Black flies are medically and ecologically significant insects. They are also interesting from an evolutionary standpoint regarding the role of chromosomal change and ecological adaptation. In this study, molecular genetic markers based on multiple gene sequences were used to assess genetic diversity and to infer phylogenetic relationships for a group of cytologically highly diverse black flies of the Simulium tuberosum species group in Thailand. Ecological affinities of the species were also
investigated. High levels of genetic diversity were found in cytological species complexes, S. tani and S. doipuiense, and also in
S. rufibasis, which was cytologically nearly monomorphic. The results highlight the necessity of integrating multilevel markers
for fully understanding black fly biodiversity. Phylogenetic relationships based on multiple gene sequences were consistent with
an existing dendrogram inferred from cytological and morphological data. Simulium tani is the most distinctive taxa among the
members of the S. tubersosum species group in Thailand based on its divergent morphological characters. Molecular data supported the monophyletic status of S. tani, S. weji, and S. yuphae, but S. doipuiense and S. rufibasis were polyphyletic, most likely due to incomplete lineage sorting and inadequate phylogenetic signals. Ecological analyses revealed that members of the S. tuberosum species group have clearly different ecological niches. The results thus supported previous views of the importance of ecology in black fly evolution.