ORIGINAL ARTICLE Phylogenetic analysis based on multiple gene sequences revealing cryptic biodiversity in Simulium multistriatum group (Diptera: Simuliidae) in Thailand Pairot PRAMUAL and Piyamas NANORK Department of Biology, Faculty of Science, Mahasarakham University, Maha Sarakham, Thailand Abstract Phylogenetic relationships among four species of the Simulium multistriatum group (Diptera: Simuliidae) in Thailand were examined based on two mitochondrial genes (COI, COII) and one nuclear gene (18S/ITS1). Simulium takense was found to be genetically divergent (>20.3% for COI) from the other species, consistent with their distinctive morphology. Simulium chainarongi and S. chaliowae were monophyletic but were included in paraphyletic S. fenestratum. Simulium fenestratum was divided into three distinct lineages with high levels of genetic divergence. This suggests that S. fenestratum is a species complex. Neither morphological nor cytological examinations revealed evidence of sibling species. The clades derived from phylogenetic analyses were found to be correlated with the ecological conditions of larval habitat. Therefore, ecological adaptation may have played a role in black fly diversification and evolution. These results suggest the use of integrated, multidisciplinary approaches for fully understanding black fly biodiversity and systematics. Key words: black fly, DNA barcode, species complex. INTRODUCTION It has long been recognized that ecological adaptation is an important cause of species evolution (Schluter 1998). For Simuliidae, several studies have revealed that dif- ferent species are associated with different ecological conditions (Adler & McCreadie 1997). For example, different sibling species of the Simulium damnosum complex were associated with different ecoregions of Africa (Boakye et al. 1998). Larval ecological shifts, such as from cascade to river habitat, were thought to be a primary driving force in speciation of S. oviceps and S. dussertorum in the Society Islands, French Polynesia (Joy et al. 2007), suggesting that ecological conditions in larval habitat can play an important role in black fly evolution. There are nine species of the S. multistriatum group in Thailand (Takaoka & Saito 1996; Takaoka & Choochote 2004, 2005a,b,c; Adler & Crosskey 2010). Phayuhasena et al. (2010) examined five species (S. chainarongi, S. chaliowae, S. fenestratum, S. takense and S. triglobus) of this group using multiple gene sequences (COI, ND4, 16S rRNA and 28S rRNA). They revealed the following points: (i) the S. multistriatum group was not monophyletic; (ii) S. takense was posi- tioned at the base of the clade consisting of S. multistria- tum and two other species groups; (iii) S. chainarongi and S. chaliowae were nested within polyphyletic S. fenestratum; and (iv) S. fenestratum might be a species complex. The S. multistriatum group is interesting because species of this group are associated with particular larval habitats or geographically restricted to a few locations (Takaoka & Choochote 2005a,b,c). Among the members of this group, S. fenestratum is the most geo- graphically and ecologically widespread. In terms of distribution, this species has been recorded throughout Thailand and from Indonesia (Adler & Crosskey 2010). Simulium fenestratum also occurs in a wide range of ecological conditions, being recorded from low eleva- tion (<50 m a.s.l.) up to 2300 m a.s.l. (Kuvangkadilok et al. 1999a). It has been suggested that species which are geographically widespread and use a wide range of ecological conditions are prone to be species complexes (Adler & McCreadie 1997). Other species of the Correspondence: Pairot Pramual, Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150, Thailand. Email: pairot.p@msu.ac.th Received 9 May 2011; accepted 21 August 2011. Entomological Science (2011) doi:10.1111/j.1479-8298.2011.00491.x © 2011 The Entomological Society of Japan