61
南太平洋海域調査研究報告 No.54（2014年12月）
OCCASIONAL PAPERS No.54（December 2014）

Species Diversity of Black Fly in Southeast Asia:
Phylogenetic Analysis of Three Subgenera, Asiosimulium,Daviesellum, and Wallacellum, of the Genus Simulium s. l.
OTSUKA Yasushi
Research Center for the Pacific Islands, Kagoshima University

Abstract
Black flies (Diptera: Simuliidae) are distributed worldwide. The biting habits of black flies are known
to cause medical and veterinary problems including transmissions of filarial diseases of humans andanimals. In Southeast Asia (Thailand, Malaysia, Philippines, and Indonesia), eight subgenera (Asiosimulium,Daviesellum, Gomphostilbia, Montisimulium, Morops, Nevermannia, Simulium s. str., and Wallacellum) of
genus Simulium s. l. are known. Of these subgenera, three subgenera (Asiosimulium, Daviesellum, andWallacellum) are small, endemic only in the Oriental region (except one species of Wallacellum), andrecently established. To confirm the subgeneric status, phylogenetic analysis was conducted using
mitochondrial 16S rRNA sequences. As results, the three subgenera were clearly separated from othersubgenera. Their subgeneric status was confirmed both by phylogenetic analysis and morphologicalcharacters.
Keywords: Asiosimulium, black fly, Daviesellum, phylogeny, Simulium, Wallacellum

Introduction
In a revision of supraspecific taxa of the genus Simulium Latreille s. l. which includes
about 10 subgenera in the Oriental region, two subgenera: Nevermannia Enderlein andHimalayum Lewis have already been evaluated by phylogenetic analysis of mitochondorial
16S ribosomal RNA (rRNA) gene sequences (OTSUKA et al. 2001, 2003). Genus Simulium s. l.
is the largest genus in the family Simuliidae, having 1,745 species in the 37 subgenera(ADLER and CROSSKEY 2014). Among these, ten subgenera are reported from the Orientalregion. Moreover, in Southeast Asia (Thailand, Malaysia, Philippines, and Indonesia), eightsubgenera (Asiosimulium Takaoka and Choochote, Daviesellum Takaoka and Adler,
Gomphostilbia Enderlein, Montisimulium Rubtsov, Morops Enderlein, Nevermannia,

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OTSUKA Yasushi

Simulium Latreille s. str., and Wallacellum Takaoka) are known (Table 1). Of these subgenera,
three subgenera (Asiosimulium, Daviesellum, and Wallacellum) are small, endemic only in theOriental region (except one species of Wallacellum), and recently established (Fig. 1). To
confirm the subgeneric status, phylogenetic analysis was conducted using mitochondrial 16SrRNA sequences.

Table 1. Number of black flies in countries of Southeast Asia.
Genus
Simulium

Subgenus
Asiosimulium
Daviesellum
Gomphostilbia
Montisimulium
MoropsNevermannia
Simulium
Wallacellum

Thailand
3223
602
430Malaysia
0126007310

Philippines
00330043414

Indonesia
0044026
8402Fig. 1. Distribution of three subgenera, Asiosimulium, Daviesellum, and Wallacellum.

63Species Diversity of Black Fly in Southeast Asia: Phylogenetic Analysis of Three Subgenera, Asiosimulium, Daviesellum, and Wallacellum, of the Genus Simulium s. l.

Materials and Methods
Phylogenetic analysis was conducted using the 16S mitochondrial sequences of 28species (OTSUKA et al. 2001, 2003, 2007) (Table 2). The sequences were aligned by theprogram CLUSTAL W, and the alignment was then adjusted manually. Sites containingalignment gaps were excluded in the following analysis. The number of nucleotide
substitution per site was estimated between each pair of the sequences, using Jukes-Cantor
methods. Construction and bootstrap probability estimation of the neighbor-joining tree wereperformed by MEGA6 (TAMURA et al. 2013).
Table 2. Black fly species used for the phylogenetic analysis.

Species

Locality

Simulium (Asiosimulium ) oblongum
S. (Daviesellum ) courtneyi
S. (D.) pahangense

Muk Da Han, Thailand
Doi Phu Kha, Thailand
Peninsular Malaysia

AB334089
AB334090
AB334091

S. (Gomphostilbia ) palauense
S. (G. ) whartoniS. (Hellichiella ) chiharuae
S. (Montisimulium ) kobayashii
S. (Mon. ) merga

Palau
Peninsular Malaysia
Kyoto, JapanMikurajima, JapanDoi Inthanon, Thailand

AB056742
AB056743
AB334092
AB334093
AB334094

S. (Morops ) farciminis
S. (Nevermannia ) aureohirtum
S. (N. ) feuerborni
S. (N. ) ornatipes
S. (N. ) uchidai

Irian Jaya, Indonesia
Peninsular Malaysia
Peninsular Malaysia
Irian Jaya, Indonesia
Oita, Japan

AB056744
AB056736
AB056729
AB056737
AB056740

S. (Simulium ) eximium
S. (S. ) malayense
S. (S. ) nobile
S. (S. ) tani

Flores, Indonesia
Peninsular Malaysia
Peninsular Malaysia
Peninsular Malaysia

AB093100
AB093112
AB093115AB093123

S. (Wallacellum ) cabrerai

Luzon, Philippines

AB093128

S. (W. ) carinatum
S. (W. ) celebesense
S. (W. ) claveriaense
S. (W. ) recurvum

Luzon, Philippines
Sulawesi, Indonesia
Luzon, Philippines
Luzon, Philippines

AB093129
AB334095
AB334096
AB334097

S. (W. ) spinosibranchium
S. (W. ) suyoense
S. (W. ) tuyense
S. (W. ) yonakuniense
Austrosimulium bancrofti

Luzon, Philippines
Luzon, Philippines
Luzon, Philippines
Yonakuni, Japan
Australia

AB334098
AB334099
AB334100
AB334101
AB093130

Prosimulium kiotoense
Oita, JapanThe sequenes were determined in OTSUKA et al . (2001, 2003, 2007).

Accession number

AB056747

64OTSUKA Yasushi

Results and Discussions
Sequences of the mitochondrial 16S rRNA gene region of the 26 species of genus
Simulium s. l. were aligned with the two species from other genera as outgroup (Table 2). To
reveal the evolutionary relationship among the species of the genus Simulium s. l., aneighbor-joining tree was made (Fig. 2). The tree shows that all subgenera, except forsubgenus Nevermannia, occur in a clade, and supports the independence of the subgeneraAsiosimulium, Daviesellum, and Wallacellum, which have been recently established. OTSUKA
et al. (2001) reported that ruficorne species-group of Nevermannia, which includes S.
aureohirtum Brunetti and S. ornatipes Skuse, was genetically and morphologically different
from the other species-groups, and suggested that revision of the definition of Nevermannia
was needed.

Fig. 2. Phylogenetic tree obtained by neighbor-joining method for a partial mitochondrial 16S rRNA
sequence of 28 black fly species. The values at branch points indicate the percentage support for aparticular node after 1,000 bootstrap replicates were performed. Values less than 50% are notshown.

65Species Diversity of Black Fly in Southeast Asia: Phylogenetic Analysis of Three Subgenera, Asiosimulium, Daviesellum, and Wallacellum, of the Genus Simulium s. l.

Asiosimulium was recently established based on a unique species, S. (A.) oblongum
Takaoka and Choochote, found in Thailand, and was reported to have more similarities
morphologically to three subgenera, namely: Boreosimulium Rubtsov and Yankovsky in theHolarctic Region, Inseliellum Rubtsov in Micronesia and Polynesia, and Nevermannia, acosmopolitan heterogeneous taxon (TAKAOKA and CHOOCHOTE 2005). Daviesellum is also asmall subgenus, represented by only two species, S. (D.) pahangense Takaoka and Davies,and S. (D.) courtneyi Takaoka and Adler, collected from Peninsular Malaysia and Thailand,
and has distinctive genitalia in both sexes, suggesting no close relationship to other subgeneraof the genus Simulium s. l. (TAKAOKA and ADLER 1997). On the other hand, Wallacellum,
represented by 11 species mostly from the Philippines, seems to have a close relationship toan Australasian subgenus Morops by having hairs on both katepisternum and pleural
membrane (TAKAOKA 2003). However, the phylogenetic tree in this study did not show aclose relationship between Wallacellum and Morops. Phylogenetic analysis of black flies
using the mitochondrial 16S rRNA region can be a tool in determining the identity of thesubgenus of Simulium s. l., but does not seem to resolve relationships among the subgenera
due to low bootstrap values. More information on other genes as well as cytogenetic study isneeded to clarify the relationships among subgenera of Simulium s. l.

References
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Comprehensive Revision of the Taxonomic and Geographical Inventory [2014], 122 pp.
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OTSUKA, Y., AOKI, C., CHOOCHOTE, W., DE LA LLAGAS, L. and TAKAOKA, H. 2007.
Phylogenetic Analysis of Three Subgenera: Asiosimulium, Daviesellum and Wallacellum,of the Genus Simulium s. l. Endemic in the Oriental Region. Medical Entomology andZoology, 58: 329-233.
OTSUKA, Y., AOKI, C., SUZUKI, H., SAITO, K., HADI, U. K. and TAKAOKA, H. 2001.
Phylogenetic Analyses of a Blackfly Subgenus Simulium (Nevermannia) Based on
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Mitochondrial 16S rRNA Gene Sequences. Medical Entomology and Zoology, 54:113-120.

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TAKAOKA, H. 2003. The Black Flies (Diptera: Simuliidae) of Sulawesi, Maluku and Irian Jaya,xxii+581 pp., Kyushu University Press, Fukuoka, Japan.
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