Who is biting you? DNA barcodes reveal cryptic diversity in human-biting black flies (Diptera: Simuliidae)

Accepted Manuscript Title: Who is biting you? DNA barcodes reveal cryptic diversity in human-biting black flies (Diptera: Simuliidae) Authors: Panya Jomkumsing, Ubon Tangkawanit, Komgrit Wongpakam, Pairot Pramual PII: DOI: Reference:

S0001-706X(19)30372-9 https://doi.org/10.1016/j.actatropica.2019.05.001 ACTROP 5010

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

21 March 2019 1 May 2019 1 May 2019

Please cite this article as: Jomkumsing P, Tangkawanit U, Wongpakam K, Pramual P, Who is biting you? DNA barcodes reveal cryptic diversity in human-biting black flies (Diptera: Simuliidae), Acta Tropica (2019), https://doi.org/10.1016/j.actatropica.2019.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Who is biting you? DNA barcodes reveal cryptic diversity in human-biting black flies (Diptera:

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Simuliidae)

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Panya Jomkumsinga, Ubon Tangkawanitb, Komgrit Wongpakamc, Pairot Pramuala*

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Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai

District, Maha Sarakham 44150, Thailand

Department of Entomology and Plant Pathology, Faculty of Agriculture,

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Khon Kaen University, Khon Kaen, 40002 Thailand Walai Rukhavej Botanical Research Institute, Mahasarakham University, Kantharawichai

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District, Maha Sarakham, 44150 Thailand

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*Corresponding author: Pairot Pramual, Ph.D. Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150 Thailand Phone: 66(0)43 754245

2 Fax: 66(0)43 754245 E-mail: [email protected]

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Graphical abstract

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Genetic diversity of three human-biting black fly species was examined. DNA barcoding sequences indicate cryptic diversity in these human-biting black flies. New records for human-biting black fly species in Thailand were revealed based on DNA barcode.

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Abstract Black flies (Simuliidae) are important biting insects and vectors of diseases agents of humans and livestock. Thus, understanding the taxonomy and biodiversity of these insects is crucial for control and management of these diseases. In this study, we used mitochondrial

3 cytochrome c oxidase I sequences to examine genetic diversity of three human-biting and possible vector black fly taxa; the Simulium asakoae species-complex, S. chamlongi and S. nigrogilvum. High levels of genetic diversity (>3.5% intraspecific genetic divergence) were found in all three taxa. Phylogenetic analyses indicated that the S. asakoae complex can be divided into seven groups with the largest group consisting of specimens from Thailand,

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Malaysia and Myanmar. This group most likely represents true S. asakoae. The remaining

haplotypes formed groups with conspecific haplotypes or with other closely related species.

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Among these groups, one including S. monglaense and another including S. myanmarense

suggest that certain specimens identified as S. asakoae most likely belong to those species.

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Therefore, they constitute new locality records for Thailand and also represent new records of

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anthropophily. Members of S. chamlongi are not monophyletic as its clade also included S.

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hackeri. A median joining network revealed strong geographic associations of the haplotypes

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of S. nigrogilvum suggesting limitation of gene flow. Because this species occurs mainly in

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high elevation habitats, low land areas could present a barrier to gene flow.

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Keywords: COI; genetic diversity; Simulium; vector

4 Introduction Black flies (Diptera: Simuliidae) are significant pests and vectors of certian disease agents of both humans and animals (Crosskey, 1990; Adler et al., 2004). Onchocerciasis, the disease caused by a filarial nematode, Onchocerca volvulus, is among the most well-known

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diseases transmitted by black flies. Many black fly species can also transmit parasites such as Leucocytozoon and Trypanosoma among wild and captive animals. Black fly biting also

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causes nuisance and irritation problems for both human and livestock in many parts of the world (Crosskey, 1990; Adler et al., 2004).

In Thailand, a total of 109 black fly species have been recorded (Adler, 2019;

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Takaoka et al., 2018a, b, c, d; Tangkawanit et al., 2018). Among these, seven are human-

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biting species; S. asakoae Takaoka & Davies complex, S. chamlongi Takaoka & Suzuki, S.

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doipuiense Takaoka & Choochote complex, S. nodosum Puri, S. nigrogilvum Summers, S.

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rufibasis Brunetti and S. umphangense Takaoka, Srisuka & Saeung (Choochote et al., 2005;

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Pramual et al., 2016; Takaoka et al., 2017a). Three species (S. asakoae complex, S. nodosum and S. nigrogilvum) are potential vectors of Onchocerca sp. (Fukuda et al., 2003; Takaoka et

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al., 2003) and two species of the subgenus Gomphostilbia (S. asakoae complex and S. chumpornense Takaoka & Kuvangkadilok) are possibly the vectors of Leucocytozoon sp.

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transmitted among birds and domestic chickens in Thailand (Jampato et al., 2019). Genetic diversity and DNA barcoding of wild adults of three human-biting black fly

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species in Thailand; S. nodosum, S. nigrogilvum and S. doipuiense complex have been investigated previously (Pramual et al., 2016) and considerable levels of intraspecific genetic diversity in these black fly species was revealed. Cryptic diversity has also been revealed in the S. doipuiense complex where four divergent lineages were detected (Pramual et al., 2016). Members of a species complex can differ in aspects of their biology such as vector

5 capacity and biting habit. Thus, understanding genetic diversity and taxonomic status are crucial for further control and management of these potential vectors (Tabachnick and Black, 1995). In this study, we examine genetic diversity and DNA barcoding based on wild adult specimens of human-biting species, namely, the S. asakoae complex, S. nigrogilvum and S.

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chamlongi. The S. asakoae complex belongs to the S. asakoae species-group (Takaoka, 2012).

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There are 36 morphospecies assigned into this group of which three species occur in Thailand (Adler, 2019). The S. asakoae complex is geographically widespread being recorded in Malaysia, Vietnam, China and throughout Thailand (Pramual et al., 2012; Adler, 2019). Wide

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geographic distribution of this species complex may be related to its ability to utilize diverse

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habitats. The S. asakoae complex occurs over a wide elevation range from 250 m to 1,600 m

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above sea level, in diverse stream sizes, velocities and water conductivities (Jitklang et al.,

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2008; Pramual et al., 2012). Cytogenetic study found that larvae of specimens morphologically identified as S. asakoae were comprised of at least four lineages

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characterized by many chromosome rearrangements (Jitklang et al., 2008). Molecular examination based on DNA barcoding sequences also detected a high level of genetic

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diversity (Pramual et al., 2011; Pramual and Adler, 2014) supporting the cytogenetic results.

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There are also geographic variations in the biting habit of the S. asakoae complex. Species in this complex were first reported as human-biter in Doi Saket District, Chiangmai Province, northern Thailand (Choochote et al., 2005). However, there are no reports of this species

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complex biting humans from other geographic regions of Thailand, although both immature and adults stages have been collected throughout the country. Given that the S. asakoae complex is geographically and ecologically widespread and with great genetic diversity at the chromosomal level; it would be useful to examine diversity at the molecular level. Although there are reports on the genetic variations of this species based on DNA barcoding sequences

6 (Pramual et al., 2011; Pramual and Adler, 2014), only a limited number of specimens were investigated and none of these studies used wild adult specimens. In addition, many closely related species of the S. asakoae species-group with very similar morphological characteristics have been described recently along with their DNA barcoding sequences (Takaoka et al., 2014, 2017b). Thus, it will be very useful to compare DNA barcoding

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sequences of these species.

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Simulium chamlongi belong to the S. variegatum species-group with 56

morphospecies recorded globally, of which five species occur in Thailand (Adler, 2019). Simulium chamlongi is found only at high elevation areas (>900 m above sea level) and is

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often found at very low abundance compared with co-existing species in the same habitat

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(Srisuka et al., 2015). A previous DNA barcoding study based on specimens of immature

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stages (larva and pupa) revealed high intraspecific genetic variation (max. K2P genetic

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distance 5.33%) despite small sample size (n = 3) and sampling from a single location (Pramual and Adler, 2014). Therefore, extending geographic sampling and increasing sample

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size will be useful to determine the level of genetic variation of this rare species.

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Simulium nigrogilvum belongs to the S. indicum species-group with only four species assigned to it globally, and three of these occur in Thailand (Adler, 2019). Simulium

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nigrogilvum is among the most well-known black fly species for local people in northern Thailand because of its biting habit. The vernacular name in northern Thailand for S.

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nigrogilvum is “khun leuang” meaning “yellow black fly” in reference to the conspicuous yellow thorax of adults. DNA barcoding studies have revealed a considerable level of genetic variation within this species with maximum intraspecific genetic divergence of 3.87%. There were also some indication of geographic isolation of populations from eastern and western sides of the country (Pramual et al., 2016), however the limited number of specimens available precluded more thorough analysis. Therefore, we made additional collections of

7 actively human-biting wild adults to determine genetic variation of this important humanbiting species. Materials and Methods Specimen collection and identification

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Wild adult black fly specimens were collected in the immediate vicinity of humans from 10 sites in north and northeastern Thailand (Table 1) using a sweep net. Specimens were also

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collected directly into sample tubes while in the act of biting humans. Specimens were

preserved in 80% ethanol and stored on ice during field work and subsequently transferred to

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a laboratory refrigerator maintained at -20° C until processing. Identifications of the

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specimens followed the keys and descriptions of black flies in Thailand and nearby countries

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(Takaoka and Choochote 2004; Takaoka et al., 2014, 2017a, b).

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DNA extraction, amplification and sequencing

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Genomic DNA was extracted from the entire fly using Vivantis GF-1 tissue DNA extraction kit (Selangor Darul Ehsan, Malaysia). A fragment of approximately 650 bp of the

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mitochondrial cytochrome c oxidase subunit I (COI) was amplified using the primers LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198: (5'-

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TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al., 1994). Polymerase chain reaction (PCR) methods followed those of Rivera & Currie (2009). The PCR products were

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checked with a 1% agarose gel and cleaned using the HiYield Gel/PCR DNA Extraction Kit (RBC Bioscience). Purified PCR products were sent for sequencing at First BASE Laboratories Sdn. Bhd. sequencing services (Seri Kembangan, Selangor, Malaysia).

8 Data analysis A total of 166 sequences (GenBank accession numbers: MK628740-MK628904) were obtained from three morphologically identified human-biting black fly species (Table 1). The COI sequences of these species from previous publications (Pramual and Adler, 2014; Pramual et al., 2016) and from public databases (e.g. NCBI GenBank) were also

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included for data analyses (Table 1). Haplotype diversity and nucleotide diversities were

calculated using the Kimura 2-parameter (K2P) model in Arlequin version 3.5.1.2 (Excoffier

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and Lischer, 2010).

To examine relationships in the collected specimens, between human-biting black fly

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species of the same species-group, three phylogenetic methods were used: neighbor-joining

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(NJ), maximum likelihood (ML) and Bayesian analysis (BA). Sequences of the other species

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belonging to the S. asakoae species-group and S. variegatum species-group were obtained

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from the NCBI database. The NJ tree analysis was performed in MEGA X (Kumar et al., 2018) based on Kimura 2-parameter (K2P) model. Branch support was estimated using the

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bootstrapping method with 1,000 replicates. The ML tree analysis was inferred in the RAxML web server version (https://raxml-ng.vital-it.ch) (Kozlov et al., 2018). Bayesian

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analysis was performed with MrBayes 3.2 (Ronquist et al., 2012), and was run for 2,000,000 generations with sampling sequences of 100 generations. Relationships between species

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within the S. indicum species-group were not inferred because there are no reports of COI sequences of other members of this group. Instead, we estimated the intraspecific genetic

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relationships using the median-joining (MJ) method (Bandelt et al., 1999) in NETWORK ver. 5.0.0.3 (http://www.fluxus-engineering.com).

9 Results Genetic diversity, structure and phylogenetic relationships Simulium asakoae complex A total of 103 sequences (98 sequences from this study and five from Pramual and Adler, (2014)) were included in the intraspecific genetic diversity analysis. Forty-six

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haplotypes were identified. Haplotype diversity was 0.8747 and nucleotide diversity was 0.0299 with a maximum intraspecific genetic divergence of 8.4718% (Table 2).

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We retrieved all available COI sequences (nine species) of the members of S. asakoae

species-group from the NCBI database to infer genetic relationships. The S. asakoae complex

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resolved into two distinct clades based on the NJ tree analysis. Most specimens of S. asakoae

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complex were included in clade I with strong support (Fig. 1). Sequences of S. asakoae from

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Malaysia and Thailand retrieved from public databases also belong to this clade. The remaining specimens were in clade II where they clustered into 10 groups. Specimens of the

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S. asakoae complex belonged to six groups (II-1, II-4 – 8), with the other four groups

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consisting of other closely related species. Group II-1 comprised nine haplotypes of the S. asakoae complex from diverse locations in Thailand and S. monglaense from Myanmar

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although with very low (<50%) support. One haplotype of the S. asakoae complex from Thailand formed group II-4 with S. myanmarense and this group was strongly supported.

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Group II-5 and II-7, each with strong support and comprised of two haplotypes of the S. asakoae complex from Thailand. One haplotype of the S. asakoae complex from Thailand

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formed a group with S. tanahrataense (II-6) and another haplotype with S. brinchangense (II8); albeit with long branch length and was not significantly (<50%) supported. Both S. tanahrataense and S. brinchangense were originally described from Malaysia (Takaoka et al., 2014).

10 Simulium chamlongi A total of 23 sequences were included in genetic diversity analysis and only five haplotypes were identified with haplotype diversity of 0.5810 and nucleotide diversity of 0.0064. Despite low haplotype and nucleotide diversity, the maximum intraspecific genetic divergence within species was high (4.36%) based on K2P model (Table 2).

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We obtained COI sequences of four species of the S. variegatum species-group from

NCBI GenBank; S. argyreatum Meigen, S. variegatum Meigen, S. monticola Friederichs and

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S. hackeri Edwards. The NJ tree (Fig. 2) revealed two main clades. Simulium argyreatum, S. variegatum and S. monticola formed clade I while S. chamlongi and S. hackeri comprised

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clade II. The five haplotypes of S. chamlongi from Thailand are not monophyletic. One

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haplotype (KF289467) was retrieved in a sister-group relationship with the group that

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included S. chamlongi and S. hackeri from Malaysia. This haplotype was genetically highly

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different from conspecific haplotypes (>4.0% K2P genetic divergence). Three haplotypes formed another group within clade II with strong support. Sequences of S. hackeri from

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Simulium nigrogilvum

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Malaysia formed a monophyletic clade with strong support.

A total of 93 sequences were included in genetic variation analysis for S. nigrogilvum;

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47 were obtained in this study and the others were from our previous publication (Pramual et al., 2016). Fifty-four haplotypes were identified with the haplotype diversity of 0.9596,

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nucleotide diversity of 0.0183 and maximum intraspecific genetic divergence of 3.5541% (Table 2). The median-joining network (Fig. 3) revealed two clusters of S. nigrogilvum that were mostly geographically associated. All of the haplotypes from the western side of the northern region (CM1, CM2, KP, MS) formed one cluster and haplotypes from the eastern side of the

11 northern region (UD) formed the other cluster. There were two exceptions where haplotypes from the eastern side were connected to the western group. To further examine genetic structure, we used a population genetic approach based on the population pairwise FST analysis. The population pairwise FST values were calculated in Arlequin software. Because of the small sample size (n = 2) for the population from Ban Pang Fan, Doi Saket District,

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Chiangmai Province (see Table 1) this location was excluded from FST analysis. Population

pairwise FST (Table 3) revealed that the UD population was genetically significantly different

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from all other populations. The KP population is also genetically significantly different from all other populations except that from the nearby location (MS). No significant genetic

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differentiation was found between Chiangmai populations (CM1 and CM2) and the Mae

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Hong Son (MS) populations.

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Discussion

Several previous studies using molecular approaches have revealed hidden diversity

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in many insect species including black flies (e.g. Pramual et al., 2011; Pramual and Adler, 2014). In this study, the COI barcoding region yet again revealed cryptic diversity in the three

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human-biting taxa that are potential vectors of Onchocerca sp. and Leucocytozoon sp. in

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Thailand. Genetic diversity of each human-biting black fly species is discussed below. Genetic diversity of Simulium asakoae complex The results support previous cytological analysis that found that S. asakoae is a

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species complex comprised of four chromosomal lineages (Jitklang et al., 2008). A previous molecular analysis based on COI barcoding sequences: (albeit with a limited number of specimens), divided S. asakoae into two divergent lineages (Pramual and Adler, 2014). This study, based on wider geographic sampling and larger sample size, provides further support that S. asakoae is a species complex. High levels of intraspecific genetic diversity (max.

12 value of 8.47%) were found among the specimens included in the analysis. The NJ tree revealed that there are two main clades among the specimens that were all morphologically identified as S. asakoae. The largest clade most likely represents true S. asakoae. This species was originally described from Malaysia (Takaoka and Davies, 1995) and has been recorded in Thailand, Vietnam and China (Adler, 2019). Most (29 haplotypes) of our specimens

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clustered in this lineage alongside sequences of S. asakoae from Malaysia (Low et al., 2015) and those from Thailand (Pramual et al., 2011) and Myanmar (Takaoka et al., 2017b).

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Nine haplotypes of the specimens that were morphologically identified as S. asakoae were clustered with S. monglaense and one haplotype was clustered with S. myanmarense,

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two species of the S. asakoae group recently described from Myanmar (Takaoka et al.,

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2017b). There are three possible explanations. Firstly, Simulium monglaense, S.

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myanmarense and S. asakoae are the same biological species. Secondly, they are full

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biological species but DNA barcode sequences could not differentiate them. Or more likely, our specimens of morphological species comprise more than one species (i.e. some

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specimens are misidentified). The first and second possibilities are unlikely because these species possess distinct morphological characteristic at some life stages although females are

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morphologically very similar (Takaoka et al., 2017b). DNA barcode analysis also found clear distinction with large genetic distances between S. asakoae, S. monglaense and S.

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mynmarense (Takaoka et al., 2017b). Genetic distances between our specimens compared with S. monglaense are between 0.17% and 2.62% and fall in the range of intraspecific

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genetic divergence reported for tropical black fly species (Pramual et al., 2011; Pramual and Adler, 2014). Comparisons between these clades revealed them to be genetically highly different, with the K2P genetic distance between 6.48 and 8.14%. The level of genetic differentiation between S. monglaense and S. asakoae is similar to those reported by Takaoka et al. (2017b) (7.00 – 7.68%). In addition, a haplotype clustered with S. myanmarense showed

13 a low level of genetic differentiation (0.33%) but was very high when compare to the S. asakoae clade (7.31 – 8.14%). Therefore, the most likely explanation is that some of our specimens that were morphologically identified as belonging to the S. asakoae complex are actually S. monglaense or S. myanmarense. Although these species can be readily differentiated based on morphological characteristics, this is only feasible with certain life

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stages. Adult females are morphologically very similar (Takaoka et al., 2017b). The results of this study therefore, indicate the necessity for the integration of multiple characters for

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species identification. They also support previous findings that COI DNA barcoding

sequences are very effective for species identification of black flies in tropical regions

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(Pramual et al., 2011; Pramual and Adler, 2014). Simulium monglaense and S. myanmarense,

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which were originally described from Myanmar, are reported here for the first time from

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Thailand. This is the first report of their human-biting habits.

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Two haplotypes each form a clade with S. tanahrataense and S. bringchangense, both described from Malaysia (Takaoka et al., 2014). The level of genetic differentiation (2.66%

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compared to S. tanahrataense and 1.99% compared to S. bringchangense) are not high compared to known interspecific genetic divergence among tropical black fly species. These

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levels of genetic differentiation are within the range of intraspecific genetic variation recorded for black fly species in Thailand. Further morphological examination is required to

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assess whether some closely related haplotypes that were morphologically identified as S. asakoae in Thailand are S. tanahrataense or S. bringchangense. Note that the female of S.

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tanahrataense is unknown (Takaoka et al., 2014) so comparison is not possible.

Genetic diversity of Simulium chamlongi Previous DNA barcoding studies detected high levels of intraspecific genetic diversity in S. chamlongi despite a limited number of specimens (only three specimens from a single

14 location) (Pramual and Adler, 2014). In contrast, additional specimens from other locations in the present study revealed extremely low diversity. Only two haplotypes were identified among 20 specimens obtained in the present study. Low haplotype diversity in S. chamlongi populations could be influenced by several factors such as small population size or sampling of consanguineous individuals. The latter explanation is unlikely given that other species

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collected using the same method did not show signs of low haplotype diversity. Simulium

chamlongi is a high elevation species found only at >900 m above sea level (this study and

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Srisuka et al., 2015). The immature stages (larva and pupa) are often found with much lower abundance in stream habitats compared to other co-existing species (Srisuka et al., 2015).

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Therefore, we hypothesize that low haplotype diversity of S. chamlongi is due to small

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effective population size. Further investigation of the biology and ecology of this species will

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be useful to test this hypothesis.

Genetic diversity of Simulium nigrogilvum

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Genetic variation in human-biting specimens of S. nigrogilvum has been reported previously with maximum intraspecific genetic variation of 3.87% based on COI sequences

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(Pramual et al., 2016). In the present study, an increased number of specimens from wider geographic areas revealed a similar level of intraspecific genetic variation (max. K2P genetic

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divergence is 3.55%). Although the intraspecific genetic divergence was not high compared to other black fly species complexes in Thailand (Pramual and Adler, 2014), the

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mitochondrial genealogy revealed obvious geographic associations of the haplotypes. Geographic association of haplotypes without deeply divergent lineages is characteristic of species in which gene flow is limited in the absence of long-term isolation (Avise, 2000). This interpretation is also support by highly significant differences in genetic structure between populations from eastern and western sides of the country.

15 Simulium nigrogilvum is a habitat specialist species because it occurs mainly at high elevations (>800 m above sea level) (Pramual et al., 2016), although it occurs rarely at elevations as low as 600 m. Ecologically specialized species are geographically isolated because of the disjunct distribution of suitable habitat. Population genetic studies in other habitat specialist black fly species such as S. weji (immature stages requiring calcareous

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streams) or the S. feuerborni complex (occur only at high elevation areas) show greater levels of genetic differentiation between populations than in species that utilize wider ecological

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conditions (Pramual and Wongpakam, 2013; Pramual and Pangjanda, 2015). Species that dwell in high elevation mountain habitats are geographically isolated by the intervening

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lowlands. This potentially forms a strong barrier to gene flow, thus facilitating genetic

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differentiation (Finn and Adler 2006; Finn et al., 2006; Pramual and Wongpakam 2013).

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Conclusion

In conclusion, our results indicate that there is hidden diversity in human-biting black

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fly species as revealed by their DNA barcoding sequences. Our results also indicate the utility an integrated approach for species identification in black flies. Some species could be

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differentiated morphologically only on characters of a particular life state. In contrast, identification though DNA barcoding is independent of developmental stage. On the basis of

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DNA barcoding, we added S monglaense and S. myanmarense to the black fly fauna of

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Thailand, further recording them as human biters for the first time.

16 Acknowledgements This research has been supported by Center of Excellence on Biodiversity (BDC), Office of Higher Education Commission (BDC-PG2-160008). We would like to thank Dr. Adrian Plant

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for valuable comments on an earlier version of the manuscript.

17 References Adler, P.H. (2019) World blackflies (Diptera: Simuliidae): a comprehensive revision of the taxonomic and geographical inventory [2019]. (accessed 09.03.19) https://biomia.sites.clemson.edu/pdfs/blackflyinventory.pdf

America. Cornell University Press, Ithaca, New York, USA.

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Adler, P.H., Currie, D.C., Wood, D.M., 2004. The Black Flies (Simuliidae) of North

Avise, J. C. 2000. Phylogeography: the history and formation of species. Harvard University

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Press.

Bandelt, H. J., Forster, P., Röhl, A. 1999. Median-joining networks for inferring intraspecific

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phylogenies. Mol. Biol. Evol. 16(1), 37–48.

N

Choochote, W., Takaoka, H., Fukuda, M., Otsuka, Y., Aoki, C., Eshima, N., 2005.

A

Seasonal abundance and daily flying activity of black flies (Diptera Simuliidae)

M

attracted to human baits in Doi Inthanon National Park, northern Thailand. Med. Entomol. Zool. 56, 335–348.

ED

Crosskey, R.W., 1990. The Natural History of Blackflies. John Wiley & Sons London. Excoffier, L., Lischer, H. E. 2010. Arlequin suite ver 3.5: a new series of programs to

PT

perform population genetics analyses under Linux and Windows. Mol. Ecol. Resour. 10(3), 564–567.

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Finn, D.S., Adler, P.H. 2006. Population genetic structure of a rare high-elevation black fly, Metacnephia coloradensis, occupying Colorado lake outlet streams. Fresh. Biol. 51,

A

2240–2251.

Finn, D.S., Theobald, D.M., Black, W.C., Poff, N.L. 2006 Spatial population genetic structure and limited dispersal in a Rocky Mountain alpine stream insect. Mol.r Ecol. 15, 3553–3566. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for

18 amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Fukuda, M., Choochote, W., Bain, O., Aoki, C., Takaoka, H., 2003. Natural infections with filarial larvae in two species of black flies (Diptera: Simuliidae) in northern Thailand. Jpn. J. Trop. Med. Hyg. 31 (2), 99–102.

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Jitklang, S., Kuvangkadilok, C., Baimai, V., Takaoka, H., Adler, P. H. 2008. Cytogenetics and morphotaxonomy of the Simulium (Gomphostilbia) ceylonicum species group

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(Diptera: Simuliidae) in Thailand. Zootaxa, 1917, 1–28.

Jumpato, W., Tangkawanit, U., Wongpakam, K., Pramual, P. 2019. Molecular detection of

U

Leucocytozoon (Apicomplexa: Haemosporida) in black flies (Diptera: Simuliidae)

N

from Thailand. Acta Trop. 190, 228–234.

A

Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K. 2018. MEGA X: Molecular

M

evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549.

ED

Kozlov, A. M., Darriba, D., Flouri, T., Morel, B., Stamatakis, A. 2018. RAxML-NG: A fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inference,

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bioRxiv. doi: https://doi.org/10.1101/447110

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Low, V. L., Takaoka, H., Adler, P. H., Ya'cob, Z., Norma-Rashid, Y., Chen, C. D., SofianAzirun, M. 2015. A multi‐locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence

A

for distinct evolutionary lineages. Med. Vet. Entomol. 29(3), 330–337.

Pramual, P., Pangjanda, S. 2015. Effects of habitat specialization on population genetic structure of black fly Simulium weji Takaoka (Diptera: Simuliidae). J. Asia Pac. Entomol. 18(1), 33–37. Pramual, P., Adler, P.H., 2014. DNA barcoding of tropical black flies (Diptera: Simuliidae)

19 of Thailand. Mol. Ecol. Resour. 14, 262–271. Pramual, P., Kuvangkadilok, C., Jitklang, S., Tangkawanit, U., Adler, P. H. 2012. Geographical versus ecological isolation of closely related black flies (Diptera: Simuliidae) inferred from phylogeny, geography, and ecology. Org. Divers. Evol. 12(2), 183–195.

flies (Diptera: Simuliidae) in Thailand. Acta Trop. 164, 33–40.

IP T

Pramual, P., Thaijarern, J., Wongpakam, K., 2016. DNA barcoding of human-biting black

SC R

Pramual, P., Wongpakam, K., Adler, P.H., 2011. Cryptic biodiversity and phylogenetic

relationships revealed by DNA barcoding of Oriental black flies in the subgenus

U

Gomphostilbia (Diptera: Simuliidae). Genome 54, 1–9.

N

Pramual, P., Wongpakam, K. 2013. Population genetics of the high elevation black fly

A

Simulium (Nevermannia) feuerborni Edwards in Thailand. Entomol. Sci.16, 298–308.

M

Rivera, J., Currie, D.C., 2009. Identification of Nearctic black flies using DNA barcodes (Diptera: Simuliidae). Mol. Ecol. Resour. 9, 224–236.

ED

Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Hӧhna, S., Larget, B., Liu, L., Suchard, M. A., Huelsenbeck, J. P. 2012. MrBayes 3.2:

PT

efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61(3), 539–542.

CC E

Srisuka, W., Takaoka, H., Otsuka, Y., Fukuda, M., Thongsahuan, S., Taai, K., Choochote, W., Saeung, A. 2015. Seasonal biodiversity of black flies (Diptera: Simuliidae) and

A

evaluation of ecological factors influencing species distribution at Doi Pha Hom Pok National Park, Thailand. Acta Trop. 149, 212–219.

Tabachnick, W.J., Black, W.C., 1995. Making a case for molecular population genetic studies of arthropod vectors. Parasitol. Today 11, 27–30. Takaoka, H. 2012. Morphotaxonomic revision of Simulium (Gomphostilbia) (Diptera:

20 Simuliidae) in the Oriental Region. Zootaxa, 3577, 1–42. Takaoka, H., Choochote, W., 2004. A list of and keys to black flies (Diptera: Simuliidae) in Thailand. Trop. Med. Health 32, 189–197. Takaoka, H., Choochote, W., Aoki, C., Fukuda, M., Bain, O., 2003. Black flies (Diptera: Simuliidae) attracted to humans and water buffalos and natural infections

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with filarial larvae, probably Onchocerca sp., in northern Thailand. Parasite 10 (1), 3–8.

SC R

Takaoka, H., Davies, D. M. 1995. The Black Flies (Diptera: Simuliidae) of West Malaysia. Kyushu University Press, Fukuoka.

Takaoka, H., Sofian-Azirun, M., Ya’cob, Z., Hashim, R. 2014. Two new species of Simulium

N

U

(Gomphostilbia) (Diptera: Simuliidae) from Cameron’s Highlands, Peninsular

A

Malaysia, with keys to 21 species of the Simulium asakoae species-group. Zootaxa,

M

3765(1), 54–68.

Takaoka, H., Srisuka, W., Saeung, A. 2017a. A new human-biting black fly species of

945–948.

ED

Simulium (Simulium) (Diptera: Simuliidae) from Thailand. J. Med. Entomol. 54(4),

PT

Takaoka, H., Srisuka, W., Low, V. L., Maleewong, W., Saeung, A. 2017b. Two new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Myanmar, and their

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phylogenetic relationships with related species in the S. asakoae species-group. Acta Trop. 176, 39–50.

A

Takaoka, H., Srisuka, W., Low, V.L., Saeung, A., 2018a. A new species of the Simulium (Simulium) striatum species group (Diptera: Simuliidae) from Thailand, and its differentiation from two related species based on a fast-evolving nuclear gene. J. Med. Entomol. 55 (3), 561–568. Takaoka, H., Srisuka, W., Low, V.L., Saeung, A., 2018b. Five new species of the Simulium

21 decuplum subgroup of the Simulium (Gomphostilbia) batoense species-group (Diptera: Simuliidae) from Thailand and their phylogenetic relationships. Acta Trop. 182, 271–284. Takaoka, H., Srisuka, W., Low, V.L., Saeung, A., 2018c. A new species and a new record of the Simulium (Gomphostilbia) gombakense species-group (Diptera: Simuliidae) from

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Thailand. Acta Trop. 185, 156–166.

Takaoka, H., Srisuka, W., Saeung, A., 2018d. A new species of Simulium (Asiosimulium)

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(Diptera: Simuliidae) from Thailand. J. Med. Entomol. 55 (3), 569–574.

Takaoka, H., Suzuki, H., 1984. The black flies (Diptera: Simuliidae) from Thailand.

U

Jpn. J. Sanit. Zool. 35, 7–45.

N

Tangkawanit, U., Wongpakam, K., Pramual, P., 2018. A new black fly (Diptera:

A

Simuliidae) species of the subgenus Asiosimulium Takaoka Choochote from Thailand.

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ED

M

Zootaxa, 4388 (1), 111–122.

A

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PT

ED

M

A

N

U

SC R

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22

Figure 1 Neighbor-joining tree constructed based on mitochondrial COI sequences for 46 haplotypes of Simulium asakoae complex and members of the S. asakoae species-group. Bootstrap values for neighbor joining, maximum likelihood and posterior probability of Bayesian analysis are shown above or near the branches.

A

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PT

ED

M

A

N

U

SC R

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23

Figure 2 Neighbor-joining tree based on mitochondrial COI sequences for four haplotypes of Simulium chamlongi and four species members of the S. variegatum species-group. Bootstrap values for neighbor joining, maximum likelihood and posterior probability of Bayesian analysis are shown above or near the branches.

U

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24

N

Figure 3 Median-joining network of 93 sequences of Simulium nigrogilvum in Thailand.

A

Circles represent haplotypes and sizes and are relative to the number of individuals sharing

A

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site. Black, west; white, east.

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the specific haplotype. Haplotypes labeled according to geographic location of the sampling

25 Table 1 Details of the collection sites of black fly specimens used in this study. Species

Location

n

Latitude / Longitude

(Code) S. asakoae

Song Khon

complex

waterfall, Phu

17°21′13″N/101°24′23″E

25

Elevation

Collection

(m)

date

773

27 May 2017

Loei Province Ban Song Khon, 2

17°21'38"N/101°23′50′′E

681

2016

District, Loei Province Lert Phop

17°29'59"N/101°20′10′′E

3

N

Ruea District, Loei Province

18°59'03"N /99°20′07′′E

District,

ED

Chiang Mai

990

8

18◦59′20″N/99◦20′23″E

22 Jan 2018

1,050

PT

Road to Ban

23 Mar 2016

M

A

51

1, Doisaket

Province

1,134

U

waterfall, Phu

Ban Pang Bong

23 Mar

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, Phu Ruea

IP T

Ruea District,

Pang Bong,

22 Jan 2018

Doisaket

CC E

District,

Chiangmai Province

A

Song Khwae

3

19°21′11″N/100°33′44″E

722

District, Nan

21 Jan 2018

Province Khao Kho District, Phetchabun

1

16°39′44″N/101°01′26″E

920

24 Mar 2018

26 Species

Location

n

Latitude / Longitude

(Code)

Elevation

Collection

(m)

date

671

31 Mar

Province Suan Hom

4

17°02′49″N/101°45′42″E

Waterfall, Nong

2018

Hin District,

Pa Pae, Mae

1

19°07'35"N /98°45'14"E

703

Taeng District,

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Province 1** 17°29'54"N /101°20'07"E

Province

2008

8 Apr 2008

A

M

1**

ED

Chiangmai

Ban Na Ngew,

1,156

/101°20'05"E

Province

Province

10 Feb

N

1** 17°29'35"N

waterfall, Loei

Ban Pha Mon,

1,152

U

waterfall, Loei

Hin Sam Chan

17 Jan 2015

Chaing Mai

Huai Toei

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Loei Province

2** 19°49'35"N /98°02'05"E

814

PT

Mae Hong Son Province

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Total S. chamlongi

Ban Pang Bong,

103 19

Doisaket

18°59'03" N /99°20′07′′

990

E

22 Jan 2018

A

District, Chiangmai Province Road to Ban Pang Bong, Doisaket District,

1

18°59′20″ N/99°20′23″ E

1,050

22 Jan 2018

27 Species

Location

n

Latitude / Longitude

(Code)

Elevation

Collection

(m)

date

1,142

19 Nov

Chiangmai Province Ban Nam Dan,

3** 19°11′19″ N/101°04′40″

Phukha District,

E

2008

Total

23

S.

Ban Pang Bong,

42

nigrogilvum

Doi saket

18°59'03"N /99°20′07′′ E

990

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Nan Province

22 Jan

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2018

District, Chiang Mai Province (CM1)

18◦59′20″N/99°20′23″ E

5

U

Road to Ban

22 Jan 2018

N

Pang Bong,

1,050

A

Doisaket District, Chiang

M

Mai Province (CM2) 4*

ED

Chong Yen,

16°05′53′′N/99°06′24″ E

1,276

Kamphaeng

20 Feb 2014

PT

Phet Province (KP)

3*

19°26′12′′N/98°29′17″ E

849

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Ban Pang Peak, Pang Mapha,

17 Jan 2015

Mae Hong

A

Son Province (MS)

Phu Soi Dao, Uttaradit

37* 17°44′12′′N/100°59′18″

1,589

E

27 Oct 2015

Province (UT) Ban Pang Fan, Doi saket

2** 19°01′00′′N/99°18′13″ E

618

23 Nov 2008

28 Species

Location

n

Latitude / Longitude

(Code)

Elevation

Collection

(m)

date

District, Chiang Mai Province Total

93

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*Sequences from Pramual et al. (2016), **Sequences from Pramual and Adler (2014)

Species

n

Haplotype

Nucleotide diversity

103 (46)

93 (54)

PT

S. nigrogilvum

23 (5)

A

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*P < 0.01, **P < 0.001

Intraspecific genetic divergence (mean) (%) 0 – 8.4718 (2.8563 )

0.014842

0.5810 +/-

0.006358 +/-

0.0930

0.003793

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S. chamlongi

0.0240

0.029955 +/-

M

complex

0.8747 +/-

A

S. asakoae

N

U

(haplotype) diversity

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Table 2 Genetic variation indices of the human-biting black fly species in Thailand.

0 – 4.3619 (0.8060)

0.9596 +/-

0.018287 +/-

0 – 3.5541 (0 –

0.0136

0.009321

1.6990)

29 Table 3 Population pairwise FST between populations of Simulium nigrogilvum in Thailand. CM2

UD

KP

CM1

0

CM2

0.04906

0

UD

0.44501*

0.66828*

0

KP

0.15091*

0.26056*

0.79255*

0

MS

-0.10996

0.01993

0.68704*

0.07416

A

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PT

ED

M

A

N

U

*P < 0.05; Details of population code are provided in Table 1.

MS

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CM1

0

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Population