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Genetic diversity and differentiation in the blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia
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Genetic Diversity and Differentiation in the Blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia Soosai Peranathan Pavitra , Zubaidah Ya’cob , Tiong Kai Tan , Yvonne Ai Lian Lim , Van Lun Low PII: DOI: Reference:
S0001-706X(19)31723-1 https://doi.org/10.1016/j.actatropica.2020.105415 ACTROP 105415
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Acta Tropica
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7 December 2019 19 February 2020 19 February 2020
Please cite this article as: Soosai Peranathan Pavitra , Zubaidah Ya’cob , Tiong Kai Tan , Yvonne Ai Lian Lim , Van Lun Low , Genetic Diversity and Differentiation in the Blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia, Acta Tropica (2020), doi: https://doi.org/10.1016/j.actatropica.2020.105415
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Highlights
High levels of genetic diversity and genetic differentiation were observed among three common species S. vanluni, S. cheongi and S.jeffreyi in Peninsular Malaysia. All three species revealed an intermediate level of gene flow among the populations. The expansion time of S. vanluni was estimated at about 233 112 years ago, followed by S. jeffreyi (232, 948 years ago) and S. cheongi (191, 845 years ago).
Genetic Diversity and Differentiation in the Blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia Soosai Peranathan Pavitraa , Zubaidah Ya’cobb , Tiong Kai Tana , Yvonne Ai Lian Lima & Van Lun Lowb a
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur,
Malaysia b
Higher Institution Centre of Excellence (HiCOE), Tropical Infectious Diseases Research and
Education Centre (TIDREC), University of Malaya, Kuala Lumpur, Malaysia *Corresponding author: [email protected] ABSTRACT The population genetic structure of S. vanluni, S. cheongi and S. jeffreyi were determined from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) across different states in Peninsular Malaysia. High levels of genetic diversity and genetic differentiation were observed among three species in Peninsular Malaysia. All three species revealed an intermediate level of gene flow among the populations. Negative values of Fu’s Fs and low values of Raggedness index supported the hypothesis of population expansion in S. vanluni, S. cheongi and S. jeffreyi. Keywords: Blackflies, population genetic structure, genetic diversity, genetic differentiation.
1. Introduction Blackflies belonging to the family Simuliidae of the order Diptera, are ecologically and medically important insects (Adler et al., 2004; Currie & Adler, 2008). Blackflies are present in almost all zoogeographical regions over a wide range of elevations except in the Antarctica, deserts and islands without running waters. To date, there are 2,328 species (2,310 living and 18 fossils) of blackflies (Adler & Crosskey, 2019). Of these, a total of 63 species of blackflies are found in Peninsular Malaysia (Takaoka et al., 2019). Adult female blackflies are the vectors of many parasitic diseases across the globe and their immature stages play an important role in stream ecology (Sriphirom et al., 2014). Population genetics of blackflies is the study of genetic variation within and among the blackfly populations and the evolutionary factors that have shaped their genetic variation (Lynch & Walsh, 1998; Hartl & Sunderland, 2000; Crow, 2008; Nielsen & Slatkin, 2013). Population genetic study can reveal morphologically cryptic species as well as evolutionary history in blackflies populations (Low et al., 2014; 2016). Several genetic studies of blackflies have been conducted in Malaysia including the genetic diversity and genetic differentiation in Simulium tani along an elevational gradient (Low et al., 2014), description of a new cryptic species of Simulium vanluni (Ya’cob, 2017), phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups (Low et al., 2015) and characterization of Simulium (Simulium) hackeri Edwards through DNA barcoding (Ya’cob et al., 2018). However, only limited study on population genetic study of blackflies has been conducted in Malaysia. Simulium cheongi Takaoka and Davies 1995, Simulium jeffreyi Takoaka and Davies, 1995 and Simulium vanluni Ya’cob, Takaoka and Sofian-Azirun, 2017 are commonly found in Peninsular Malaysia (Ya’cob et al., 2016a; Pavitra et al., 2019). Two of them belong to
Simulium (s.str.); Simulium vanluni in the S. nobile species-group and Simulium jeffreyi in the S. striatum species-group, while Simulium cheongi in the S. epistum species-group of Gomphostilbia. In view of their ample distribution and abundance in Peninsular Malaysia, these three species of blackflies were selected for a comparative study. Simulium cheongi was first described from Janda Baik, Pahang, Malaysia (Takaoka & Davies, 1995), and subsequently reported in various locations in Peninsular Malaysia (Ya’cob et al., 2016b), Thailand (Takaoka et al., 2009) and Indonesia (Sumatra and Kalimantan) (Takaoka et al., 2000; Takaoka et al., 2017). While, Simulium jeffreyi was originally described from Peninsular Malaysia (Takaoka and Davies 1995). Its distribution includes various localities in Peninsular Malaysia (Ya’cob et al., 2016b), and Lang Son Province in northern Vietnam (Pham, 1999). On the other hand, Simulium vanluni from Peninsular Malaysia was previously treated as Simulium nobile (Crosskey, 1973). A molecular DNA barcoding analysis has revealed three cryptic species, each from Borneo, Java and mainland of Southeast Asia (southern Thailand and Peninsular Malaysia) (Low et al., 2016a). The cryptic species from mainland of Southeast Asia was then formally described as Simulium vanluni by using an integrated morpho-taxonomical and genetic approach. (Ya’cob et al., 2017). Simulim cheongi, S. jeffreyi and S. vanluni may have different evolutionary relationships. To test this hypothesis, the current study aimed to determine their genetic diversity and to evaluate the degree of genetic differentiation, based on the mitochondriaencoded cytochrome c oxidase subunit I (COI) gene.
2. Material and method 2.1. Black fly specimens Blackfly pupae were collected from streams across different states in Peninsular Malaysia: Central: Selangor (S. vanluni, n = 10; S. cheongi, n = 5; S. jeffreyi, n = 10), Northern: Perak (S. vanluni, n = 10; S. cheongi, n = 8; S. jeffreyi, n = 5) and Kedah (S. vanluni, n = 3; S. cheongi, n = 9; S. jeffreyi, n = 9), Southern: Johor (S. vanluni, n = 6; S. cheongi, n = 4), and Eastern: Kelantan (S. vanluni, n = 9; S. cheongi, n = 8), Terengganu (S. vanluni, n = 9; S. cheongi, n = 8) and Pahang (S. vanluni, n = 9; S. jeffreyi, n = 9) (Fig. 1.). The pupae attached on aquatic substrates such as leaves, stems, grasses, plant roots, rocks and twigs were collected by using fine forceps (McCreadie and Colbo, 1991; McCreadie et al., 2005) and stored alive in vials individually for adult emergence. Species of blackflies were identified following the keys and descriptions of Takaoka, (2003) and Ya’cob et al, (2017). 2.2. DNA extraction and PCR amplification DNAs of S. vanluni, S. cheongi and S. jeffreyi were extracted from all reared adult specimens using the G-spinTM Total DNA Extraction Mini Kit according to the manufacturer's instructions. The PCR amplifications were performed by Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems, Inc., Foster City, CA, USA). Amplifications of the COI genes were performed in a final volume of 25 µL containing 0.5–1.0 mg genomic DNA, 2X ExPrime Taq Master Mix (GENETBIO, South Korea), and 10 pmol of each forward and reverse primer. The primers used in this study were LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) (Folmer, 1994). The parameters of PCR amplifications of the COI gene was 1 min at 96oC, followed by 35 cycles of denaturation at
94oC for 1 min; annealing at 55oC for 1 min; extension at 72oC for 1 min 30 s, and a final extension at 72oC for 7 min (Rivera & Currie, 2009). 2.3. DNA sequencing and data analysis The amplified PCR products were viewed in 2% agarose gel pre-stained with SYBR Safe (Invitrogen Corp., Carlsbad, CA, U.S.A.). The PCR products were sequenced and analyzed using an ABI PRISM 377 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA, U.S.A.). Sequencing data were analyzed and edited using ChromasPro 1.7.6 (Technelysium Pty Ltd., Qld, Australia) and BioEdit 7.0.9.0 (Hall, 1999). Representative sequences generated from this study were deposited in the National Center for Biotechnology Information (NCBI) GenBank under the accession numbers MN514637-MN514767. The haplotype networks of S. vanluni, S. cheongi and S. jeffreyi were analyzed using a medianjoining algorithm (Bandelt et al., 1999) in the program Network 4.6. Haplotype diversity (Hd), nucleotide diversity (pi), genetic differentiation (FST), and gene flow (N m) values were performed with the program DnaSP 5.0 (Librado & Rozas, 2009). The levels of genetic differentiation can be categorized as FST> 0.25 (great differentiation), 0.15 to 0.25 (moderate differentiation), and FST< 0.05 (negligible differentiation) (Wright, 1978). The levels of gene flow can be categorized as Nm > 1 (high gene flow), 0.25 to 0.99 (intermediate gene flow), and Nm< 0.25 (low gene flow) (Govindajuru, 1989). Average pairwise distances among different populations based on the Kimura-2-parameter model were calculated using MEGA 7 software. Tajima’s D (Tajima, 1989), Fu’s Fs (Fu, 1997), and mismatch distribution test, Harpending’s raggedness index (Harpending, 1994) were performed with the program DnaSP 5.0 to test for population equilibrium and signature of population expansion. The expansion time was determined assuming 12 generations a year
for tropical blackflies (Pramual et al. 2005) and a divergence rate of 2.3% per million years for insect mitochondrial DNA (Brower 1994). 3. Results The median-joining network of 56 individuals of S. vanluni, 42 individuals of Simulium cheongi and 33 individuals of S. jeffreyi aligned as 531 characters of the COI gene revealed high levels of genetic diversity among populations from different states in Peninsular Malaysia. Simulium vanluni and Simulium cheongi demonstrated a well dispersed haplotypes across all states. Of the 56 individuals of S. vanluni, 24 distinct haplotypes were discovered, six of which were shared among populations whereas S. cheongi had 23 distinct haplotypes with four shared haplotypes from 42 individuals. For S. jeffreyi, a total of 13 distinct haplotypes were identified from 33 individuals and only one was shared between two populations (Fig. 2.). In total data estimates, S. cheongi revealed higher haplotype diversity (0.9310) and nucleotide diversity (0.0081) than S. vanluni and S. jeffreyi. The haplotype diversity of S. vanluni ranged from 0.5333 (Perak population) to 1.0000 (Selangor population) with an average of 0.9240, whereas the nucleotide diversity ranged from 0.0013 (Kedah population) to 0.0057 (Kelantan population) with an average of 0.0060. For S. cheongi, the haplotype diversity ranged from 0.6667 (Johor population) to 1.0000 (Selangor population) with an average of 0.9310, whereas the nucleotide diversity ranged from 0.0013 (Johor population) to 0.0067 (Terengganu population) with an average of 0.0081. Additionally, haplotype diversity of S. jeffreyi ranged from 0.4667 (Selangor population) to 0.8611 (Pahang population) with an average of 0.9130, whereas the nucleotide diversity ranged from 0.0025 (Kedah population) to 0.0077 (Pahang population) with an average of 0.0067. Both S. vanluni and S. cheongi populations originating from Selangor and
S. jeffreyi population from Pahang exhibited highest haplotype diversity (Hd > 0.9). Moderate nucleotide diversity was recorded for S. vanluni from Kelantan, S. cheongi from Terengganu and S. jeffreyi from Pahang. According to neutrality test results, the Fu’s Fs tests revealed negative values for all three species while a negative Tajima’s D value was recorded only in S. vanluni (Table 1). The genetic differentiation (FST) and gene flow (Nm) of S. vanluni, S. cheongi and S. jeffreyi from different states in Peninsular Malaysia are presented in Tables 2, 3 and 4. The majority of the population pairs of all three species showed FST> 0.25, indicating great differentiation. The highest FST value with great genetic differentiation for S. vanluni, S. cheongi and S. jeffreyi were between Kedah- Johor (FST= 0.7852), Kelantan- Johor (FST= 0.8365) and Perak- Kedah (FST= 0.7328), respectively. In contrast, the least differentiation (FST<0.05) for S. vanluni, S. cheongi and S. jeffreyi were found between SelangorTerengganu (FST= 0.0363), Kelantan- Kedah (FST= -0.0325) and Pahang- Selangor (FST= 0.0254). All three species revealed an intermediate level of gene flow (N m= 0.38-0.64) among the populations. An average pairwise distances based on the Kimura 2-parameter model were calculated (Tables 5, 6 and 7). S. vanluni, S. cheongi and S. jeffreyi had greatest genetic distance between Perak- Pahang (0.96), Terengganu- Johor (1.80) and Perak- Kedah (1.00) and lowest genetic distance between Perak- Kedah (0.21), Kelantan- Kedah (0.35) and PerakSelangor (0.50), each respectively. Low values of the Raggedness index were recorded for all three blackflies species in the mismatch distribution analysis (Fig. 3.). The expansion times of S. vanluni and S. jeffreyi are estimated to be 233 000 and S. cheongi with 192 000 years ago.
4. Discussion Population genetics of S. vanluni, S. cheongi and S. jeffreyi was conducted for the first time in Malaysia. This study revealed high levels of genetic diversity among populations from different states in Peninsular Malaysia. The median-joining network demonstrated the most common haplotype (H9 in S. vanluni and H7 in S. cheongi) as the ancestral haplotype in S. vanluni and S. cheongi due to its central position in the network with various lineages extending from it and its significant number of individuals in all population (Posada and Crandall 2001). This haplotype also categorized under stable haplotype because of its various environmental adaptabilities (Xu et al., 2019). Moreover, high level of haplotype diversity recorded for all three species of blackflies (Hd > 0.9) showed strong adaptability to different environmental conditions. S. vanluni, S. cheongi and S. jeffreyi are called as generalist species because of their high tolerance level over an extensive set of physicochemical characteristics which allowed them to extend their distribution across different eco-environmental conditions (Brown, 1984; Pavitra et al., 2019). This was consistent with a previous study conducted in Malaysia that revealed high haplotype diversity in S. tani along different elevation gradient (Low et al., 2014). The results of Fu’s Fs test revealed negative values for all three blackfly species (S. vanluni, S. cheongi and S. jeffreyi), indicating that the populations are undergoing demographic expansion while a negative Tajima’s D value was recorded only in S. vanluni population. It has been shown that Fu’s Fs test is more powerful than Tajima’s D in detecting the population expansion (Fu, 1997), and this would explain that the populations provide strong evidence for recent population expansion in this study. Furthermore, maximum genetic differentiation was found in S. vanluni population between Kedah- Johor and S. cheongi population between Kelantan- Johor. This is attributed
to the large geographical distance between these states; Kedah- Johor with 769.8 km and Kelantan- Johor with 692.9 km. In S. jeffreyi population, the most differentiation was showed between Perak- Kedah (236.1 km). Despite the closer geographical distance between PerakKedah, other anthropological (deforestation, urbanization, and habitat destruction) factors also can affect the gene flow in these states and lead to population differentiation (SQI, 2010; Bashir-Ali, 2014). This corroborates with other previous studies that indicated high levels of genetic differentiation in S. feuerborni and S. angulistylum complexes (Pramual & Kuvangkadilok, 2012; Pramual & Wongpakam, 2013). In contradistinction, low levels of genetic differentiation were observed among populations of S. tani along an elevational gradient in Cameron Highlands, Malaysia (Low et al., 2014). High genetic differentiation between populations can decrease the process of gene flow (Pramual et al., 2011). This hypothesis is consistent with our finding of high genetic differentiation with intermediate gene flow in all three species. Simulium vanluni, S. cheongi and S. jeffreyi that were reported to be distributed at low altitude areas (<500 m) in Peninsular Malaysia (Ya’cob et al., 2016). The restriction to low altitude areas could promote population separation due to the effective gene flow barrier in high altitude areas. Additionally, the values of pairwise genetic distance range from 0.2 to 1.0 in S. vanluni, 0.3 to 1.8 in S. cheongi and 0.5 to 1.0 in S. jeffreyi, further supported the high genetic differentiation between different populations in Peninsular Malaysia. The intraspecific genetic distances for all three species were less than 3% (the cut-off point for species boundary), suggesting the absence of cryptic species in the examined populations. (Low et al., 2016b; Pramual et al., 2016). This hypothesis was further supported by the morphological observation where no distinct characters was observed within the species.
The star-like shapes of S. vanluni and S. cheongi were consistent with the sudden expansion of the mismatch distribution, indicating that population experienced recent demographic expansion. While, S. jeffreyi showed a dominant left-hand peak that indicates a very recent expansion. The expansion time of both S. vanluni and S. jeffreyi were estimated at about 233 000 years ago, followed by S. cheongi (192, 000 years ago). The population expansion of other blackflies has been reported in recent times such as Simulium angulistylum (930,000 years ago) (Pramual & Kuvangkadilok, 2012), Simulium tani (500,000 years ago) (Pramual et al., 2005), Simulium siamense (120,000 years ago) (Pramual et al., 2011) and Simulium aureohirtum (18,000 years ago) (Thaijarern et al., 2014) and Simuliun nodosum (2,600–5,200 years ago) (Chaiyasan & Pramual, 2016). In conclusion, this study has revealed high levels of genetic diversity and genetic differentiation in S. vanluni, S. cheongi and S. jeffreyi across different states in Peninsular Malaysia using mitochondria-encoded cytochrome c oxidase subunit I (COI) gene; however, in-depth studies are still needed. More number of blackflies from different geographical areas and use of multiple genetic markers involving both mitochondrial-encoded and nuclearencoded genes will be useful in unfolding the hidden diversity of the blackfly populations. Acknowledgements This work was supported by research grants from the University of Malaya (Project no. BKP009-2018 and RP021C-16SUS) and Ministry of Energy, Science, Technology, Environment and Climate Change (Project no. FP024-2019A) as well as funding from the Ministry of Education, Malaysia for niche area research under the Higher Institution Centre of Excellence (HICoE) program (Project no. MO002-2019). This project is a part of the first author's PhD research at the University of Malaya, Kuala Lumpur.
Author agreement We declare that this is our original work. It has not been published elsewhere and we have no conflicts of interest concerning the work reported in this paper. All authors have contributed to this study throughout the study design, data collection, data analyses and interpretation. The authors have read and approved the manuscript. Declaration of competing interests We have no conflict of interest to declare.
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Xu, Y., Mai, J., Yu, B., Hu, H., Yuan, L., Jashenko, R, Ji1, R., 2019. Study on the genetic differentiation of geographic populations of Calliptamus italicus (Orthoptera: Acrididae) in Sino-Kazakh border areas based on mitochondrial COI and COII genes. J. Econ. Entomol. 112(4), 1912–1919. Ya’cob, Z., Takaoka, H., Pramual, P., Low, V.L., Sofian-Azirun, M., 2016a. Breeding habitat preference of preimaginal black flies (Diptera: Simuliidae) in Peninsular Malaysia. Acta Trop. 153, 57-63. Ya’cob, Z., Takaoka, H., Pramual, P., Low, V.L., Sofian-Azirun, M., 2016b. Distribution pattern of black fly (Diptera: Simuliidae) assemblages along an altitudinal gradient in Peninsular Malaysia. Parasite Vector, 9, 219. Ya’cob, Z., Takaoka, H., Low, V.L., Sofian-Azirun, M., 2017. First description of a new cryptic species, Simulium vanluni from Peninsular Malaysia: An integrated morphotaxonomical and genetic approach for naming cryptic species in the Family Simuliidae. Acta Trop. 165, 31- 39. Ya’cob, Z., Takaoka, H., Low, V. L., Sofian-Azirun, M., 2018. Characterization of Simulium (Simulium) hackeri Edwards (Diptera: Simuliidae) from Malaysia: Morphological description of the pupa and larva, and DNA barcoding. Acta Tropica, 185, 110- 114.
List of figures
Fig. 1. Map showing the location of sampling points in different states in Peninsular Malaysia.
Fig. 2. Median joining networks from different populations of Simulium vanluni, Simulium cheongi and Simulium jeffreyi. Each haplotype is represented by a circle. Relative sizes of the circles indicate haplotype frequency. Circles of the same colour represent haplotypes from the same population (red = Selangor, blue = Pahang, green = Kelantan, grey = Perak, black = Kedah, brown = Johor and yellow = Terengganu).
Fig. 3. Observed and expected mismatch distributions for Simulium vanluni, Simulium cheongi and Simulium jeffreyi based on COI sequences.
List of tables Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7
Table 1 Haplotype diversity (Hd), nucleotide diversity (pi), Tajima’s D (D) and Fu’s Fs (Fs) tests based on COI DNA sequences of Simulium vanluni, Simulim cheongi and Simulum jeffreyi. Populations Simulium vanluni Selangor Pahang Kelantan Perak Kedah Johor Terengganu Total Simulium cheongi Terengganu Kelantan Johor Kedah Perak Selangor Total Simulium jeffreyi Perak Kedah Pahang Selangor Total
Hd
pi
D
Fs
1.0000 0.6944 0.8056 0.5333 0.6667 0.8000 0.9444 0.9240
0.0045 0.0027 0.0057 0.0020 0.0013 0.0024 0.0050 0.0060
-0.6528 -0.0754 -0.4328 1.6415 0.0000 -0.1854 -1.6677 -1.1732
-9.5150 -0.2860 0.0470 2.3380 0.0000 -1.3500 -2.9520 -14.3090
0.7857 0.7500 0.6667 0.7222 0.9643 1.0000 0.9310
0.0067 0.0034 0.0013 0.0038 0.0040 0.0064 0.0081
-1.1876 -0.3355 1.6330 -0.4103 -0.3449 0.0830 0.5094
0.0590 -0.0730 0.5400 -0.8480 -4.3030 -2.0040 -11.8080
0.7000 0.6944 0.8611 0.4667 0.9130
0.0030 0.0025 0.0077 0.0035 0.0067
-1.0938 -0.3823 0.5463 1.2293 0.0910
0.2760 -0.4500 0.7510 3.7790 -2.9280
Table 2 Genetic differentiation (FST) and gene flow (Nm) based on COI DNA sequences of Simulium vanluni from 7 states (Selangor, Pahang, Kelantan, Perak, Kedah, Johor and Terengganu). Population 1
Population 2
Selangor Selangor Selangor Selangor Selangor Selangor Pahang Pahang Pahang Pahang Pahang Kelantan Kelantan Kelantan Kelantan Perak Perak Perak Kedah Kedah Johor
Pahang Kelantan Perak Kedah Johor Terengganu Kelantan Perak Kedah Johor Terengganu Perak Kedah Johor Terengganu Kedah Johor Terengganu Johor Terengganu Terengganu
*p value<0.05, **p value<0.01
COI FST 0.1083 0.0654 0.6199 0.6230 0.1406 0.0363 0.2969 0.7500** 0.7564* 0.4326 0.2920 0.4058* 0.4438 0.1830 0.0452 0.2353 0.7586** 0.6239* 0.7852 0.6042 0.1051 Total Nm
Nm 2.06 3.57 0.15 0.15 1.53 6.63 0.59 0.08 0.08 0.33 0.61 0.37 0.31 1.12 5.28 0.81 0.08 0.15 0.07 0.14 2.13 0.56
Table 3 Genetic differentiation (FST) and gene flow (Nm) based on COI DNA sequences of Simulium cheongi from 6 states (Selangor, Kelantan, Perak, Kedah, Johor and Terengganu). Population 1
Population 2
Terengganu Terengganu Terengganu Terengganu Terengganu Kelantan Kelantan Kelantan Kelantan Johor Johor Johor Kedah Kedah Perak
Kelantan Johor Kedah Perak Selangor Johor Kedah Perak Selangor Kedah Perak Selangor Perak Selangor Selangor
COI FST 0.5700* 0.7759 0.5461* 0.4460 0.2813 0.8365* -0.0325 0.1619 0.1897 0.8273* 0.7957 0.7663 0.0679 0.1196 0.0605 Total Nm
Nm 0.38 0.14 0.42 0.62 1.28 0.10 -15.90 2.59 2.14 0.10 0.13 0.15 6.87 3.68 7.76 0.38
*p value<0.05, **p value<0.01
Table 4 Genetic differentiation (FST) and gene flow (Nm) based on COI DNA sequences of Simulium jeffreyi from 4 states (Selangor, Perak, Kedah and Pahang). Population 1 Perak Perak Perak Kedah Kedah Pahang
Population 2 Kedah Pahang Selangor Pahang Selangor Selangor
*p value<0.05, **p value<0.01
COI FST 0.7328* 0.2485 0.3434** 0.4228* 0.6087** 0.0254** Total Nm
Nm 0.18 1.51 0.96 0.68 0.32 19.21 0.64
Table 5 Average pairwise distances among Simulium vanluni population based on the Kimura-2-parameter model.
Selangor Pahang Johor Terengganu Kelantan Kedah Perak
1 0.41 0.40 0.49 0.55 0.77 0.87
2
3
4
5
6
0.45 0.54 0.60 0.82 0.96
0.41 0.50 0.85 0.92
0.56 0.87 0.93
0.63 0.65
0.21
Table 6 Average pairwise distances among Simulium cheongi population based on the Kimura-2-parameter model.
Terengganu Selangor Perak Kedah Kelantan Johor
1 0.92 0.97 1.18 1.16 1.80
2
3
4
5
0.56 0.61 0.58 1.67
0.44 0.42 1.31
0.35 1.43
1.48
Table 7 Average pairwise distances among Simulium jeffreyi population based on the Kimura-2-parameter model.
Perak Selangor Pahang Kedah
1 0.50 0.72 1.00
2
3
0.58 0.78
0.90
Genetic Diversity and Differentiation in the Blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia Soosai Peranathan Pavitra , Zubaidah Ya’cob , Tiong Kai Tan , Yvonne Ai Lian Lim , Van Lun Low PII: DOI: Reference:
S0001-706X(19)31723-1 https://doi.org/10.1016/j.actatropica.2020.105415 ACTROP 105415
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
7 December 2019 19 February 2020 19 February 2020
Please cite this article as: Soosai Peranathan Pavitra , Zubaidah Ya’cob , Tiong Kai Tan , Yvonne Ai Lian Lim , Van Lun Low , Genetic Diversity and Differentiation in the Blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia, Acta Tropica (2020), doi: https://doi.org/10.1016/j.actatropica.2020.105415
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Highlights
High levels of genetic diversity and genetic differentiation were observed among three common species S. vanluni, S. cheongi and S.jeffreyi in Peninsular Malaysia. All three species revealed an intermediate level of gene flow among the populations. The expansion time of S. vanluni was estimated at about 233 112 years ago, followed by S. jeffreyi (232, 948 years ago) and S. cheongi (191, 845 years ago).
Genetic Diversity and Differentiation in the Blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia Soosai Peranathan Pavitraa , Zubaidah Ya’cobb , Tiong Kai Tana , Yvonne Ai Lian Lima & Van Lun Lowb a
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur,
Malaysia b
Higher Institution Centre of Excellence (HiCOE), Tropical Infectious Diseases Research and
Education Centre (TIDREC), University of Malaya, Kuala Lumpur, Malaysia *Corresponding author: [email protected] ABSTRACT The population genetic structure of S. vanluni, S. cheongi and S. jeffreyi were determined from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) across different states in Peninsular Malaysia. High levels of genetic diversity and genetic differentiation were observed among three species in Peninsular Malaysia. All three species revealed an intermediate level of gene flow among the populations. Negative values of Fu’s Fs and low values of Raggedness index supported the hypothesis of population expansion in S. vanluni, S. cheongi and S. jeffreyi. Keywords: Blackflies, population genetic structure, genetic diversity, genetic differentiation.
1. Introduction Blackflies belonging to the family Simuliidae of the order Diptera, are ecologically and medically important insects (Adler et al., 2004; Currie & Adler, 2008). Blackflies are present in almost all zoogeographical regions over a wide range of elevations except in the Antarctica, deserts and islands without running waters. To date, there are 2,328 species (2,310 living and 18 fossils) of blackflies (Adler & Crosskey, 2019). Of these, a total of 63 species of blackflies are found in Peninsular Malaysia (Takaoka et al., 2019). Adult female blackflies are the vectors of many parasitic diseases across the globe and their immature stages play an important role in stream ecology (Sriphirom et al., 2014). Population genetics of blackflies is the study of genetic variation within and among the blackfly populations and the evolutionary factors that have shaped their genetic variation (Lynch & Walsh, 1998; Hartl & Sunderland, 2000; Crow, 2008; Nielsen & Slatkin, 2013). Population genetic study can reveal morphologically cryptic species as well as evolutionary history in blackflies populations (Low et al., 2014; 2016). Several genetic studies of blackflies have been conducted in Malaysia including the genetic diversity and genetic differentiation in Simulium tani along an elevational gradient (Low et al., 2014), description of a new cryptic species of Simulium vanluni (Ya’cob, 2017), phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups (Low et al., 2015) and characterization of Simulium (Simulium) hackeri Edwards through DNA barcoding (Ya’cob et al., 2018). However, only limited study on population genetic study of blackflies has been conducted in Malaysia. Simulium cheongi Takaoka and Davies 1995, Simulium jeffreyi Takoaka and Davies, 1995 and Simulium vanluni Ya’cob, Takaoka and Sofian-Azirun, 2017 are commonly found in Peninsular Malaysia (Ya’cob et al., 2016a; Pavitra et al., 2019). Two of them belong to
Simulium (s.str.); Simulium vanluni in the S. nobile species-group and Simulium jeffreyi in the S. striatum species-group, while Simulium cheongi in the S. epistum species-group of Gomphostilbia. In view of their ample distribution and abundance in Peninsular Malaysia, these three species of blackflies were selected for a comparative study. Simulium cheongi was first described from Janda Baik, Pahang, Malaysia (Takaoka & Davies, 1995), and subsequently reported in various locations in Peninsular Malaysia (Ya’cob et al., 2016b), Thailand (Takaoka et al., 2009) and Indonesia (Sumatra and Kalimantan) (Takaoka et al., 2000; Takaoka et al., 2017). While, Simulium jeffreyi was originally described from Peninsular Malaysia (Takaoka and Davies 1995). Its distribution includes various localities in Peninsular Malaysia (Ya’cob et al., 2016b), and Lang Son Province in northern Vietnam (Pham, 1999). On the other hand, Simulium vanluni from Peninsular Malaysia was previously treated as Simulium nobile (Crosskey, 1973). A molecular DNA barcoding analysis has revealed three cryptic species, each from Borneo, Java and mainland of Southeast Asia (southern Thailand and Peninsular Malaysia) (Low et al., 2016a). The cryptic species from mainland of Southeast Asia was then formally described as Simulium vanluni by using an integrated morpho-taxonomical and genetic approach. (Ya’cob et al., 2017). Simulim cheongi, S. jeffreyi and S. vanluni may have different evolutionary relationships. To test this hypothesis, the current study aimed to determine their genetic diversity and to evaluate the degree of genetic differentiation, based on the mitochondriaencoded cytochrome c oxidase subunit I (COI) gene.
2. Material and method 2.1. Black fly specimens Blackfly pupae were collected from streams across different states in Peninsular Malaysia: Central: Selangor (S. vanluni, n = 10; S. cheongi, n = 5; S. jeffreyi, n = 10), Northern: Perak (S. vanluni, n = 10; S. cheongi, n = 8; S. jeffreyi, n = 5) and Kedah (S. vanluni, n = 3; S. cheongi, n = 9; S. jeffreyi, n = 9), Southern: Johor (S. vanluni, n = 6; S. cheongi, n = 4), and Eastern: Kelantan (S. vanluni, n = 9; S. cheongi, n = 8), Terengganu (S. vanluni, n = 9; S. cheongi, n = 8) and Pahang (S. vanluni, n = 9; S. jeffreyi, n = 9) (Fig. 1.). The pupae attached on aquatic substrates such as leaves, stems, grasses, plant roots, rocks and twigs were collected by using fine forceps (McCreadie and Colbo, 1991; McCreadie et al., 2005) and stored alive in vials individually for adult emergence. Species of blackflies were identified following the keys and descriptions of Takaoka, (2003) and Ya’cob et al, (2017). 2.2. DNA extraction and PCR amplification DNAs of S. vanluni, S. cheongi and S. jeffreyi were extracted from all reared adult specimens using the G-spinTM Total DNA Extraction Mini Kit according to the manufacturer's instructions. The PCR amplifications were performed by Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems, Inc., Foster City, CA, USA). Amplifications of the COI genes were performed in a final volume of 25 µL containing 0.5–1.0 mg genomic DNA, 2X ExPrime Taq Master Mix (GENETBIO, South Korea), and 10 pmol of each forward and reverse primer. The primers used in this study were LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) (Folmer, 1994). The parameters of PCR amplifications of the COI gene was 1 min at 96oC, followed by 35 cycles of denaturation at
94oC for 1 min; annealing at 55oC for 1 min; extension at 72oC for 1 min 30 s, and a final extension at 72oC for 7 min (Rivera & Currie, 2009). 2.3. DNA sequencing and data analysis The amplified PCR products were viewed in 2% agarose gel pre-stained with SYBR Safe (Invitrogen Corp., Carlsbad, CA, U.S.A.). The PCR products were sequenced and analyzed using an ABI PRISM 377 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA, U.S.A.). Sequencing data were analyzed and edited using ChromasPro 1.7.6 (Technelysium Pty Ltd., Qld, Australia) and BioEdit 7.0.9.0 (Hall, 1999). Representative sequences generated from this study were deposited in the National Center for Biotechnology Information (NCBI) GenBank under the accession numbers MN514637-MN514767. The haplotype networks of S. vanluni, S. cheongi and S. jeffreyi were analyzed using a medianjoining algorithm (Bandelt et al., 1999) in the program Network 4.6. Haplotype diversity (Hd), nucleotide diversity (pi), genetic differentiation (FST), and gene flow (N m) values were performed with the program DnaSP 5.0 (Librado & Rozas, 2009). The levels of genetic differentiation can be categorized as FST> 0.25 (great differentiation), 0.15 to 0.25 (moderate differentiation), and FST< 0.05 (negligible differentiation) (Wright, 1978). The levels of gene flow can be categorized as Nm > 1 (high gene flow), 0.25 to 0.99 (intermediate gene flow), and Nm< 0.25 (low gene flow) (Govindajuru, 1989). Average pairwise distances among different populations based on the Kimura-2-parameter model were calculated using MEGA 7 software. Tajima’s D (Tajima, 1989), Fu’s Fs (Fu, 1997), and mismatch distribution test, Harpending’s raggedness index (Harpending, 1994) were performed with the program DnaSP 5.0 to test for population equilibrium and signature of population expansion. The expansion time was determined assuming 12 generations a year
for tropical blackflies (Pramual et al. 2005) and a divergence rate of 2.3% per million years for insect mitochondrial DNA (Brower 1994). 3. Results The median-joining network of 56 individuals of S. vanluni, 42 individuals of Simulium cheongi and 33 individuals of S. jeffreyi aligned as 531 characters of the COI gene revealed high levels of genetic diversity among populations from different states in Peninsular Malaysia. Simulium vanluni and Simulium cheongi demonstrated a well dispersed haplotypes across all states. Of the 56 individuals of S. vanluni, 24 distinct haplotypes were discovered, six of which were shared among populations whereas S. cheongi had 23 distinct haplotypes with four shared haplotypes from 42 individuals. For S. jeffreyi, a total of 13 distinct haplotypes were identified from 33 individuals and only one was shared between two populations (Fig. 2.). In total data estimates, S. cheongi revealed higher haplotype diversity (0.9310) and nucleotide diversity (0.0081) than S. vanluni and S. jeffreyi. The haplotype diversity of S. vanluni ranged from 0.5333 (Perak population) to 1.0000 (Selangor population) with an average of 0.9240, whereas the nucleotide diversity ranged from 0.0013 (Kedah population) to 0.0057 (Kelantan population) with an average of 0.0060. For S. cheongi, the haplotype diversity ranged from 0.6667 (Johor population) to 1.0000 (Selangor population) with an average of 0.9310, whereas the nucleotide diversity ranged from 0.0013 (Johor population) to 0.0067 (Terengganu population) with an average of 0.0081. Additionally, haplotype diversity of S. jeffreyi ranged from 0.4667 (Selangor population) to 0.8611 (Pahang population) with an average of 0.9130, whereas the nucleotide diversity ranged from 0.0025 (Kedah population) to 0.0077 (Pahang population) with an average of 0.0067. Both S. vanluni and S. cheongi populations originating from Selangor and
S. jeffreyi population from Pahang exhibited highest haplotype diversity (Hd > 0.9). Moderate nucleotide diversity was recorded for S. vanluni from Kelantan, S. cheongi from Terengganu and S. jeffreyi from Pahang. According to neutrality test results, the Fu’s Fs tests revealed negative values for all three species while a negative Tajima’s D value was recorded only in S. vanluni (Table 1). The genetic differentiation (FST) and gene flow (Nm) of S. vanluni, S. cheongi and S. jeffreyi from different states in Peninsular Malaysia are presented in Tables 2, 3 and 4. The majority of the population pairs of all three species showed FST> 0.25, indicating great differentiation. The highest FST value with great genetic differentiation for S. vanluni, S. cheongi and S. jeffreyi were between Kedah- Johor (FST= 0.7852), Kelantan- Johor (FST= 0.8365) and Perak- Kedah (FST= 0.7328), respectively. In contrast, the least differentiation (FST<0.05) for S. vanluni, S. cheongi and S. jeffreyi were found between SelangorTerengganu (FST= 0.0363), Kelantan- Kedah (FST= -0.0325) and Pahang- Selangor (FST= 0.0254). All three species revealed an intermediate level of gene flow (N m= 0.38-0.64) among the populations. An average pairwise distances based on the Kimura 2-parameter model were calculated (Tables 5, 6 and 7). S. vanluni, S. cheongi and S. jeffreyi had greatest genetic distance between Perak- Pahang (0.96), Terengganu- Johor (1.80) and Perak- Kedah (1.00) and lowest genetic distance between Perak- Kedah (0.21), Kelantan- Kedah (0.35) and PerakSelangor (0.50), each respectively. Low values of the Raggedness index were recorded for all three blackflies species in the mismatch distribution analysis (Fig. 3.). The expansion times of S. vanluni and S. jeffreyi are estimated to be 233 000 and S. cheongi with 192 000 years ago.
4. Discussion Population genetics of S. vanluni, S. cheongi and S. jeffreyi was conducted for the first time in Malaysia. This study revealed high levels of genetic diversity among populations from different states in Peninsular Malaysia. The median-joining network demonstrated the most common haplotype (H9 in S. vanluni and H7 in S. cheongi) as the ancestral haplotype in S. vanluni and S. cheongi due to its central position in the network with various lineages extending from it and its significant number of individuals in all population (Posada and Crandall 2001). This haplotype also categorized under stable haplotype because of its various environmental adaptabilities (Xu et al., 2019). Moreover, high level of haplotype diversity recorded for all three species of blackflies (Hd > 0.9) showed strong adaptability to different environmental conditions. S. vanluni, S. cheongi and S. jeffreyi are called as generalist species because of their high tolerance level over an extensive set of physicochemical characteristics which allowed them to extend their distribution across different eco-environmental conditions (Brown, 1984; Pavitra et al., 2019). This was consistent with a previous study conducted in Malaysia that revealed high haplotype diversity in S. tani along different elevation gradient (Low et al., 2014). The results of Fu’s Fs test revealed negative values for all three blackfly species (S. vanluni, S. cheongi and S. jeffreyi), indicating that the populations are undergoing demographic expansion while a negative Tajima’s D value was recorded only in S. vanluni population. It has been shown that Fu’s Fs test is more powerful than Tajima’s D in detecting the population expansion (Fu, 1997), and this would explain that the populations provide strong evidence for recent population expansion in this study. Furthermore, maximum genetic differentiation was found in S. vanluni population between Kedah- Johor and S. cheongi population between Kelantan- Johor. This is attributed
to the large geographical distance between these states; Kedah- Johor with 769.8 km and Kelantan- Johor with 692.9 km. In S. jeffreyi population, the most differentiation was showed between Perak- Kedah (236.1 km). Despite the closer geographical distance between PerakKedah, other anthropological (deforestation, urbanization, and habitat destruction) factors also can affect the gene flow in these states and lead to population differentiation (SQI, 2010; Bashir-Ali, 2014). This corroborates with other previous studies that indicated high levels of genetic differentiation in S. feuerborni and S. angulistylum complexes (Pramual & Kuvangkadilok, 2012; Pramual & Wongpakam, 2013). In contradistinction, low levels of genetic differentiation were observed among populations of S. tani along an elevational gradient in Cameron Highlands, Malaysia (Low et al., 2014). High genetic differentiation between populations can decrease the process of gene flow (Pramual et al., 2011). This hypothesis is consistent with our finding of high genetic differentiation with intermediate gene flow in all three species. Simulium vanluni, S. cheongi and S. jeffreyi that were reported to be distributed at low altitude areas (<500 m) in Peninsular Malaysia (Ya’cob et al., 2016). The restriction to low altitude areas could promote population separation due to the effective gene flow barrier in high altitude areas. Additionally, the values of pairwise genetic distance range from 0.2 to 1.0 in S. vanluni, 0.3 to 1.8 in S. cheongi and 0.5 to 1.0 in S. jeffreyi, further supported the high genetic differentiation between different populations in Peninsular Malaysia. The intraspecific genetic distances for all three species were less than 3% (the cut-off point for species boundary), suggesting the absence of cryptic species in the examined populations. (Low et al., 2016b; Pramual et al., 2016). This hypothesis was further supported by the morphological observation where no distinct characters was observed within the species.
The star-like shapes of S. vanluni and S. cheongi were consistent with the sudden expansion of the mismatch distribution, indicating that population experienced recent demographic expansion. While, S. jeffreyi showed a dominant left-hand peak that indicates a very recent expansion. The expansion time of both S. vanluni and S. jeffreyi were estimated at about 233 000 years ago, followed by S. cheongi (192, 000 years ago). The population expansion of other blackflies has been reported in recent times such as Simulium angulistylum (930,000 years ago) (Pramual & Kuvangkadilok, 2012), Simulium tani (500,000 years ago) (Pramual et al., 2005), Simulium siamense (120,000 years ago) (Pramual et al., 2011) and Simulium aureohirtum (18,000 years ago) (Thaijarern et al., 2014) and Simuliun nodosum (2,600–5,200 years ago) (Chaiyasan & Pramual, 2016). In conclusion, this study has revealed high levels of genetic diversity and genetic differentiation in S. vanluni, S. cheongi and S. jeffreyi across different states in Peninsular Malaysia using mitochondria-encoded cytochrome c oxidase subunit I (COI) gene; however, in-depth studies are still needed. More number of blackflies from different geographical areas and use of multiple genetic markers involving both mitochondrial-encoded and nuclearencoded genes will be useful in unfolding the hidden diversity of the blackfly populations. Acknowledgements This work was supported by research grants from the University of Malaya (Project no. BKP009-2018 and RP021C-16SUS) and Ministry of Energy, Science, Technology, Environment and Climate Change (Project no. FP024-2019A) as well as funding from the Ministry of Education, Malaysia for niche area research under the Higher Institution Centre of Excellence (HICoE) program (Project no. MO002-2019). This project is a part of the first author's PhD research at the University of Malaya, Kuala Lumpur.
Author agreement We declare that this is our original work. It has not been published elsewhere and we have no conflicts of interest concerning the work reported in this paper. All authors have contributed to this study throughout the study design, data collection, data analyses and interpretation. The authors have read and approved the manuscript. Declaration of competing interests We have no conflict of interest to declare.
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List of figures
Fig. 1. Map showing the location of sampling points in different states in Peninsular Malaysia.
Fig. 2. Median joining networks from different populations of Simulium vanluni, Simulium cheongi and Simulium jeffreyi. Each haplotype is represented by a circle. Relative sizes of the circles indicate haplotype frequency. Circles of the same colour represent haplotypes from the same population (red = Selangor, blue = Pahang, green = Kelantan, grey = Perak, black = Kedah, brown = Johor and yellow = Terengganu).
Fig. 3. Observed and expected mismatch distributions for Simulium vanluni, Simulium cheongi and Simulium jeffreyi based on COI sequences.
List of tables Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7
Table 1 Haplotype diversity (Hd), nucleotide diversity (pi), Tajima’s D (D) and Fu’s Fs (Fs) tests based on COI DNA sequences of Simulium vanluni, Simulim cheongi and Simulum jeffreyi. Populations Simulium vanluni Selangor Pahang Kelantan Perak Kedah Johor Terengganu Total Simulium cheongi Terengganu Kelantan Johor Kedah Perak Selangor Total Simulium jeffreyi Perak Kedah Pahang Selangor Total
Hd
pi
D
Fs
1.0000 0.6944 0.8056 0.5333 0.6667 0.8000 0.9444 0.9240
0.0045 0.0027 0.0057 0.0020 0.0013 0.0024 0.0050 0.0060
-0.6528 -0.0754 -0.4328 1.6415 0.0000 -0.1854 -1.6677 -1.1732
-9.5150 -0.2860 0.0470 2.3380 0.0000 -1.3500 -2.9520 -14.3090
0.7857 0.7500 0.6667 0.7222 0.9643 1.0000 0.9310
0.0067 0.0034 0.0013 0.0038 0.0040 0.0064 0.0081
-1.1876 -0.3355 1.6330 -0.4103 -0.3449 0.0830 0.5094
0.0590 -0.0730 0.5400 -0.8480 -4.3030 -2.0040 -11.8080
0.7000 0.6944 0.8611 0.4667 0.9130
0.0030 0.0025 0.0077 0.0035 0.0067
-1.0938 -0.3823 0.5463 1.2293 0.0910
0.2760 -0.4500 0.7510 3.7790 -2.9280
Table 2 Genetic differentiation (FST) and gene flow (Nm) based on COI DNA sequences of Simulium vanluni from 7 states (Selangor, Pahang, Kelantan, Perak, Kedah, Johor and Terengganu). Population 1
Population 2
Selangor Selangor Selangor Selangor Selangor Selangor Pahang Pahang Pahang Pahang Pahang Kelantan Kelantan Kelantan Kelantan Perak Perak Perak Kedah Kedah Johor
Pahang Kelantan Perak Kedah Johor Terengganu Kelantan Perak Kedah Johor Terengganu Perak Kedah Johor Terengganu Kedah Johor Terengganu Johor Terengganu Terengganu
*p value<0.05, **p value<0.01
COI FST 0.1083 0.0654 0.6199 0.6230 0.1406 0.0363 0.2969 0.7500** 0.7564* 0.4326 0.2920 0.4058* 0.4438 0.1830 0.0452 0.2353 0.7586** 0.6239* 0.7852 0.6042 0.1051 Total Nm
Nm 2.06 3.57 0.15 0.15 1.53 6.63 0.59 0.08 0.08 0.33 0.61 0.37 0.31 1.12 5.28 0.81 0.08 0.15 0.07 0.14 2.13 0.56
Table 3 Genetic differentiation (FST) and gene flow (Nm) based on COI DNA sequences of Simulium cheongi from 6 states (Selangor, Kelantan, Perak, Kedah, Johor and Terengganu). Population 1
Population 2
Terengganu Terengganu Terengganu Terengganu Terengganu Kelantan Kelantan Kelantan Kelantan Johor Johor Johor Kedah Kedah Perak
Kelantan Johor Kedah Perak Selangor Johor Kedah Perak Selangor Kedah Perak Selangor Perak Selangor Selangor
COI FST 0.5700* 0.7759 0.5461* 0.4460 0.2813 0.8365* -0.0325 0.1619 0.1897 0.8273* 0.7957 0.7663 0.0679 0.1196 0.0605 Total Nm
Nm 0.38 0.14 0.42 0.62 1.28 0.10 -15.90 2.59 2.14 0.10 0.13 0.15 6.87 3.68 7.76 0.38
*p value<0.05, **p value<0.01
Table 4 Genetic differentiation (FST) and gene flow (Nm) based on COI DNA sequences of Simulium jeffreyi from 4 states (Selangor, Perak, Kedah and Pahang). Population 1 Perak Perak Perak Kedah Kedah Pahang
Population 2 Kedah Pahang Selangor Pahang Selangor Selangor
*p value<0.05, **p value<0.01
COI FST 0.7328* 0.2485 0.3434** 0.4228* 0.6087** 0.0254** Total Nm
Nm 0.18 1.51 0.96 0.68 0.32 19.21 0.64
Table 5 Average pairwise distances among Simulium vanluni population based on the Kimura-2-parameter model.
Selangor Pahang Johor Terengganu Kelantan Kedah Perak
1 0.41 0.40 0.49 0.55 0.77 0.87
2
3
4
5
6
0.45 0.54 0.60 0.82 0.96
0.41 0.50 0.85 0.92
0.56 0.87 0.93
0.63 0.65
0.21
Table 6 Average pairwise distances among Simulium cheongi population based on the Kimura-2-parameter model.
Terengganu Selangor Perak Kedah Kelantan Johor
1 0.92 0.97 1.18 1.16 1.80
2
3
4
5
0.56 0.61 0.58 1.67
0.44 0.42 1.31
0.35 1.43
1.48
Table 7 Average pairwise distances among Simulium jeffreyi population based on the Kimura-2-parameter model.
Perak Selangor Pahang Kedah
1 0.50 0.72 1.00
2
3
0.58 0.78
0.90