Ecology of black flies (Diptera: Simuliidae) in streams of northern and southern Thailand: Factors associated with larval and pupal distributions.

The ecology of black flies in Thailand was investigated, based on 19,451 larvae and pupae collected from 65 stream sites in 10 northern provinces during the rainy, cool, and hot seasons, and 1,906 larvae and pupae collected from 18 sites in 9 southern provinces during the cool season. Twenty-seven black fly species were identified from northern Thailand, of which 26 were found in the cool season, when richness was greatest. Significant regressions between species richness and elevation fit a unimodal model in the rainy season but a linear model in the cool and hot seasons. Twenty-two species occurred in all seasons. Species in the subgenera Gomphostilbia and Nevermannia were most common in the hot season, whereas species in the subgenus Simulium were predominant in the cool season. Some species (e.g., S. nakhonense) were geographically widespread, whereas others (e.g., S. chaliowae and S. weji) were restricted to particular localities. Eighteen species and species complexes were found in southern Thailand. The S. tani complex was the most widely distributed taxon, occurring at 66.7% of the sites in the South. Ecological analyses revealed that water temperature, elevation, conductivity, dissolved oxygen, and stream size were among the significant factors associated with the distributions of black flies in both regions of Thailand-the same factors associated with simuliid distributions in other areas of the world.

Integrated cytogenetic, ecological, and DNA barcode study reveals cryptic diversity in Simulium (Gomphostilbia) angulistylum (Diptera: Simuliidae).

An integrated approach based on cytogenetics, molecular genetics, and ecology was used to examine diversity in the black fly Simulium angulistylum Takaoka & Davies in Thailand. Cytological analysis revealed three cytoforms (A, B, and C) of S. angulistylum differentiated by fixed chromosome inversions. Distributions of these cytoforms were associated with ecology. Cytoforms A and B were found in low-altitude habitats (<600 m above sea level), whereas cytoform C occurred at high altitudes (>1000 m above sea level). Mitochondrial DNA sequences of the cytochrome oxidase subunit I barcoding region revealed significant genetic differentiation among the cytoforms. The mitochondrial DNA haplotype network revealed divergent lineages within cytoforms, indicating additional hidden diversity. Therefore, integrated approaches are necessary for fully understanding black fly biodiversity. Population genetic analysis revealed high genetic structuring that could be due to the habitat preferences of S. angulistylum. Phylogeographic analyses indicated population demographic expansion at the mid-Pleistocene (900 000 years ago), which is older than for other black flies and insects in the Southeast Asian mainland. The high level of genetic structure and diversity, therefore, could also be due to the long demographic history of S. angulistylum.

Phylogenetic relationships among the black fly species (Diptera: Simuliidae) of Thailand based on multiple gene sequences.

Simulium is a very speciose genus of the black fly family Simuliidae that includes many important pests of humans and animals. Cytotaxonomic and morphological studies have made substantial progress in Simulium systematics. 16S rRNA and ITS-1 DNA sequence studies have assisted this progress. Intensive multi-gene molecular systematic investigations will, however, be required for a comprehensive understanding of the genus' taxonomy and evolution. Our research was conducted to investigate the relationships of Thai Simulium at the subgeneric, species group and species levels. We also examined the possibility of using mitochondrial DNA sequences to facilitate Simulium species identification. Data were collected from three mitochondrial genes (COI, ND4 and 16S rRNA) and two segments of the nuclear 28S ribosomal RNA (the D1 to D2 and the D4 expansion regions). The subgenera Simulium and Gomphostilbia were monophyletic in most analyses. Nevermannia included Montisimulium but was otherwise monophyletic in multigene analyses. In most analyses, Simulium and Nevermannia were more closely related to each other than to Gomphostilbia which was usually basal. Species groups were generally monophyletic. Within Gomphostilbia, however, the batoense species group was always paraphyletic to the other two species groups found in Thailand. Three species groups in Simulium were not monophyletic. The tendency to gill filament number reduction for some species groups in the subgenus Simulium was associated with a derived position in multigene analyses. Most species were monophyletic with two exceptions that probably represent species complexes and will present difficulties for rapid mitochondrial DNA identification.

Cytotaxonomy of Simulium siamense Takaoka and Suzuki (Diptera: Simuliidae) in Thailand.

Larval polytene chromosomes of 1824 individuals of Simulium siamense Takaoka and Suzuki from 32 locations in northern, northeastern, eastern, and central Thailand were examined. Differences in sex chromosomes, fixed inversions, and the kind and frequency of floating inversions revealed five cytoforms (A, B, C, D, and E). In contrast to most Simulium species, females of S. siamense are the heterogametic sex (ZW) and males are the homogametic sex (ZZ). Cytoform A differs from the others by having 23 floating inversions and undifferentiated sex chromosomes (W0 was undifferentiated from Z0). Cytoforms B, C, and D differ from each other by having different W chromosomes (W1, W2, and W3) in females. Cytoform E is characterized by having two fixed inversions, IS-1 and IIIS-2. Cytoform A is widely distributed throughout the geographic ranges of the species in northern, northeastern, eastern, and central Thailand, whereas cytoforms B, C, and D are restricted to a few locations in central Thailand. Although the larvae of the five cytoforms are morphologically similar, they seem to occupy different ecological habitats, which should be further investigated. At present, no definite conclusion can be drawn as to the taxonomic status of the cytoforms of S. siamense. However, this study indicates that S. siamense is represented by at least five cytoforms, of which cytoform B is the most distinct by having complete sex linkage and no floating inversions.

Molecular phylogeny of black flies (Diptera: Simuliidae) from Thailand, using ITS2 rDNA.

The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 40 black fly species from Thailand, belonging to 4 subgenera of the genus Simulium, namely Gomphostilbia (12 species), Nevermannia (5 species), Montisimulium (1 species), Simulium sensu stricto (21 species), and an unknown subgenus with one species (Simulium baimaii). The length of the ITS2 ranged from 247 to 308 bp. All black fly species had high AT content, ranging from 71 to 83.8%. Intraindividual variation (clonal variation) occurred in 13 species, ranging from 0.3 to 1.1%. Large intrapopulation and interpopulation heterogeneities exist in S. feuerboni from the same and different locations in Doi Inthanon National Park, northern Thailand. Phylogenetic relationships among 40 black fly species were examined using PAUP (version 4.0b10) and MrBAYS (version 3.0B4). The topology of the trees revealed two major monophyletic clades. The subgenus Simulium and Simulium baimaii were placed in the first monophyletic clade, whereas the subgenera Nevermannia + Montisimulium were placed as the sister group to the subgenus Gomphostilbia in the second monophyletic clade. Our results suggest that S. baimaii belongs to the malyschevi-group or variegatum-group in the subgenus Simulium. The molecular phylogeny generally agrees with existing morphology-based phylogenies.

Phylogeography of the black fly Simulium tani (Diptera: Simuliidae) from Thailand as inferred from mtDNA sequences.

Intraspecific phylogeography has been used widely as a tool to infer population history. However, little attention has been paid to Southeast Asia despite its importance in terms of biodiversity. Here we used the cytochrome oxidase I gene of mitochondrial DNA (mtDNA) for a phylogeographic study of 147 individuals of the black fly Simulium tani from Thailand. The mtDNA revealed high genetic differentiation between the major geographical regions of north, east and central/south Thailand. Mismatch distributions indicate population expansions during the mid-Pleistocene and the late Pleistocene suggesting that current population structure and diversity may be due in part to the species' response to Pleistocene climatic fluctuations. The genealogical structure of the haplotypes, high northern diversity and maximum-likelihood inference of historical migration rates, suggest that the eastern and central/southern populations originated from northern populations in the mid-Pleistocene. Subsequently, the eastern region had had a largely independent history but the central/southern population may be largely the result of recent (c. 100,000 years ago) expansion, either from the north again, or from a relictual population in the central region. Cytological investigation revealed that populations from the south and east have two overlapping fixed chromosomal inversions. Since these populations also share ecological characteristics it suggests that inversions are involved in ecological adaptation. In conclusion both contemporary and historical ecological conditions are playing an important role in determining population genetic structure and diversity.

Larval polytene chromosomes of black flies (Simulium) from Thailand. I. Comparison among five species in the subgenus Gomphostilbia enderlein.

Larval polytene chromosome maps of Simulium (G.) asakoae and S. (G.) sp. g in the ceylonicum-group and S. (G.) angulistylum, S. (G.) decuplum and S. (G.) siamense in the batoense-group of the subgenus Gomphostilbia from Thailand are presented. These species have three pairs of chromosomes (2n = 6). Light stained centromeric bands were observed in the chromosomes of S. (G.) asakoae, S. (G.) sp. g, S. (G.) decuplum and S. (G.) siamense, whereas heavy dark centromeric bands were present in S. (G.) angulistylum. The best distinguishing character of Simulium species in the subgenus Gomphostilbia is the position of the nucleolar organizer in the short arm of chromosome I. The Ring of Balbiani and the double bubble are located in chromosome arm IIS in all species except for S. (G.) angulistylum, which showed these cytological markers in chromosome arm IIIS. A low chromosomal polymorphism was recorded in all species except for S. (G.) sp. g, which exhibited a standard polytene chromosome. Inversion polymorphisms found in this study conformed to Hardy-Weinberg equilibrium and were not associated with sex. These species have different specific markers and banding patterns although homologous banding sequences were found in chromosome arm IIS in S. (G.) asakoae, S. (G.) sp. g, S. (G.) decuplum and S. (G.) siamense and chromosome arm IIIS in S. (G.) angulistylum. Our results showed no evidence of a sibling species complex within any taxon.