South Pacific Studies Vol.35, No.2, 2015

Biogeographical Distribution and Phylogenetic Analysis of
Simulium (Wallacellum) (Diptera: Simuliidae) Based on the
Mitochondrial Sequences
OTSUKA Yasushi1* and TAKAOKA Hiroyuki2

1: Research Center for the Pacific Islands, Kagoshima University,
Korimoto 1-21-24, Kagoshima, 890-8580 Japan
2: Institute of Biological Sciences, Faculty of Science, University of Malaya,
Kuala Lumpur, 50603 Malaysia* Corresponding author
E-mail: yotsuka@cpi.kagoshima-u.ac.jp

Abstract
The blackfly subgenus Wallacellum of genus Simulium (Diptera: Simuliidae) is a small subgenus,
represented by 17 species, and distributed insularly in Southeast Asia. Phylognetic relationships among
Wallacellum were surveyed using mitochondrial sequences of cytochrome c oxidase subunit I (COI) and
16S ribosomal RNA (16S rRNA) regions. Combined with morphological data, Wallacellum was divided
into three groups (A, B, C). The group A has ancestral characters and is distributed widely, whereas thegroup B has derivative characters and is distributed only in the central Philippines, suggesting that thegroup A is basal and might have been distributed widely in the early period, and then species radiation of
the group B occurred in the center of the distribution of this subgenus. The interspecific and intraspecificdivergence values were not overlapped both for the COI and 16S rRNA regions, indicating that both regions
were effective to identify the species of Wallacellum.
Key words: blackfly, distribution, phylogeny, Simulium, Wallacellum

Introduction　　The subgenus Wallacellum Takaoka is one of the subgenera of the genus Simulium
Latreille s. l. (Diptera: Simuliidae) and is known to have insular distribution (TAKAOKA
1983, 2003). It is consisted of 14 species recorded from the Philippines (TAKAOKA
1983, 2006, 2009), two species from Indonesia (TAKAOKA 2003) and one species bothfrom Yonakuni Island, the Ryukyu Islands, Japan (TAKAOKA 1972) and Lanyu Island,
Taiwan (CHUNG 1986). According to TAKAOKA (1983), this subgenus is characterized
Received: 13 February, 2015
Accepted: 24 February, 2015

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South Pacific Studies Vol.35, No.2, 2015

by combination of following characters: in the adult, the enlarged calcipala and thehind tibia with a narrow ridge on the anterointernal surface along basal 1/2 or 1/3, thehaired pleural membrane and katepisternum; in the pupa, the abdominal segments 6-9
devoid of dorsal spine-combs, abdominal segments 6 and 7 each having an inner hookand lacking an outer hook ventrally on each side.
The subgeneric status of Asiosimulium Takaoka and Choochote, Daviesellum
Takaoka and Wallacellum, all recently-established small subgenera of the genus
Simulium, was confirmed by phylogenetic analysis based on mitochondrial sequences(OTSUKA et al. 2007). In the phylogenetic analysis, Wallacellum was placed basallywith a sister group of the counter clade including other eight subgenera of genus
Simulium in the Oriental Region. The subgenus Wallacellum is the only subgenus
having insular distribution in the Oriental Region. In the Pacific, two subgenera,
Hebridosimulium Grenier and Rageau and Inseliellum Rubtsov, also have insular
distribution. Hebridosimulium is endemic in Vanuatu, Fiji and Society islands.
Inseliellum is distributed in Micronesia (Guam and Chuuk islands in Federated Statesof Micronesia) and disparately in the southern central Pacific (Marquesas islands,
Society islands and Cook islands). TAKAOKA (2012) also investigated the phylogenetic
relationships of 10 subgenera distributing in Southeast Asia and the Pacific usingmorphological data. In the cladogram, Wallacellum has a sister taxon relationship with
Hebridosimulium. The clade of Wallacellum and Hebridosimulium was a sister relation
with other subgenera and clades. The analysis both from mitochondrial sequences
and morphological data suggested that Wallacellum might have been separated
from other subgenera of the Oriental Region in the early period. In this paper, wesurveyed phylogenetic relationships of 15 of the 17 species of Wallacellum basedon mitochondrial sequences, and explored the process of distribution of this insularsubgenus with morphological data.

Materials and Methods
Sample collection and DNA extraction
Pupae and larvae of each blackfly species examined in this study were collected
at localities shown in Table 1. The pupae were individually reared in tubes until adultemergence. Identification was done according to the original descriptions (TAKAOKA
1972, 1983, 2003, 2006, 2009). DNA was extracted from a single larva, pupa oradult using DNeasy blood and tissue kit (Qiagen) according to the manufacturer's
instructions. Extracted DNA was dissolved in 200μl AE provided in the kit.
PCR amplification and sequencing
The cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S
rRNA) regions were amplified by polymerase chain reaction (PCR) using thefollowing primers: LCO1490, 5’-GGTCAACAAATCATAAAGATATTGG-3’ andOTSUKA and TAKAOKA : Biogeographical Distribution of Wallacellum

47

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3URVLPXOLXPNLRWRHQVH DQG3\H]RHQVH ZHUHXVHGDVRXWJURXSLQSK\ORJHQHWLFDQDO\VLVHCO2198, 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’ for COI (F OLMER
et al. 1994); and primer A, 5’-CGCCTGTTTATCAAAAACAT-3’ and primer B,
5’-CTCCGGTTTGAACTCAGATC-3’ for 16S rRNA region (XIONG and KOCHER
1991). PCR was carried out using 20 μL volumes containing 0.5 units of Ex Taq(TaKaRa), 1X Ex Taq buffer, 2 mM of MgCl2, 0.2 mM of each dNTP, 0.25 μM of eachprimer and 1 μL of the extracted DNA. The amplified products were electrophorised
through a 1% agarose gel. PCR products were purified with the QIAquick PCR
Purification Kit (Qiagen) and directly sequenced using the PCR primers. Sequencing
reactions were performed using the BigDyeⓇ Terminator Cycle Sequencing Kit andrun on a 3130 Genetic Analyzer (Applied Biosystems). The sequence data of this paper48

South Pacific Studies Vol.35, No.2, 2015

have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence database underaccession numbers LC034954-LC035010 (Table 1).
Phylogenetic analysis
Sequences of COI and 16S rRNA of Wallacellum were aligned using the
CLUSTAL W multiple alignment program (THOMPSON et al. 1994) with deposited
sequences. Prosimulium kiotoense and P. yezoense were used as outgroups. Gap
sites were excluded from the following analysis. The Kimura two-parameter model
was employed to calculate genetic divergence (KIMURA 1980). Using the divergencevalues, construction of neighbor-joining (NJ) trees (SAITOU and NEI 1987) and thebootstrap test with 1,000 replications were performed with the MEGA version 6.0
program (TAMURA et al. 2013). Bayesian analysis was conducted with MrBayes 3.2(RONQUIST et al. 2012) by using two replicates of 1 million generations with thenucleotide evolutionary model. The best-fit model was chosen for each gene separately
using the Akaike Information Criterion in MrModeltest version 2.3 (NYLANDER 2004).
The general time reversible with gamma distribution shape parameter and invariable
sites (GTR+G+I) was selected for both regions. Bayesian posterior probabilities were
calculated from the consensus tree after excluding the first 25% trees as burn-in.

Results and Discussion
Sequence diversity
The sequences of 29 samples for COI and 24 samples of Wallacellum for 16S
rRNA region were determined (Table 1). Combined with the published sequences(OTSUKA et al. 2003, 2007) and those of the outgroup species (P. kiotoense and P.
yezoense), the sequences were aligned and compared. Means of interspecific divergencein Wallacellum were 15.79% (range 3.94-20.28%) for COI and 3.80% (range 0.786.07%) for 16S rRNA region (Table 2). Low levels of interspecific divergence wereobserved between S. tuyense and S. yonakuniense (3.94-4.59% for COI and 0.98-1.38%
for 16S rRNA) and between S. marilogense and S. ogonukii (6.08% for COI and 0.78%
for 16S rRNA). Simulium tuyense and S. yonakuniense were morphologically similar
especially in the adult and pupal stages, having slight differences in the number ofrows and hooks of the larval posterior circle (TAKAOKA 1983). Means of intraspecificOTSUKA and TAKAOKA : Biogeographical Distribution of Wallacellum

49

Fig. 1. Histograms
showing interspecific (black) and intraspecific (gray) genetic divergences for COI
and 16S rRNA regions.

divergence were 0.75% (range 0-2.49%) for COI and 0.09% (range 0-0.39%) for 16S
rRNA (Table 2). For both genes, interspecific and intraspecific divergence values
were not overlapped (Fig. 1). In COI, since most of the interspecific divergences werelarger than 10%, the peak of intraspecific divergence is clearly separated from thatof intraspecific divergence. The COI region was used for the identification of animal
species known as DNA barcoding (HEBERT et al. 2003). DNA barcoding has showed
to be effective in various taxa of animal including blackfly (R IVERA and CURRIE
2009, PRAMUAL et al. 2010, HERNÁNDEZ-TRIANA et al. 2012, PRAMUAL and ADLER
2014). In the previous works of Thai blackflies using DNA barcoding, interspecificand intraspecific divergence values overlapped in some taxa (PRAMUAL et al. 2010,
PRAMUAL and ADLER 2014), resulting difficulties of species identification. Our results
showed that DNA barcoding is effective for Wallacellum. Moreover, 16S rRNA region
was also proved to be useful for the identification of species of Wallacellum.
Phylogenetic analysis
Phylogentic analysis was conducted for COI and 16S rRNA regions by two
methods (NJ and bayes). In Figs. 2 & 3, bayesian trees were shown for COI and 16S
rRNA regions, respectively, since bayesian analysis revealed the relationships ofspecies of Wallacellum with higher confident values than NJ, although the bootstrap
values of NJ were shown in the nodes of the trees. In the analysis, S. celebesense,
S. marilogense, S. ogonukii, S. tuyense and S. yonakuniense had a clade with highconfidences without NJ of 16S rRNA region. These species are assigned to the group
A. Furthermore, S. marilogense and S. ogonukii were clearly separated from the otherspecies of the group A. Although S. marilogense and S. ogonukii were endemic onlyin Mindanao, the other species of the group A have different distribution. Simulium
tuyense is known to be distributed in many islands of the Philippines (Luzon, Mindoro,
Samar, Palawan and Mindanao). Despite S. yonakuniense in Yonakuni island, Japanand Lanyu island, Taiwan and S. celbesense in Sulawesi and Biak, Indonesia were
geographically separated, the two species were phylogenetically related. In the trees,50South Pacific Studies Vol.35, No.2, 2015

Simulium amplum, S. cabrerai, S. carinatum, S. molawinense, S. recurvum , S. resimumand S. spinosibranchium also had a clade with high confidences, all of which areassigned to the group B. Although S. resimum is endemic only in Mindanao, mostFig. 2. A bayesian tree of the species of Wallacellum based on the sequences of COI. Numbers at tree
nodes are bayesian posterior probabilities (% ) and bootstrap values (% ) of NJ analysis, withdashes indicating a lack of support for the analysis. Branch lengths are proportional to genetic
distance (scale bar).

OTSUKA and TAKAOKA : Biogeographical Distribution of Wallacellum

51

species of the group B are mainly endemic in Luzon. Simulium carinatum and S.
recurvum are also known in Negros, and were slightly related in Bayesian analysis of
16S rRNA. Three other species of Wallacellum, S. claveriaense, S. suyoense and S.

Fig. 3. A bayesian tree of the species of Wallacellum based on the sequences of 16S rRNA region.
Numbers at tree nodes are bayesian posterior probabilities（%）and bootstrap values（%）
of NJ analysis, with dashes indicating a lack of support for the analysis. Branch lengths areproportional to genetic distance (scale bar).

52

South Pacific Studies Vol.35, No.2, 2015

tenederoi, all assigned to the group C, were a monophyletic group only in bayesiananalysis of 16S rRNA region. Means of divergence between the three groups werecompared (Table 2). Means of divergence between the groups A and B were highest
both for COI (17.51%) and 16S rRNA regions (4.89%), whereas those of between thegroups A and C were lowest both for COI (14.64%) and 16S rRNA regions (3.83%).
Morphological character and distribution
The three groups also can be separated by morphological characters. The pupaeof the groups A and C bear long and slender gill filaments. Moreover, the cuticlesof the filament are thick, with numerous transvers ridges. On the other hand, pupal
gill filaments of the group B are of short, inflated tubular form, lacking transvers
ridges. In the female genitalia, paraprocts of the group B are large, strongly extending
ventroanteriorly, and thrusting the posterior margin of the ovipositor valves inward,
whereas those of the group A are not so large. The genitalia of female adult of group C
are intermediate between the groups A and B (TAKAOKA 1972, 1983, 2003, 2006, 2009).
In this work, S. alfurense and S. makilingense were not analyzed with mitochondrial
sequences. From the original descriptions, S. alfurense and S. makilingense are likely
to belong to the groups A and C, respectively (TAKAOKA 1983, 2003). The distributionof all the 17 species of Wallacellum is listed in Table 3, and the distribution of the threegroups is shown in Fig. 4. The group A is widely distributed in the Philippines, andextends northward to Taiwan and Japan and southward to Indonesia. On the other hand,
the groups B and C are endemic in the central parts of the Philippines, mainly in Luzon.
Certain morphological characters are presiomorphic in the group A and apomorphic in

TAKAOKATAKAOKAOTSUKA and TAKAOKA : Biogeographical Distribution of Wallacellum

53

Fig. 4. D istribution of Wallacellum in Southeast Asia; group A (bold line), group B (thin line), groupC (dot line). The numbers of species of each group in the island are shown under the name of
the island.

the group B (TAKAOKA 1983). Combined these results and information, it is suggested
that the group A is the basal group, having been distributed widely in the early period,
and then species radiation of the group B occurred in the center of the distribution ofthis subgenus.
Geographical history of the subgenus Inseliellum, which also has insular
distribution in the Pacific, was surveyed with phylogenies and information on island
ages of hot-spot archipelagoes (CRAIG et al. 2001). In Inseliellum, basal species andclades are widely distributed in separated old islands. Moreover, S. malardei and
S. lotti, which are basal among the species of Inseliellum in the Society Islands, arewidely spread in the islands. In contrast, derived species are limited to younger islands,
where species radiation occurred. The insular subgenera Wallacellum and Inseliellum
have similarities in their distribution; 1) basal species or clade are widely distributed,2) in some certain islands, species radiation occurred. In Sulawesi, Indonesia, theSimulium variegatum species-group of subgenus Simulium Latreille s.str. of the genus
Simulium is represented by 11 species (TAKAOKA 2003), most of which have various
shapes of inflated pupal gill filaments, like those of the group B of Wallacellum. Thisvariation in the pupal gill filaments of the S. variegatum species-group might be due
to species radiation. Although the geographical history of Inseliellium was considered54South Pacific Studies Vol.35, No.2, 2015

with evolution of running water habitats (CRAIG et al. 2001), mechanisms of radiationof blackfly species in islands still remain unknown.

Acknowledgements
We sincerely thank Dr. Lilian DE LA LLAGAS, University of the Philippines, forher kind and longstanding support. Our appreciation goes to Mr. Victor F. TENEDERO,
Manila, for his enthusiastic assistance during field surveys in the Philippines. This
study was supported by the Grant-in-aid for Oversea Research from Japan Society for
the Promotion of Science (No. 18406011; representative TAKAOKA H.) and the research
grant Malaya (RP003A-13SUS).

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