Key words. Gomphostilbia, Simulium asakoae group, Simulium ceylonicum group, mitochondrial genes, nuclear genes, phylogenetics, Malaysia.

Medical and Veterinary Entomology (2015), doi: 10.1111/mve.12120 A multi-locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages V. L. L O W 1, H. TA K AO K A 1, P. H. A D L E R 2, Z. YA C O B 1, Y. N O R M A- R A S H I D 1, C. D. C H E N 1 and M. S O F I A N- A Z I R U N 1 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia and 2 Department of Agricultural and Environmental Sciences, Clemson University, Clemson, SC, U.S.A. Abstract. A multi-locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria-encoded cytochrome c oxidase subunit I (COI), 12S rrna and 16S rrna genes, and the nuclear-encoded 18S rrna and 28S rrna genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi-locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups. Key words. Gomphostilbia, Simulium asakoae group, Simulium ceylonicum group, mitochondrial genes, nuclear genes, phylogenetics, Malaysia. Introduction Genetic studies of blackflies in the Oriental biogeographic region, notably Thailand, have been instrumental in the discovery of cryptic species and in testing the monophyly of morphologically based taxa (Phayuhasena et al., 2010; Pramual et al., 2010, 2012; Pramual & Kuvangkadilok, 2012; Pramual & Nanork, 2012; Pramual & Adler, 2014; Sriphirom et al., 2014). The Oriental subgenus Gomphostilbia is one of the three most species-rich subgenera of the genus Simulium in the world, representing about 10% of all species, arranged in 15 morphotaxonomic species groups (Takaoka, 2012). Species in the subgenus typically differ in only minor structural characters, and thus morphological data for recognizing species and inferring evolutionary relationships are limited (Jitklang et al., 2008; Pramual & Kuvangkadilok, 2012). A morphotaxonomic revision divided the former Simulium ceylonicum species group into three new species groups, represented by the Simulium asakoae, S. ceylonicum and Simulium darjeelingense groups, respectively (Takaoka, 2012). The S. asakoae and S. ceylonicum groups include species of medical and veterinary importance. Simulium asakoae Takaoka & Davies and Simulium sheilae Takaoka & Davies in Thailand and Simulium tenuistylum Datta in India are human-biting species (Datta, 1992; Choochote et al., 2005). Additionally, S. asakoae in northern Thailand has been incriminated as a vector of animal filarial parasites (Ishii et al., 2008). Elucidating the relationships among Malaysian populations is key to understanding the genetic background of the S. asakoae and S. ceylonicum groups because Malaysia is home to the type localities of all but one (Simulium trangense Jitklang et al.) of the species covered in this study. The S. asakoae group in Malaysia includes eight nominal species: S. asakoae; Simulium brinchangense Takaoka et al.; Simulium hoiseni Takaoka; Simulium izuae Takaoka et al.; Simulium lurauense Takaoka et al.; Simulium roslihashimi Takaoka Correspondence: Van Lun Low, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia. Tel.: + 60 16 560 5857; Fax: +60 3 7967 4376; E-mail: lucaslow24@gmail.com 2015TheRoyalEntomologicalSociety 1

2 V. L. Low et al. & Sofian-Azirun; Simulium sofiani Takaoka & Hashim, and Simulium tanahrataense Takaoka et al.thes. ceylonicum group in Malaysia includes five nominal members, all in the S. sheilae subgroup: Simulium capillatum Takaoka; Simulium leparense Takaoka et al.; S. sheilae; Simulium longitruncum Takaoka & Davies, and S. trangense. The phylogenetic relationships of S. asakoae in Thailand have been studied (Pramual & Adler, 2014), but monophyly of the S. asakoae species group has not been tested. This is the first phylogenetic study of the Malaysian members of the S. asakoae and S. ceylonicum species groups using mitochondria-encoded COI, 12S rrna and 16S rrna genes, and nuclear-encoded 18S rrna and 28S rrna genes to be conducted. Whether these loci can be used as genetic markers for molecular identification of the members of both species groups was tested. Material from the type localities was used for eight of the 10 nominal species analysed. Materials and methods Taxonomic sampling In view of the structural similarity among members of the S. asakoae and S. ceylonicum groups, the most distinctive life stage, the adult, was used for molecular analyses. Specimens were identified using taxonomic keys (Takaoka & Davies, 1995; Jitklang et al., 2008; Takaoka et al., 2011a, 2011b, 2013, 2014a, 2014b). The study included seven species from the S. asakoae group (S. asakoae, S. brinchangense, S. izuae, S. lurauense, S. roslihashimi, S. sofiani and S. tanahrataense) and three species from the S. ceylonicum group (S. leparense, S. sheilae and S. trangense) (Table 1). Given the challenges of identification and the frequency of cryptic biodiversity in simuliids, including those of the subgenus Gomphostilbia (Jitklang et al., 2008; Pramual et al., 2010; Pramual & Kuvangkadilok, 2012), the use of samples from the type localities provides an important means of associating each formal name with a genetic characterization. Thus, eight of the 10 species were sampled at their type locality; the exceptions were S. roslihashimi and S. trangense. One Malaysian member of the S. asakoae group (S. hoiseni) and two members of the S. ceylonicum group (S. capillatum and S. longitruncum)werenotavailable forstudy. Polymerase chain reaction and DNA sequencing DNA was extracted from each of three specimens per species, using the i-genomic CTB DNA Extraction Mini Kit (intron Biotechnology, Inc., Seongnam, South Korea). Amplifications of the mitochondria-encoded COI, 12S rrna and 16S rrna, and nuclear-encoded 18S rrna and 28S rrna genes were performed in a final volume of 50 μl containing50 100ngof genomic DNA, 25 μl of ExPrime Taq Master Mix (GENETBIO, Inc., Daejeon, South Korea), and 10 pmol of each forward and reverse primer. Polymerase chain reaction (PCR) was conducted using a 96-well Veriti Thermal Cycler (Applied Biosystems, Inc., Foster City, CA, U.S.A.). The PCR cycling parameters followed those of Rivera & Currie (2009) for COI, Conflitti et al. (2012) for 12S rrna, Phayuhasena et al. (2010) for 16S rrna, and Low et al. (2014) for 18S rrna and 28S rrna. Primers used for amplification of these five genes are summarized in Table 2. The PCR products were purified and directly sequenced using an ABI PRISM 377 Genetic Analyzer (Applied Biosystems, Inc.). Sequence alignment and DNA analyses Both forward and reverse sequences were assembled and edited using ChromasPro Version 1.7.6 (Technelysium Pty Ltd, Brisbane, Qld, Australia). All sequences were initially aligned using ClustalX and edited using BioEdit 7.0.9.0 (Hall, 1999). The COI, 12S rrna, 16S rrna, 18S rrna and 28S rrna sequences generated from this study were deposited in the National Center for Biotechnology Information (NCBI) GenBank DNA sequence database (Table 1). Congruence between separate genes was tested using a partition homogeneity test (Farris et al., 1995) with 100 replicates implemented in paup 4.0b10 (Swofford, 2002). The partition homogeneity test showed that COI, 12S rrna, 16S rrna, 28S rrna and the concatenated dataset shared the same phylogenetic information. Hence, the concatenated dataset was used for phylogenetic analysis. Uncorrected (p) pairwise genetic distances were calculated for species pairs, using paup 4.0b10. Maximum likelihood (ML) analysis was performed with 1000 bootstrap replicates using Treefinder Version October 2008 (Jobb et al., 2004). The best-fit nucleotide substitution model was determined using kakusan Version 3 (Tanabe, 2007) and evaluated using the corrected Akaike information criterion (AIC) (Akaike, 1973; Shono, 2000), with significance determined by chi-squared analysis. Maximum parsimony (MP) and neighbour-joining (NJ) analyses were performed using paup 4.0b10. The MP tree was constructed using the heuristic search option, 100 random sequence additions, tree bisection reconnection (TBR) branch swapping, and unordered and unweighted characters. Bootstrap percentage (BP) was computed with 1000 replications. Neighbour-joining bootstrap values were estimated using 1000 replicates with Kimura s two-parameter model of substitution (K2P distance). Bayesian inference (BI) analysis was performed using four chains of Markov chain Monte Carlo (MCMC) implemented in MrBayes Version 3.1.2 (Huelsenbeck & Ronquist, 2001). Four million MCMC generations were run, with convergence diagnostics calculated every 1000th generation to monitor the stabilization of log likelihood scores. Trees in each chain were sampled every 100th generation. Simulium (Simulium) tani cytoform K, a member of the Simulium tuberosum group, was used as an outgroup. Results DNA sequence variation The ranges of interspecific genetic divergence based on the COI, 12S rrna and 16S rrna genes are summarized in Tables S1 S3 (online). Interspecific divergence ranged from 0.30% (S. lurauense vs. S. sofiani) to 11.59% (S. leparense vs.

Phylogeny of S. asakoae and S. ceylonicum groups 3 Table 1. Collection details for blackflies in Malaysia, with GenBank accession numbers for COI, 12S rrna, 16S rrna, 18S rrna and 28S rrna sequences of blackflies in the Simulium subgenus Gomphostilbia. GenBank accession no. Species ID no. Locality Coordinates Altitude Collection date COI 12S rrna 16S rrna 18S rrna 28S rrna S. tani cytoform K ST1 Cameron Highlands 04 16 N, 101 19 E 235m 28/07/2012 KM410160 KM410216 KM410188 KM410244 KM410272 Simulium asakoae species group S. izuae I1 Cameron Highlands 04 34 N, 101 20 E 1315m 28/05/2012 KM410161 KM410217 KM410189 KM410245 KM410273 S. izuae I2 Cameron Highlands 04 16 N, 101 18 E 224m 24/09/2012 KM410162 KM410218 KM410190 KM410246 KM410274 S. izuae I3 Cameron Highlands 04 24 N, 101 22 E 1033m 23/10/2012 KM410163 KM410219 KM410191 KM410247 KM410275 S. roslihashimi R1 Cameron Highlands 04 22 N, 101 21 E 711m 25/06/2012 KM410164 KM410220 KM410192 KM410248 KM410276 S. roslihashimi R2 Cameron Highlands 04 14 N, 101 18 E 159m 25/07/2012 KM410165 KM410221 KM410193 KM410249 KM410277 S. roslihashimi R3 Cameron Highlands 04 24 N, 101 22 E 1033m 26/05/2012 KM410166 KM410222 KM410194 KM410250 KM410278 S. tanahrataense TR1 Cameron Highlands 04 34 N, 101 20 E 1315m 28/05/2012 KM410167 KM410223 KM410195 KM410251 KM410279 S. tanahrataense TR2 Cameron Highlands 04 34 N, 101 20 E 1315m 31/03/2012 KM410168 KM410224 KM410196 KM410252 KM410280 S. tanahrataense TR3 Cameron Highlands 04 34 N, 101 20 E 1315m 31/03/2012 KM410169 KM410225 KM410197 KM410253 KM410281 S. asakoae A1 Cameron Highlands 04 31 N, 101 24 E 1602m 22/08/2012 KM410170 KM410226 KM410198 KM410254 KM410282 S. asakoae A2 Cameron Highlands 04 31 N, 101 24 E 1602m 27/05/2012 KM410171 KM410227 KM410199 KM410255 KM410283 S. asakoae A3 Cameron Highlands 04 31 N, 101 24 E 1602m 27/07/2012 KM410172 KM410228 KM410200 KM410256 KM410284 S. brinchangense B1 Cameron Highlands 04 31 N, 101 23 E 1813m 30/03/2012 KM410173 KM410229 KM410201 KM410257 KM410285 S. brinchangense B2 Cameron Highlands 04 31 N, 101 23 E 1813m 30/03/2012 KM410174 KM410230 KM410202 KM410258 KM410286 S. brinchangense B3 Cameron Highlands 04 31 N, 101 23 E 1813m 30/03/2012 KM410175 KM410231 KM410203 KM410259 KM410287 S. lurauense L1 Janda Baik 03 18 N, 101 52 E 530m 22/02/2011 KM410176 KM410232 KM410204 KM410260 KM410288 S. lurauense L2 Janda Baik 03 18 N, 101 52 E 530m 22/02/2011 KM410177 KM410233 KM410205 KM410261 KM410289 S. lurauense L3 Janda Baik 03 18 N, 101 52 E 530m 22/02/2011 KM410178 KM410234 KM410206 KM410262 KM410290 S. sofiani S1 Cameron Highlands 04 28 N, 101 22 E 1405m 27/06/2012 KM410179 KM410235 KM410207 KM410263 KM410291 S. sofiani S2 Cameron Highlands 04 34 N, 101 20 E 1315m 28/08/2012 KM410180 KM410236 KM410208 KM410264 KM410292 S. sofiani S3 Cameron Highlands 04 34 N, 101 20 E 1315m 27/06/2012 KM410181 KM410237 KM410209 KM410265 KM410293 Simulium ceylonicum species group S. trangense T1 Cameron Highlands 04 23 N, 101 22 E 872m 28/07/2012 KM410182 KM410238 KM410210 KM410266 KM410294 S. trangense T2 Cameron Highlands 04 14 N, 101 18 E 159m 25/06/2012 KM410183 KM410239 KM410211 KM410267 KM410295 S. trangense T3 Cameron Highlands 04 14 N, 101 18 E 159m 25/06/2012 KM410184 KM410240 KM410212 KM410268 KM410296 S. sheilae SH1 Janda Baik 03 18 N, 101 52 E 530m 01/03/2011 KM410185 KM410241 KM410213 KM410269 KM410297 S. sheilae SH2 Janda Baik 03 18 N, 101 52 E 530m 01/03/2011 KM410186 KM410242 KM410214 KM410270 KM410298 S. sheilae SH3 Janda Baik 03 18 N, 101 52 E 530m 01/03/2011 KM410187 KM410243 KM410215 KM410271 KM410299 S. leparense LPR1 Jerantut 04 06 N, 102 36 E 400m 17/06/2011 KM502570 KM502573 KM502576 KM502579 KM502582 S. leparense LPR2 Jerantut 04 06 N, 102 36 E 400m 17/06/2011 KM502571 KM502574 KM502577 KM502580 KM502583 S. leparense LPR3 Jerantut 04 06 N, 102 36 E 400m 17/06/2011 KM502572 KM502575 KM502578 KM502581 KM502584 Simulium tani cytoform K was used as an outgroup.

4 V. L. Low et al. Table 2. Primers used for amplification and sequencing of the mitochondria-encoded COI, 12S rrna, 16S rrna and nuclear-encoded 18S rrna and 28S rrna sequences in blackflies of the subgenus Gomphostilbia in Malaysia. Gene Primer and sequence (5 3 ) Reference Mitochondrial COI LCO1490: GGT CAA CAA ATC ATA AAG ATA TTG G HCO2198: TAA ACT TCA GGG TGA CCA AAA AAT CA 12S rrna 12Sbi: AAG AGC GAC GGG CGA TGT GT 12Sz: AGT ATT GGT AAA ATT TGT GCC AGC 16S rrna 16S1: CTC CGG TTT GAA CTC AGA TC 16S2: CGC CTG TTT ATC AAA AAC AT Nuclear 18S rrna B18S_F: TTT TAT GCA AGC CAA GCA CA B18S_R: TGG GAA TTC CAG GTT CAT GT 28S rrna B28S_F: GAA AAG GGA AAA GTC CAG CAC B28S_R: CAC ATT TTA TGC GCT CAT GG Folmer et al. (1994) Kocher et al., (1989) Simon et al. (1994) Xiong & Kocher (1991) Low et al. (2014) Low et al. (2014) S. trangense) for COI, 0.00% (S. lurauense vs. S. sofiani) to 9.31% (S. izuae vs. S. trangense) for 12S rrna, and 0.38% (S. izuae vs. S. roslihashimi, and S. lurauense vs. S. sofiani) to 6.10% (S. sofiani vs. S. trangense) for 16S rrna. Genetic variability for 18S rrna was highly conserved, with limited interspecific variation. Differentiation among species, using 28S rrna, was ambiguous, particularly for S. brinchangense, S. tanahrataense and S. izuae (results not shown). Interspecific divergence for the concatenated dataset varied from 0.23% (S. lurauense vs. S. sofiani) to 5.90% (S. izuae vs. S. leparense) (Table 3). Phylogenetic analyses Based on the COI gene, S. trangense and S. leparense clustered with members of the S. asakoae group (Figure S1, online), although morphologically they are members of the S. ceylonicum group. Within the S. asakoae group, S. lurauense and S. sofiani formed a subclade. The 12S rrna gene revealed two monophyletic clades, corresponding to the S. asakoae and S. ceylonicum species groups (Figure S2, online). Simulium lurauense and S. sofiani represented a distinct subclade, but with no genetic support for separate species. The 16S rrna gene indicated that members of the S. asakoae group formed a monophyletic clade, but members of the S. ceylonicum group were in an unresolved polytomy, and did not distinguish S. lurauense and S. sofiani as separate species (Figure S3, online). Among four tested phylogenetic analyses for the 16S rrna gene, the BI tree differed in that S. asakoae clustered with S. sofiani and S. lurauense, with low posterior probability support (0.52) (Figure S4, online). The 28S rrna gene did not unequivocally resolve phylogenetic relationships for the S. asakoae group (Figure S5, online). However, S. leparense, S. sheilae, S. trangense and the closely related species S. lurauense and S. sofiani were distinct. Simulium leparense, S. sheilae and S. trangense formed a monophyletic clade corresponding to the S. ceylonicum group. Simulium lurauense and S. sofiani formed a separate monophyletic clade. Based on concatenated sequences of COI, 12S rrna, 16S rrna and 28S rrna genes, three phylogenetic analyses (ML, MP and NJ) produced trees with the same topology but with different bootstrap support values. The BI tree differed by grouping S. trangense with members of the S. asakoae species group, with 0.56 posterior probability support (Figure S6, online). By contrast, full bootstrap support values were recovered by ML, MP and NJ analyses for the genetic clade of the S. asakoae species group. Hence, ML, MP and NJ trees were proposed as the best hypotheses of phylogenetic relationships among the members. The ML tree for the concatenated dataset is representative of relationships and shows ML, MP and NJ node support values (Fig. 1). The phylogenetic tree was comprised of two main monophyletic clades. One clade consisted of members of the S. ceylonicum group (S. leparense, S. sheilae and S. trangense), Table 3. Ranges of interspecific genetic distance (uncorrected p,expressedas percentages)basedonthe concatenated dataset ofcoi,12srrna,16s rrna and 28S rrna genes for blackflies in the genus Simulium, subgenusgomphostilbia, frommalaysia. 1 2 3 4 5 6 7 8 9 10 1 S. izuae 2 S. roslihashimi 1.08 1.23 3 S. tanahrataense 1.58 2.00 1.50 1.85 4 S. asakoae 2.97 3.16 2.74 2.89 2.78 2.97 5 S. brinchangense 2.54 2.74 2.20 2.43 2.24 2.54 2.85 3.09 6 S. lurauense 3.39 3.62 3.20 3.35 3.08 3.24 3.13 3.36 3.08 3.39 7 S. sofiani 3.55 3.70 3.28 3.35 3.24 3.32 3.13 3.36 3.24 3.47 0.23 0.35 8 S. trangense 5.48 5.67 5.20 5.36 5.36 5.51 5.17 5.40 5.01 5.32 4.93 5.32 5.05 5.28 9 S. sheilae 5.37 5.56 5.02 5.14 5.25 5.44 4.94 5.14 5.17 5.40 4.90 5.13 4.90 5.06 4.21 4.36 10 S. leparense 5.63 5.90 5.48 5.55 5.36 5.44 5.29 5.48 5.13 5.48 5.09 5.32 5.13 5.21 4.47 4.63 4.01 4.09

Phylogeny of S. asakoae and S. ceylonicum groups 5 Fig. 1. Maximum likelihood phylogenetic tree for Simulium (Gomphostilbia) species from Malaysia, with Simulium tani as the outgroup, based on concatenated sequences of COI, 12S rrna, 16S rrna and 28S rrna genes. Bootstrap values [ML/MP/NJ] are shown on the branches. For supraspecific nodes, bootstrap and posterior probability values are shown below the arrows [ML/MP/NJ/BI]. The bar indicates substitutions per site. BI, Bayesian inference; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbour-joining. with low to high bootstrap support (ML 57%, MP 77%, NJ 89%). The second clade consisted of members of the S. asakoae group, with 100% bootstrap support. Simulium lurauense and S. sofiani formed a subclade with full bootstrap support within the S. asakoae group, but the relationships of the two species were paraphyletic. Discussion Of the five genetic markers in our study, the COI gene is more variable and informative in resolving interspecific relationships among the members of the S. ceylonicum and S. asakoae species groups. The COI gene has been the genetic marker of choice for

6 V. L. Low et al. blackflies in studies of DNA barcoding (Rivera & Currie, 2009; Pramual et al., 2010; Pramual & Adler, 2014), phylogenetics (Conflitti et al., 2010; Phayuhasena et al., 2010; Pramual et al., 2012), and phylogeography (Pramual et al., 2005; Finn & Adler, 2006; Low et al., 2014). The value of this marker in revealing the cryptic biodiversity of blackflies has been documented (Pramual et al., 2010; Pramual & Nanork, 2012). Yet, limitations of the COI gene in differentiating species of blackfly have been reported (Ilmonen et al., 2009; Pramual & Adler, 2014). Similarly, the COI gene fails to differentiate the S. lurauense and S. sofiani material, which highlights the need to test the resolving power of other loci. The 28S rrna gene provides resolution of S. lurauense and S. sofiani, althoughithaslimitedutilityfor other members of the S. asakoae group. Although 12S rrna and 28S rrna support the monophyly of the S. ceylonicum species group, COI and 16S rrna do not. Relationships within each species group are not concordant, based on COI, 12S rrna, 16S rrna or 28S rrna. These discrepancies again highlight the need for a multi-locus analysis, as recognized in previous studies of other species groups (Phayuhasena et al., 2010). At least one gene confirmed the species status of all 10 nominal taxa in our study, typically with multiple genes providing support. Note, however, that the number of specimens used in the analyses is not sufficient to infer intraspecific variation in the members of the species groups. Further, the specimens of each species in the present study were collected from a single location, despite sampling efforts in several Malaysian states, which did not allow for the assessment of possible geographic variations within species. The inadequate phylogenetic signal from mitochondrial DNA sequences for distinguishing S. lurauense and S. sofiani, even when using a multi-locus sequence dataset, demonstrates the need for multiple genetic markers. Previous studies have reported the non-monophyly of Simulium nakhonense Takaoka & Suzuki and the closely related Simulium quinquestriatum Shiraki, based on COI, although 28S rrna confirmed that they are distinct species (Phayuhasena et al., 2010; Pramual & Adler, 2014). Slight structural differences support the limited genetic evidence (28S rrna) that S. lurauense and S. sofiani are separate species (Takaoka et al., 2011a, 2011b). Ecological evidence also supports their distinct species status; S. lurauense occurs in lowlands (< 600 m a.s.l.), whereas S. sofiani occurs in highlands (> 1300 m a.s.l.) (Takaoka et al., 2011a,2011b), minimizing opportunities for hybridization. Similar altitudinal differentiation is known for other Oriental simuliids (Pramual &Pangjanda,2015).ThepresentanalysisofS. lurauense and S. sofiani is based on samples from the only sites at which they are known to occur: their respective type localities, which are 140 aerial km apart. The geographic separation of samples, whether it reflects actual species distributions or incomplete sampling, complicates the assessment of species status and is a common challenge in the Simuliidae (Adler & Şirin, 2014). The polytene chromosomes of the Simuliidae have provided arichsourceofcharactersfortestingreproductiveisolation and interpreting evolutionary relationships (Adler et al., 2010). The only chromosomal information available for the S. asakoae and S. ceylonicum groups (Jitklang et al., 2008), albeit based on material from Thailand, supports the validity of three of the species subjected to molecular analysis in the present study (S. asakoae, S. sheilae and S. trangense). The chromosomes initially revealed the cryptic species S. trangense. However, S. trangense and S. asakoae from Thailand were considered to be sister species, based on a single shared chromosomal inversion, rather than as members of separate monophyletic species groups. Lack of congruence between chromosomal and molecular data is not unprecedented (della Torre et al., 2002). There are several possible explanations for the discrepancy, such as mimic inversions between S. asakoae and S. trangense, cryptic species within S. asakoae, and unrooted chromosomal relationships. Asinglemarkerfordelineatingspeciesrelationshipscanoffer limited resolution and the estimation of species relationships can be ambiguous when phylogenies are not concordant among genes. Therefore, a multi-locus approach is often required to resolve evolutionary relationships in simuliids. The multi-locus data presented here support the morphological hypotheses of species and their relationships, confirming the value of morphology while recognizing the need for integrated character sources in the modern era of systematics. Supporting Information Additional Supporting Information may be found in the online version of this article under the DOI reference: DOI: 10.1111/mve.12120 Figure S1. Maximum likelihood phylogenetic tree of Simulium taxa from Malaysia, based on COI sequences. Bootstrap and posterior probability values (ML/MP/NJ/BI) are shown on the branches. The bar indicates substitutions per site. BI, Bayesian inference; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbour-joining. Figure S2. Maximum likelihood phylogenetic tree of Simulium taxa from Malaysia, based on 12S rrna sequences. Bootstrap and posterior probability values (ML/MP/NJ/BI) are shown on the branches. The bar indicates substitutions per site. BI, Bayesian inference; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbour-joining. Figure S3. Maximum likelihood phylogenetic tree of Simulium taxa from Malaysia, based on 16S rrna sequences. Bootstrap values (ML/MP/NJ) are shown on the branches. The bar indicates substitutions per site. ML, maximum likelihood; MP, maximum parsimony; NJ, neighbour-joining. Figure S4. Bayesian inference phylogenetic tree of Simulium taxa based on 16S rrna sequences. Posterior probability values are shown on the branches. The bar indicates substitutions per site. Figure S5. Maximum likelihood phylogenetic tree of Simulium taxa based on 28S rrna sequences. Bootstrap and posterior probability values (ML/MP/NJ/BI) are shown on the branches. The bar indicates substitutions per site. BI, Bayesian inference; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbour-joining. 2015TheRoyalEntomologicalSociety,Medical and Veterinary Entomology, DOI: 10.1111/mve.12120

Phylogeny of S. asakoae and S. ceylonicum groups 7 Figure S6. Bayesian inference phylogenetic tree of Simulium taxa based on concatenated sequences of COI, 12S rrna, 16S rrna and 28S rrna genes. Posterior probability values are shown on the branches. The bar indicates substitutions per site. Table S1. Ranges of interspecific genetic distance (uncorrected p, expressed as percentages) for species of Simulium (Gomphostilbia) frommalaysia,basedoncoigenedata. Table S2. Ranges of interspecific genetic distance (uncorrected p,expressedaspercentages)forspeciesofsimulium (Gomphostilbia) frommalaysia,basedon12srrnagenedata. Table S3. Ranges of interspecific genetic distance (uncorrected p,expressedaspercentages)forspeciesofsimulium (Gomphostilbia) frommalaysia,basedon16srrnagenedata. Acknowledgements This work was supported by research grants from the University of Malaya (RP003C-13SUS and RP003A-13SUS) and by the Fundamental Research Grant Scheme (FP016-2012A). 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