091–0.102(0.096) 0.110–0.115 (0.113) 0.090–0.118 (0.104) 0.002–0.012(0.007) Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.004 (0.001) S. chainarongi (CN) 0.088–0.090 (0.088) 0.00–0.002(0.001) S. chaliowae (CW) 0.092–0.097 (0.094) 0.036–0.042 (0.038) 0.000–0.004 (0.001) S. daoense (DA) 0.128–0.166 (0.145) 0.077–0.158 (0.112) 0.066–0.170 (0.112) 0.000–0.101 (0.056) S. fenestratum (FN) 0.083–0.095 (0.090) 0.021–0.042 (0.033) 0.006–0.032 (0.016) 0.058–0.202 (0.110) 0.004–0.026 (0.015) S. lampangense (LP) 0.086–0.095(0.092) 0.028–0.038(0.033) 0.004–0.019 (0.013) 0.056–0.170 (0.106) 0.002–0.026 (0.013) 0.000–0.016 (0.009) S. malayense cytoform A (MYA) 0.063–0.073 (0.068) 0.101–0.110 (0.105) 0.103–0.115 (0.109) 0.125–0.161 (0.144) 0.101–0.147 (0.110) 0.103–0.117 (0.108) 0.006–0.014 (0.013) S. malayense cytoform C(MYC) 0.021–0.050(0.026) 0.083–0.110(0.097) 0.088–0.114 (0.096) 0.123–0.187 (0.147) 0.077–0.112(0.091) 0.081–0.112(0.092) 0.058–0.097 (0.071) 0.004–0.031(0.014) S. triglobus (TB) 0.092–0.094(0.093) 0.093–0.102(0.097) 0.093–0.102(0.097) 0.093–0.172 (0.124) 0.088–0.106(0.097) 0.091–0.102(0.096) 0.110–0.115 (0.113) 0.090–0.118 (0.104) 0.002–0.012(0.007) View Large The sequence length of the COII gene was 697 bp, with 235 variable sites, of which 184 were parsimony informative. Maximum intraspecific genetic divergence based on the COII sequence (Table 7) was in S. daoense (3.30%), and the lowest intraspecific genetic divergence was in S. chaliowae (0.40%). The maximum interspecific genetic divergence was between S. lampangense and S. malayense cytoform A, and S. malayense cytoform A and S. malayense cytoform C (16.00%), and the minimum interspecific genetic divergence was between S. chaliowae and S. lampangense (1.20%). Table 7. Range (mean) of intraspecific and interspecific genetic distances for mitochondrial cytochrome c oxidase II (COII) sequences of nine taxa in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam, based on Kimura 2-parameter Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.012 (0.007) S. chainarongi (CN) 0.092–0.137 (0.105) 0.002–0.038 (0.019) S. chaliowae (CW) 0.074–0.087 (0.081) 0.034–0.074 (0.048) 0.000–0.006 (0.004) S. daoense (DA) 0.121–0.147 (0.134) 0.054–0.090 (0.067) 0.082–0.135 (0.107) 0.029–0.060 (0.033) S. fenestratum (FN) 0.081–0.119 (0.095) 0.016–0.063 (0.033) 0.010–0.053 (0.029) 0.044–0.118 (0.072) 0.004–0.040 (0.019) S. lampangense (LP) 0.084–0.103 (0.095) 0.018–0.067 (0.038) 0.003–0.020 (0.012) 0.046–0.083 (0.061) 0.014–0.044 (0.025) 0.000–0.028 (0.010) S. malayense cytoform A (MYA) 0.094–0.115 (0.105) 0.114–0.134 (0.123) 0.148–0.167 (0.158) 0.145–0.170 (0.158) 0.116–0.168 (0.138) 0.154–0.167 (0.160) 0.006–0.017 (0.012) S. malayense cytoform C (MYC) 0.032–0.055 (0.046) 0.096–0.137 (0.110) 0.098–0.119 (0.109) 0.072–0.089 (0.081) 0.094–0.123 (0.106) 0.092–0.116 (0.105) 0.154–0.167 (0.160) 0.010–0.034 (0.020) S. triglobus (TB) 0.114–0.152(0.127) 0.072–0.105 (0.090) 0.098–0.132 (0.111) 0.072–0.098 (0.083) 0.078–0.123 (0.098) 0.087–0.130 (0.106) 0.132–0.161 (0.144) 0.114–0.146 (0.127) 0.002–0.026 (0.015) Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.012 (0.007) S. chainarongi (CN) 0.092–0.137 (0.105) 0.002–0.038 (0.019) S. chaliowae (CW) 0.074–0.087 (0.081) 0.034–0.074 (0.048) 0.000–0.006 (0.004) S. daoense (DA) 0.121–0.147 (0.134) 0.054–0.090 (0.067) 0.082–0.135 (0.107) 0.029–0.060 (0.033) S. fenestratum (FN) 0.081–0.119 (0.095) 0.016–0.063 (0.033) 0.010–0.053 (0.029) 0.044–0.118 (0.072) 0.004–0.040 (0.019) S. lampangense (LP) 0.084–0.103 (0.095) 0.018–0.067 (0.038) 0.003–0.020 (0.012) 0.046–0.083 (0.061) 0.014–0.044 (0.025) 0.000–0.028 (0.010) S. malayense cytoform A (MYA) 0.094–0.115 (0.105) 0.114–0.134 (0.123) 0.148–0.167 (0.158) 0.145–0.170 (0.158) 0.116–0.168 (0.138) 0.154–0.167 (0.160) 0.006–0.017 (0.012) S. malayense cytoform C (MYC) 0.032–0.055 (0.046) 0.096–0.137 (0.110) 0.098–0.119 (0.109) 0.072–0.089 (0.081) 0.094–0.123 (0.106) 0.092–0.116 (0.105) 0.154–0.167 (0.160) 0.010–0.034 (0.020) S. triglobus (TB) 0.114–0.152(0.127) 0.072–0.105 (0.090) 0.098–0.132 (0.111) 0.072–0.098 (0.083) 0.078–0.123 (0.098) 0.087–0.130 (0.106) 0.132–0.161 (0.144) 0.114–0.146 (0.127) 0.002–0.026 (0.015) View Large Table 7. Range (mean) of intraspecific and interspecific genetic distances for mitochondrial cytochrome c oxidase II (COII) sequences of nine taxa in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam, based on Kimura 2-parameter Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.012 (0.007) S. chainarongi (CN) 0.092–0.137 (0.105) 0.002–0.038 (0.019) S. chaliowae (CW) 0.074–0.087 (0.081) 0.034–0.074 (0.048) 0.000–0.006 (0.004) S. daoense (DA) 0.121–0.147 (0.134) 0.054–0.090 (0.067) 0.082–0.135 (0.107) 0.029–0.060 (0.033) S. fenestratum (FN) 0.081–0.119 (0.095) 0.016–0.063 (0.033) 0.010–0.053 (0.029) 0.044–0.118 (0.072) 0.004–0.040 (0.019) S. lampangense (LP) 0.084–0.103 (0.095) 0.018–0.067 (0.038) 0.003–0.020 (0.012) 0.046–0.083 (0.061) 0.014–0.044 (0.025) 0.000–0.028 (0.010) S. malayense cytoform A (MYA) 0.094–0.115 (0.105) 0.114–0.134 (0.123) 0.148–0.167 (0.158) 0.145–0.170 (0.158) 0.116–0.168 (0.138) 0.154–0.167 (0.160) 0.006–0.017 (0.012) S. malayense cytoform C (MYC) 0.032–0.055 (0.046) 0.096–0.137 (0.110) 0.098–0.119 (0.109) 0.072–0.089 (0.081) 0.094–0.123 (0.106) 0.092–0.116 (0.105) 0.154–0.167 (0.160) 0.010–0.034 (0.020) S. triglobus (TB) 0.114–0.152(0.127) 0.072–0.105 (0.090) 0.098–0.132 (0.111) 0.072–0.098 (0.083) 0.078–0.123 (0.098) 0.087–0.130 (0.106) 0.132–0.161 (0.144) 0.114–0.146 (0.127) 0.002–0.026 (0.015) Species BUL CN CW DA FN LP MYA MYC TB S. bullatum (BUL) 0.000–0.012 (0.007) S. chainarongi (CN) 0.092–0.137 (0.105) 0.002–0.038 (0.019) S. chaliowae (CW) 0.074–0.087 (0.081) 0.034–0.074 (0.048) 0.000–0.006 (0.004) S. daoense (DA) 0.121–0.147 (0.134) 0.054–0.090 (0.067) 0.082–0.135 (0.107) 0.029–0.060 (0.033) S. fenestratum (FN) 0.081–0.119 (0.095) 0.016–0.063 (0.033) 0.010–0.053 (0.029) 0.044–0.118 (0.072) 0.004–0.040 (0.019) S. lampangense (LP) 0.084–0.103 (0.095) 0.018–0.067 (0.038) 0.003–0.020 (0.012) 0.046–0.083 (0.061) 0.014–0.044 (0.025) 0.000–0.028 (0.010) S. malayense cytoform A (MYA) 0.094–0.115 (0.105) 0.114–0.134 (0.123) 0.148–0.167 (0.158) 0.145–0.170 (0.158) 0.116–0.168 (0.138) 0.154–0.167 (0.160) 0.006–0.017 (0.012) S. malayense cytoform C (MYC) 0.032–0.055 (0.046) 0.096–0.137 (0.110) 0.098–0.119 (0.109) 0.072–0.089 (0.081) 0.094–0.123 (0.106) 0.092–0.116 (0.105) 0.154–0.167 (0.160) 0.010–0.034 (0.020) S. triglobus (TB) 0.114–0.152(0.127) 0.072–0.105 (0.090) 0.098–0.132 (0.111) 0.072–0.098 (0.083) 0.078–0.123 (0.098) 0.087–0.130 (0.106) 0.132–0.161 (0.144) 0.114–0.146 (0.127) 0.002–0.026 (0.015) View Large COII was more effective than COI for differentiating members of the S. multistriatum species group (Table 8). Identifications based on best match for COI were 73.9% correct (51 of 69 sequences), with 14.5% (ten of 69 sequences) misidentifications and 11.6% (eight of 69 sequences) ambiguous identifications. Identifications based on COII were 97.1% (67 of 69 sequences) correct, with 2.9% (two of 69 sequences) misidentifications and no ambiguous identifications. Table 8. Nucleotide sequence statistics based on COI and COII of nine taxa of black flies in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam Gene Number of taxa Number of sequences Variable sites (V) Parsimony-informative sites (Pi) Length of sequences (bp) Percentage of correct identifications (N) Percentage of misidentifications (N) Percentage ambiguous (N) COI 9 69 168 143 581 73.9% (51) 14.5% (10) 11.6% (8) COII 9 69 235 184 697 97.1% (67) 2.9% (2) 0 Gene Number of taxa Number of sequences Variable sites (V) Parsimony-informative sites (Pi) Length of sequences (bp) Percentage of correct identifications (N) Percentage of misidentifications (N) Percentage ambiguous (N) COI 9 69 168 143 581 73.9% (51) 14.5% (10) 11.6% (8) COII 9 69 235 184 697 97.1% (67) 2.9% (2) 0 Percentage of correct identification is based on best match method in TaxonDNA (Meier et al. 2006). View Large Table 8. Nucleotide sequence statistics based on COI and COII of nine taxa of black flies in the Simulium multistriatum species group in Thailand, Malaysia and Vietnam Gene Number of taxa Number of sequences Variable sites (V) Parsimony-informative sites (Pi) Length of sequences (bp) Percentage of correct identifications (N) Percentage of misidentifications (N) Percentage ambiguous (N) COI 9 69 168 143 581 73.9% (51) 14.5% (10) 11.6% (8) COII 9 69 235 184 697 97.1% (67) 2.9% (2) 0 Gene Number of taxa Number of sequences Variable sites (V) Parsimony-informative sites (Pi) Length of sequences (bp) Percentage of correct identifications (N) Percentage of misidentifications (N) Percentage ambiguous (N) COI 9 69 168 143 581 73.9% (51) 14.5% (10) 11.6% (8) COII 9 69 235 184 697 97.1% (67) 2.9% (2) 0 Percentage of correct identification is based on best match method in TaxonDNA (Meier et al. 2006). View Large Molecular phylogenetic relationships Phylogenetic analyses were conducted for COI, COII and the combined dataset (COI + COII). All phylogenetic analysis methods (NJ, MP, ML and Bayesian) revealed similar tree topologies; thus, only NJ trees are shown. The NJ tree based on COI sequences revealed two major clades (Fig. 16). Simulium chainarongi, S. chaliowae, S. daoense, S. fenestratum, S. lampangense and S. triglobus formed clade I. Simulium fenestratum and S. lampangense were paraphyletic, whereas S. chainarongi and S. chaliowae were monophyletic, although they nested within S. fenestratum. All members of S. daoense from Thailand and Vietnam were clustered in the same clade with moderate bootstrap support (> 71%). Clade II comprised S. bullatum, S. malayense cytoform A from Thailand and S. malayense cytoform C from Malaysia. Simulium bullatum and S. malayense cytoforms A and C in clade II were monophyletic with strong support. Simulium malayense cytoform B was not available for molecular analysis. Figure 16. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on COI sequences. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. Figure 16. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on COI sequences. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. The phylogenetic analysis based on the COII gene (Fig. 17) provided better resolution than did the COI gene. The COII sequences revealed two main clades. Clade I comprised six species, namely S. chainarongi, S. chaliowae, S. daoense, S. fenestratum, S. lampangense and S. triglobus. All species in this clade were monophyletic, except S. chaliowae and S. lampangense. Simulium chainarongi nested within S. fenestratum. All specimens of S. lampangense formed a single well-supported clade with S. chaliowae. Clade II was composed of S. bullatum, S. malayense cytoform A from Thailand and cytoform C from Malaysia. Figure 17. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on COII sequences. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. Figure 17. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on COII sequences. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. The phylogenetic tree based on the combined data (Fig. 18) showed two main clades similar to the trees derived from the COI and COII sequences. Simulium chainarongi, S. chaliowae, S. daoense, S. fenestratum, S. lampangense and S. triglobus each formed a monophyletic cluster within clade I. Simulium lampangense formed a single well-supported subclade with S. chaliowae in clade I. Simulium chainarongi, however, nested within S. fenestratum. Simulium bullatum and S. malayense cytoforms A and C formed a monophyletic cluster within clade II. Figure 18. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on combined (COI + COII) data set. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. Figure 18. View largeDownload slide Neighbor-joining (NJ) tree for nine species in Simulium multistriatum species group from Thailand, based on combined (COI + COII) data set. Bootstrap values of neighbor-joining, maximum parsimony (MP) and maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BA) are shown above or near branches. Scale bar represents 0.01 substitutions per nucleotide position. Chromosomal identification key for members of the Simulium multistriatum species group in Thailand and Malaysia 1. Centromere bands associated in > 50% of nuclei (e.g. Fig. 10). IL-9 absent (Fig. 3), IIIL-10 present (Fig. 9B) 2 Centromere bands associated in < 10% of nuclei. IL-9 present, IIIL-10 absent 5 2. True chromocentre with extra heterochromatin present (Fig. 10A) Simulium bullatum Partial chromocentre (Fig. 10B) or ectopic pairing present 3 3. Ectopic pairing of all combinations of centromere bands (CI, CII and CIII) Simulium malayense cytoform B (Thailand) Partial chromocentre or ectopic pairing of only two centromere bands 4 4. IS-2 present (Fig. 2B). Ectopic pairing involving centromeres I and III in 1–60% of nuclei. …………………… Simulium malayense cytoform A (Thailand) IS-2 absent. Partial chromocentre (Fig. 10B) present, with extra heterochromatin, involving centromeres CI and CII. .Simulium malayense cytoform C (Malaysia) 5. Centromere bands well defined, darkly stained (Fig. 12A) Simulium chainarongi Centromere bands weakly defined, lightly stained (Fig. 12B) 6 6. Flaring of ends of IL, IIL, IIIL and IIIS > 1.5 times wider than respective indicator bands (Fig. 11D) Simulium triglobus Flaring of ends of all chromosome arms < 1.5 times wider than respective indicator bands (Fig. 11C) 7 7. ‘2 blocks’ marker appearing as 3 blocks in at least some nuclei (Fig. 2C). IIIL-2 fixed (Fig. 8A) Simulium chaliowae, Simulium lampangense ‘2 blocks’ marker standard in all nuclei (Fig. 2B). IIIL-2 polymorphic 8 8. IIIL-1 present (Fig. 9A) Simulium daoense IIIL-1 absent Simulium fenestratum DISCUSSION Taxonomic status of chromosomal entities Our chromosomal study recognizes ten distinct taxa among eight nominal morphospecies in the S. multistriatum species group. Chromosomal analyses confirm reproductive isolation (species status), via absence of hybrids, of eight species, reveal two undescribed taxa (S. malayense cytoforms A and B), and suggest possible conspecificity of at least two nominal species (S. chaliowae and S. lampangense) and possible cryptic species in another (S. fenestratum). The species status of S. bullatum separate from all other studied species is supported chromosomally by the presence of a large chromocentre. Morphologically, the large unpigmented organ of its pupal gill is unique (Takaoka & Choochote, 2005b). Molecular analyses also support its distinction from other species, with K2P genetic distances ranging from 2.60 to 14.50% for COI and from 4.60 to 15.60% for COII. Molecular phylogenetic analysis shows that all specimens of S. bullatum form a monophyletic group, in agreement with a previous molecular study based on the COI and ECP1 genes (Thaijarern et al., 2017). Species status is tentatively suggested for the three cytoforms of S. malayense. The presence of IS-2 in homozygous and standard arrangements (with no heterozygotes), coupled with unique centromeric associations, in sympatry suggests that cytoforms A and B are distinct species. This suggestion, however, is tempered by an inadequate sample size. Molecular analysis corroborates chromosomal data that S. malayense cytoform C is genetically distinct from A. However, the allopatric distribution of A relative to C does not permit a strict evaluation of species status; A and B are ~1605 km distant from C. The collection site of cytoform C is < 100 km from the type locality of S. malayense and, therefore, is the best candidate for typical S. malayense. Simulium chainarongi is homosequential with S. fenestratum and S. triglobus but has unique centromeric expression and polymorphisms. Differential expression of centromere bands is not uncommon in closely related species of black flies (Bedo, 1975). Although S. chainarongi is difficult to separate morphologically from some species in the S. multistriatum species group (Takaoka & Kuvangkadilok, 1999), its unique centromeric expression, coupled with molecular and ecological data (i.e. low-elevation habitats, < 250 m), support its species status. Gene sequences indicate that S. chainarongi is monophyletic, with interspecific genetic distances ranging from 3.30 to 11.20% for COI and from 3.30 to 12.30% for COII. Its status as a species distinct from S. fenestratum, however, is questionable based on COI and COII sequences, which depict it nesting within S. fenestratum. Given the geographical separation of our samples of the two taxa (~330 km), additional study is needed to test their current status as distinct species. Simulium daoense differs by only one fixed inversion (IIIL-1) from S. chainarongi, S. fenestratum and S. triglobus. Our chromosomal analysis shows the presence of S. daoense in Thailand (Chiang Mai Province) for the first time. The Thai population is chromosomally identical with S. daoense from the type locality in Vietnam ~700 km distant. The type locality of S. daoense is at high elevation (1315 m), similar to the location in Thailand (1304 m). The stream width at the type locality is small (0.5 m) and the water temperature cold (7.0 °C), whereas in Thailand, the stream is wider (5 m) and the water temperature warmer (15.7 °C). The COI and COII sequences place S. daoense from Thailand and Vietnam together, with maximum intraspecific genetic divergence of 5.60% for COI and 3.30% for COII. The chromosomes suggest that the two populations are a single species, but environmental effects and geographical distance might be driving the molecular differentiation between the two populations. Geographical distance is the main factor limiting gene flow between populations of many black fly species (Pramual et al., 2005), particularly those inhabiting high-elevation areas (Finn & Adler, 2006; Finn et al., 2006; Pramual & Wongpakam, 2013). The banding pattern of S. triglobus is homosequential with that of S. chainarongi and S. fenestratum, but differs from that of S. chainarongi by standard centromere band expression and from both species by enhanced terminal flaring of the chromosome arms. Typically, the chromosomal ends would be heterochromatinized via duplication processes related to repeat DNA elements and perhaps heterochromatic genes. Thus, when the ends flare, some form of expression is probably occurring with regard to these elements or genes. Gene expression is necessary to produce the appropriate proteins for development in particular environments (Gottlieb, 1998). The enhanced flaring of S. triglobus might be associated with conditions in calcareous streams, such as high calcium carbonate, pH and conductivity. Although the effect of calcium carbonate on genomic expression has not been investigated for black flies, some evidence suggests an indirect effect on the structure of polytene chromosomes in larval chironomids via surface adsorption of heavy metals on marl surfaces (Jabłońska-Barna, Szarek-Gwiazda & Michailova, 2013). If terminal flaring of chromosomes is environmentally influenced, no chromosomal evidence is available to support species status of S. triglobus. Morphological support for the species status of S. triglobus separate from S. chainarongi and S. fenestratum is based primarily on the more branched thoracic trichomes and corbicular cocoon of the pupa, lack of dorsolateral protuberances on the larval abdomen, and three (vs. one) spermathecae in females of S. triglobus (Takaoka & Kuvangkadilok, 1999). Molecular analysis based on COI and COII support the unique, albeit weak chromosomal feature (flaring); all specimens of S. triglobus from the type locality form a monophyletic group with strong support (100%), agreeing with a previous DNA barcode tree by Pramual & Wongpakam (2014). This species also shows a high level of interspecific molecular genetic differentiation (K2P genetic distance: 9.30–12.40% for COI and 8.30–14.40% for COII). The banding patterns and chromosomal characteristics of S. chaliowae and S. lampangense are identical, and both species are known only from limestone streams. The unique expression of the ‘2 blocks’ marker is positively related to the degree of polytenization and might represent gene expression related to environmental influence from the calcareous streams they inhabit, as discussed for S. triglobus. The COII sequence and the combined COI + COII data set show a close relationship between S. chaliowae and S. lampangense, with low interspecific genetic divergence (1.20% for COII). The results agree with those based on the ECP1 gene, which found that S. chaliowae and S. lampangense are closely related but fall into separate clusters (Thaijarern et al., 2017). The molecular difference might be a site effect, rather than a species effect. Known locations of S. chaliowae are ~130–150 km from those of S. lampangense. If these taxa are restricted to calcareous streams, which have patchy distributions (Pramual & Pangjanda, 2015), the implication is that females tend to return to their natal streams to oviposit, enhancing the build-up of location-specific genetic differences. Morphologically, the larvae differ only by the presence of dorsal protuberances on abdominal segments two to six in S. chaliowae vs. three to seven in S. lampangense (Thaijarern et al., 2017); however, this character varies intraspecifically in some members of the S. multistriatum species group (Takaoka & Kuvangkadilok, 1999; Takaoka & Choochote, 2005a). For example, some populations of S. fenestratum have dorsal protuberances, whereas others do not (J. Thaijarern and P. Pramual, unpublished data). The cocoon of S. lampangense is fenestrated and either slipper or shoe shaped, whereas that of S. chaliowae is unfenestrated and shoe shaped (Takaoka & Kuvangkadilok, 1999; Takaoka & Choochote, 2005a). However, the presence of windows in the cocoon can vary intraspecifically in other simuliid species (Adler et al., 2004). Differences in the adults are based on minor characteristics, such as the length-to-width ratio of the anal lobe (Takaoka & Kuvangkadilok, 1999; Takaoka & Choochote, 2005a). Morphological differences might also be location specific. Thus, the evidence that S. chaliowae and S. lampangense are distinct species is weak, suggesting that S. lampangense might be a junior synonym of S. chaliowae. Until evidence to the contrary can be adduced, we continue to recognize S. chaliowae and S. lampangense as separate species, based on recovery of distinct clusters in molecular phylogenies using the ECP1 (Thaijarern et al., 2017) and COII genes, and minor morphological differences. Thus, they would be nearly homosequential cryptic species. The chromosomes of S. chaliowae and S. lampangense are fundamentally the same as those in populations of S. fenestratum homozygous for the IIIL-2 inversion. Larvae and pupae of S. chaliowae and S. lampangense have conventionally been distinguished from S. fenestratum by body colour, dorsal protuberances on the abdomen, and shape of the cocoon (Takaoka & Kuvangkadilok, 1999). These characters, however, are subject to intraspecific variation. The male of S. chaliowae is similar to that of S. fenestratum but is distinguished by the horn-like basal protuberance of the gonostylus with many teeth along the anterior margin (Takaoka & Kuvangkadilok, 1999), and S. lampangense can be distinguished from S. fenestratum by having a bare radial vein in females and several spines on the basal protuberance of the gonostylus (Takaoka & Choochote, 2005a); S. fenestratum has one apical spine on the basal protuberance (Takaoka, 1977). Although the ECP1 gene separates S. fenestratum from S. chaliowae and S. lampangense with strong support, the COI gene groups some populations of S. fenestratum in a clade with S. chaliowae (Pramual & Wongpakam, 2014; Thaijarern et al., 2017). Our chromosomal results provide some indication that S. fenestratum consists of cryptic species. A previous report of cryptic species (i.e. chromocentric individuals) in S. fenestratum (Pramual & Nanork, 2012) in fact pertains to S. bullatum. Our evidence for cryptic species of S. fenestratum involves the IIIL-2 inversion. The existence of populations homozygous standard, homozygous inverted and polymorphic for this inversion presents four hypotheses: (1) a single polymorphic species, with IIIL-2 perhaps reflecting local adaptation; (2) two species, one fixed for IIIL-2 and one polymorphic for IIIL-2; (3) two species, one homozygous standard and one polymorphic for IIIL-2; and (4) three species, one fixed for IIIL-2, one fixed for standard and one polymorphic. This same scenario has been found in the S. tani complex in Thailand, with all four possibilities (although involving other inversions), representing different cytoforms (Tangkawanit et al., 2009). A population in Trat Province where individuals homozygous for IIIL-2 and for standard were found, with a dearth of heterozygotes, might provide a test for cryptic species if larger samples are available to test the inversion for Hardy–Weinberg equilibrium or to conduct a molecular evaluation. The biogeographical pattern for IIIL-2 in S. fenestratum is similar to that for inversions in Simulium aureohirtum and Simulium tani s.l., which increase in frequency with latitude (Pramual et al., 2005; Pramual, Wongpakam & Kuvangkadilok, 2008). Phylogenetic relationships Monophyly of the S. multistriatum species group, based on species from Thailand, is demonstrated on the basis of four shared chromosomal rearrangements (IIS-2, IIIL-3, IIIL-5 and IIIL-6), providing a framework for further testing of the other 25 known species (Adler & Crosskey, 2017) in the group. The chromosomal characters should prove especially useful for evaluating group membership of species such as S. takense, which does not cluster with the S. multistriatum species group on the basis of molecular evidence (COI, COII and 18S/ITS; Pramaul & Nanork, 2012; Pramual & Adler, 2014). Chromosomal characters suggest that the S. multistriatum group is most closely related to the S. striatum group and that these two groups, in turn, are related to the S. malyschevi-reptans and S. nobile groups. This cluster of species groups finds morphological support based on the presence of fenestrated cocoons. Although the fenestra can be lost in some species (Moulton & Adler, 1995), their presence is strongly associated with these groups. If this structural character has phylogenetic value, we would expect the S. griseifrons species group s.l. (including its recent divisions; Takaoka, 2017) also to be a member of this clade; to date, no chromosomal information is available for S. griseifrons and its relatives. The chromosomal phylogeny showing that species of the S. multistriatum species group in Thailand fall into two distinct groups based on three fixed chromosome inversions (IL-9, IIIL-4 and IIIL-10) and centromere associations agrees with phylogenetic analysis based on COI and COII gene sequences: S. bullatum and S. malayense cytoforms A, B and C form one clade, and S. chainarongi, S. chaliowae, S. daoense, S. fenestratum, S. lampangense and S. triglobus form another clade. These results agree with a previous DNA-barcode tree (Pramual & Wongpakam, 2014). A paucity of shared structural characters does not permit inference of a morphological phylogeny. The chromosomal evidence that the S. multistriatum group is most closely related to the S. striatum species group agrees with some molecular analyses (Thanwisai et al., 2006) but not others (Otsuka et al., 2003; Phayuhasena et al., 2010; Pramual & Adler, 2014). Speciation in the S. multistriatum species group Limited morphological differentiation is found among the species in the S. multistriatum group, consistent with the low levels of cytogenetic differentiation. Only 30 rearrangements, other than those common to the basic sequence, have been found in this species group in Thailand. This number is low compared with other Southeast Asian species groups, such as the S. tuberosum species group in Vietnam, with 88 rearrangements (Adler et al., 2016a). Speciation in the Simuliidae is typically associated with chromosomal phenomena, particularly coadaptation of sex chromosomes, cooption of individual rearrangements for different roles in different lineages and, more rarely, larger genomic restructuring events (Rothfels, 1989; Adler et al., 2016c). Speciation in the S. multistriatum group, however, is largely not associated with the typical chromosomal phenomena in the Simuliidae. The sex chromosomes, for example, are undifferentiated in all taxa in our study. Only one inversion, IIIL-2, functions in multiple roles: fixed in S. chaliowae and S. lampangense and polymorphic in S. fenestratum. Sharing of the IIIL-2 inversion suggests that S. fenestratum, S. chaliowae and S. lampangense are derived from a common ancestor. Accordingly, this inversion would have been polymorphic in the ancestor, remained so in S. fenestratum, and become fixed in S. chaliowae and S. lampangense. The latter two species occur only in a specific habitat, calcareous streams; thus, fixation of IIIL-2 might be associated with habitat specialization. Chromosomal and molecular study of another calcareous stream specialist, S. weji, in Thailand suggests that females return to their natal sites to oviposit (Pramual & Pangjanda, 2015). If this scenario also applies to S. chaliowae and S. lampangense, inbreeding could occur and facilitate the fixation of IIIL-2 in the populations. The low molecular diversity in the COI and COII genes of S. chaliowae and S. lampangense supports this possibility. Rather than the typical chromosomal rearrangements associated with simuliid speciation (e.g. inversions), other chromosomal phenomena occur in the S. multistriatum species group, including centromere associations with and without heterochromatinization (S. bullatum and S. malayense cytoforms A, B and C). Centromere associations occur throughout the Simuliidae in > 12% of all species (Adler et al., 2010). Heterochromatinization within the genome can serve as a driving force in speciation (Ferree & Barbasha, 2009). The chromosomal and molecular phylogenetic clustering of the three types of centromeric associations (i.e. ectopic pairing, partial chromocentre and chromocentre) suggests a common origin. The simplest form of centric association, ectopic pairing, is a frequent phenomenon in diverse species, and is often restricted to certain populations of a species, although it is not necessarily expressed in all individuals or even in all nuclei of an individual (Rothfels & Freeman, 1977). The origin of heterochromatinization is inferred to be a result of over-replication of repetitive elements (Thapa et al., 2014). An occasional tendency for centromeres to associate ectopically might represent the first step in acquisition of a permanent, species-specific chromocentre or partial chromocentre, with the next step being addition of heterochromatin. A similar progression from non-chromocentric to fully chromocentric, via ectopic pairing (pseudochromocentre), has also been proposed for some members of the Simulium vernum species group, of which at least one species exhibits chromocentre polymorphism (Brockhouse, Bass & Straus, 1989). The partial chromocentric state, which is typically a species-specific trait, might result from the loss of repetitive DNA sequences responsible for the chromocentre from only one of the three chromosomes (Brockhouse et al., 1989). ACKNOWLEDGEMENTS This work was funded by the Thailand Research Fund (TRF) special programme for the Royal Golden Jubilee (RGJ) Ph.D. Program and Mahasarakham University. We thank C. E. Beard of the Clemson University Cherry Farm Insectary, where J.T. worked in the laboratory of P.H.A. for 11 months in 2016–2017. We are grateful to Zubaidah Ya’cob (University of Malaya) for providing specimens of S. malayense C from Malaysia and to Professor Hiroyuki Takaoka for graciously providing larvae of S. daoense from Vietnam. REFERENCES Adler PH , Cheke RA , Post RJ . 2010 . Evolution, epidemiology, and population genetics of black flies (Diptera: Simuliidae) . Infection, Genetics and Evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 10 : 846 – 865 . Google Scholar CrossRef Search ADS Adler PH , Crosskey RW . 2015 . Cytotaxonomy of the Simuliidae (Diptera): a systematic and bibliographic conspectus . Zootaxa 3975 : 1 – 139 . Google Scholar CrossRef Search ADS Adler PH , Crosskey RW . 2017 . World blackflies (Diptera: Simuliidae): a comprehensive revision of the taxonomic and geographical inventory [2017] . Available at: https://biomia.sites.clemson.edu/pdfs/blackflyinventory.pdf Adler PH , Currie DC , Wood DM . 2004 . The black flies (Simuliidae) of North America . New York : Cornell University Press . Adler PH , Huang YT . 2011 . Integrated systematics of the Simuliidae (Diptera): evolutionary relationships of the little-known Palearctic black fly Simulium acrotrichum . Canadian Entomologist 143 : 612 – 628 . Google Scholar CrossRef Search ADS Adler PH , Kachvorian EA . 2001 . Cytogenetics of the Holarctic black fly Simulium noelleri (Diptera: Simuliidae) . Canadian Journal of Zoology 79 : 1972 – 1979 . Google Scholar CrossRef Search ADS Adler PH , Kúdelová T , Kúdela M , Seitz G , Ignjatović-Ćupina A . 2016a . Cryptic biodiversity and the origins of pest status revealed in the macrogenome of Simulium colombaschense (Diptera: Simuliidae), history’s most destructive black fly . PLoS ONE 11 : e0147673 . Google Scholar CrossRef Search ADS Adler PH , Takaoka H , Sofian-Azirun M , Low VL , Ya’cob Z , Chen CD , Lau KW , Pham XD . 2016b . Vietnam, a hotspot for chromosomal diversity and cryptic species in black flies (Diptera: Simuliidae) . PLoS ONE 11 : e0163881 . Google Scholar CrossRef Search ADS Adler PH , Yadamsuren O , Procunier WS . 2016c . Chromosomal translocations in black flies (Diptera: Simuliidae)—facilitators of adaptive radiation ? PLoS ONE 11 : e0158272 . Google Scholar CrossRef Search ADS Anisimova M , Gascuel O . 2006 . Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative . Systematic Biology 55 : 539 – 552 . Google Scholar CrossRef Search ADS Bedo DG . 1975 . Polytene chromosomes of three species of blackflies in the Simulium pictipes group (Diptera:Simuliidae) . Canadian Journal of Zoology 53 : 1147 – 1164 . Google Scholar CrossRef Search ADS Bedo DG . 1979 . Cytogenetics and evolution of Simulium ornatipes Skuse (Diptera: Simuliidae). II. Temporal variation in chromosomal polymorphisms and homosequential sibling species . Evolution; international journal of organic evolution 33 : 296 – 308 . Google Scholar CrossRef Search ADS Brockhouse C , Bass JAB , Straus NA . 1989 . Chromocentre polymorphism in polytene chromosomes of Simulium costatum (Diptera: Simuliidae) . Genome 32 : 510 – 515 . Google Scholar CrossRef Search ADS Conflitti IM , Kratochvil MJ , Spironello M , Shields GF , Currie DC . 2010 . Good species behaving badly: non-monophyly of black fly sibling species in the Simulium arcticum complex (Diptera: Simuliidae) . Molecular Phylogenetics and Evolution 57 : 245 – 257 . Google Scholar CrossRef Search ADS Ferree PM , Barbash DA . 2009 . Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila . PLoS Biology 7 : e1000234 . Google Scholar CrossRef Search ADS Finn DS , Adler PH . 2006 . Population genetic structure of a rare high‐elevation black fly, Metacnephia coloradensis, occupying Colorado lake outlet streams . Freshwater Biology 51 : 2240 – 2251 . Google Scholar CrossRef Search ADS Finn DS , Theobald DM , Black WC 4th , Poff NL . 2006 . Spatial population genetic structure and limited dispersal in a Rocky Mountain alpine stream insect . Molecular Ecology 15 : 3553 – 3566 . Google Scholar CrossRef Search ADS 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 . Molecular Marine Biology and Biotechnology 3 : 294 – 299 . Gottlieb G . 1998 . Normally occurring environmental and behavioral influences on gene activity: from central dogma to probabilistic epigenesis . Psychological Review 105 : 792 – 802 . Google Scholar CrossRef Search ADS Guindon S , Dufayard JF , Lefort V , Anisimova M , Hordijk W , Gascuel O . 2010 . New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0 . Systematic Biology 59 : 307 – 321 . Google Scholar CrossRef Search ADS Henderson CAP . 1986 . Homosequential species 2a and 2b within the Prosimulium onychodactylum complex (Diptera): temporal heterogeneity, linkage disequilibrium, and Wahlund effect . Canadian Journal of Zoology 64 : 859 – 866 . Google Scholar CrossRef Search ADS Huang YT , Adler PH , Takaoka H . 2011 . Polytene chromosomes of Simulium arakawae, a pest species in the Simulium venustum group (Diptera: Simuliidae) from Japan . Tropical Biomedicine 28 : 376 – 381 . Huelsenbeck JP , Ronquist F , Nielsen R , Bollback JP . 2001 . Bayesian inference of phylogeny and its impact on evolutionary biology . Science (New York, N.Y.) 294 : 2310 – 2314 . Google Scholar CrossRef Search ADS Ilmonen J , Adler PH , Malmqvist B , Cywinska A . 2009 . The Simulium vernum group (Diptera: Simuliidae) in Europe: multiple character sets for assessing species status . Zoological Journal of the Linnean Society 156 : 847 – 863 . Google Scholar CrossRef Search ADS Jabłońska-Barna I , Szarek-Gwiazda E , Michailova P . 2013 . Environmental agents in Lake Łuknajno (Poland) affecting the genome of Chironomus melanotus Keyl, 1961 (Diptera, Chironomidae)—a new species of Polish fauna . Oceanological and Hydrobiological Studies 42 : 164 – 172 . Google Scholar CrossRef Search ADS Meier R , Shiyang K , Vaidya G , Ng PK . 2006 . DNA barcoding and taxonomy in Diptera: a tale of high intraspecific variability and low identification success . Systematic Biology 55 : 715 – 728 . Google Scholar CrossRef Search ADS Moulton JK , Adler PH . 1995 . Revision of the Simulium jenningsi species-group (Diptera: Simuliidae) . Transactions of the American Entomological Society 121 : 1 – 57 . Otsuka Y , Takaoka H , Aoki C , Choochote W . 2003 . Phylogenetic analysis of the subgenus Himalayum within the genus Simulium sl (Diptera: Simuliidae) using mitochondrial 16S rRNA gene sequences . Medical Entomology and Zoology 54 : 113 – 120 . Google Scholar CrossRef Search ADS Phayuhasena S , Colgan DJ , Kuvangkadilok C , Pramual P , Baimai V . 2010 . Phylogenetic relationships among the black fly species (Diptera: Simuliidae) of Thailand based on multiple gene sequences . Genetica 138 : 633 – 648 . Google Scholar CrossRef Search ADS Pramual P . 2006 . Population genetic structure of black flies (Diptera: Simuliidae) from Thailand inferred from mitochondrial DNA sequences . Ph.D. Thesis, Mahidol University , Bangkok, Thailand . Pramual P , Adler PH . 2014 . DNA barcoding of tropical black flies (Diptera: Simuliidae) of Thailand . Molecular Ecology Resources 14 : 262 – 271 . Google Scholar CrossRef Search ADS Pramual P , Kuvangkadilok C . 2012 . Integrated cytogenetic, ecological, and DNA barcode study reveals cryptic diversity in Simulium (Gomphostilbia) angulistylum (Diptera: Simuliidae) . Genome 55 : 447 – 458 . Google Scholar CrossRef Search ADS Pramual P , Kuvangkadilok C , Baimai V , Walton C . 2005 . Phylogeography of the black fly Simulium tani (Diptera: Simuliidae) from Thailand as inferred from mtDNA sequences . Molecular Ecology 14 : 3989 – 4001 . Google Scholar CrossRef Search ADS Pramual P , Nanork P . 2012 . Phylogenetic analysis based on multiple gene sequences revealing cryptic biodiversity in Simulium multistriatum group (Diptera: Simuliidae) in Thailand . Entomological Science 15 : 202 – 213 . Google Scholar CrossRef Search ADS Pramual P , Pangjanda S . 2015 . Effects of habitat specialization on population genetic structure of black fly Simulium weji Takaoka (Diptera: Simuliidae) . Journal of Asia-Pacific Entomology 18 : 33 – 37 . Google Scholar CrossRef Search ADS Pramual P , Wongpakam K . 2011 . Cytogenetics of Simulium siamense Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand . Aquatic Insects 33 : 171 – 184 . Google Scholar CrossRef Search ADS Pramual P , Wongpakam K . 2013 . Population genetics of the high elevation black fly Simulium (Nevermannia) feuerborni Edwards in Thailand . Entomological Science 16 : 298 – 308 . Google Scholar CrossRef Search ADS Pramual P , Wongpakam K . 2014 . Association of black fly (Diptera: Simuliidae) life stages using DNA barcode . Journal of Asia-Pacific Entomology 17 : 549 – 554 . Google Scholar CrossRef Search ADS Pramual P , Wongpakam K , Kuvangkadilok C . 2008 . Cytogenetics of the black fly Simulium aureohirtum Brunetti from Thailand . Cytologia 73 : 293 – 304 . Google Scholar CrossRef Search ADS Rivera J , Currie DC . 2009 . Identification of Nearctic black flies using DNA barcodes (Diptera: Simuliidae) . Molecular Ecology Resources 9 ( Suppl s1 ): 224 – 236 . Google Scholar CrossRef Search ADS Rothfels KH . 1979 . Cytotaxonomy of black flies (Simuliidae) . Annual Review of Entomology 24 : 507 – 539 . Google Scholar CrossRef Search ADS Rothfels KH . 1988 . Cytological approaches to black fly taxonomy . In: Kim KC , Merritt RW , eds. Black flies: ecology, population management, and annotated world list . University Park, PA : Pennsylvania State University Press , 39 – 52 . Rothfels K . 1989 . Speciation in black flies . Genome 32 : 500 – 509 . Google Scholar CrossRef Search ADS Rothfels K , Feraday R , Kaneps A . 1978 . A cytological description of sibling species of Simulium venustum and S. verecundum with standard maps for the subgenus Simulium Davies (Diptera) . Canadian Journal of Zoology 56 : 1110 – 1128 . Google Scholar CrossRef Search ADS Rothfels KH , Freeman DM . 1977 . The salivary gland chromosomes of seven species of Prosimulium (Diptera, Simuliidae) in the mixtum (IIIL-1) group . Canadian Journal of Zoology 55 : 482 – 507 . Google Scholar CrossRef Search ADS Swofford DL . 2002 . PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4.b.10 . Sunderland : Sinauer . Takaoka H . 1977 . Studies on black flies of the Nansei Islands, Japan (Simuliidae; Diptera): III. On six species of the subgenus Simulium Latreille . Medical Entomology and Zoology 28 : 193 – 217 . Google Scholar CrossRef Search ADS Takaoka H . 2017 . Morphotaxonomic revision of species-groups of Simulium (Simulium) (Diptera: Simuliidae) in the Oriental Region . Zootaxa 4353 : 425 – 446 . Google Scholar CrossRef Search ADS Takaoka H , Choochote W . 2004 . A list of and keys to black flies (Diptera: Simuliidae) in Thailand . Tropical Medicine and Health 32 : 189 – 197 . Google Scholar CrossRef Search ADS Takaoka H , Choochote W . 2005a . Two new species of black flies (Diptera: Simuliidae) from northern Thailand . Medical Entomology and Zoology 56 : 319 – 333 . Google Scholar CrossRef Search ADS Takaoka H , Choochote W . 2005b . Two new species of Simulium Latreille (Diptera: Simuliidae) from northwestern Thailand . Medical Entomology and Zoology 56 : 123 – 133 . Google Scholar CrossRef Search ADS Takaoka H , Choochote W . 2007 . A new species of the multistriatum species group of Simulium (Simulium) (Diptera: Simuliidae) from Northern Thailand . Tropical Medicine and Health 35 : 19 – 22 . Google Scholar CrossRef Search ADS Takaoka H , Davies DM . 1995 . The black flies (Diptera: Simuliidae) of West Malaysia . Fukuoka, Japan : Kyushu University Press . Takaoka H , Kuvangkadilok C . 1999 . Four new species of black flies (Diptera: Simuliidae) from Thailand . Japanese Journal of Tropical Medicine and Hygiene 27 : 497 – 509 . Google Scholar CrossRef Search ADS Tamura K , Stecher G , Peterson D , Filipski A , Kumar S . 2013 . MEGA6: molecular evolutionary genetics analysis version 6.0 . Molecular Biology and Evolution 30 : 2725 – 2729 . Google Scholar CrossRef Search ADS Tangkawanit U , Kuvangkadilok C , Baimai V , Adler PH . 2009 . Cytosystematics of the Simulium tuberosum group (Diptera: Simuliidae) in Thailand . Zoological Journal of the Linnean Society 155 : 289 – 315 . Google Scholar CrossRef Search ADS Tangkawanit U , Kuvangkadilok C , Trinachartvanit W , Baimai V . 2011 . Cytotaxonomy, morphology and ecology of the Simulium nobile species group (Diptera: Simuliidae) in Thailand . Cytogenetic and Genome Research 134 : 308 – 318 . Google Scholar CrossRef Search ADS Thaijarern J , Pramual P , Adler PH . 2017 . Life-stage association of black flies, using a fast-evolving nuclear gene sequence, and description of the larva of Simulium lampangense Takaoka & Choochote (Diptera: Simuliidae) from Thailand . Zootaxa 4299 : 263 – 270 . Google Scholar CrossRef Search ADS Thanwisai A , Kuvangkadilok C , Baimai V . 2006 . Molecular phylogeny of black flies (Diptera: Simuliidae) from Thailand, using ITS2 rDNA . Genetica 128 : 177 – 204 . Google Scholar CrossRef Search ADS Thapa S , Procunier W , Henry W , Chhetri S . 2014 . Heterochromatin and sibling species of Simulium praelargum s.l. (Diptera: Simuliidae) . Genome 57 : 223 – 232 . Google Scholar CrossRef Search ADS © 2018 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Zoological Journal of the Linnean SocietyOxford University Press

Published: Apr 30, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off