Chaliow Kuvangkadilok

Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction

The Anopheles dirus complex of mosquitoes contains some of the most important vectors of malaria in Southeast Asia. To distinguish five species of the complex that occur in Thailand, a method using the polymerase chain reaction (PCR) was developed. The method utilizes allele‐specific amplification to detect fixed differences between the species in the DNA sequence of the ribosomal DNA internal transcribed spacer 2. Primers were designed to amplify fragments of diagnostic length from the DNA of the different species. The method was tested on 179 mosquitoes of the An. dirus complex from many parts of Thailand and shown to be effective. Every specimen was unambiguously identified as species A, B, C, D or F (i.e. An. dirus s.s. species B, C, D or An. nemophilous, respectively) by the PCR method, with confirmation of 58/61 identifications from polytene chromosome characteristics. For the other three specimens (3/44 from Kanchanaburi 5 locality), there was disagreement between the PCR and chromosomal methods of species identification (probably due to errors in the chromosomal identifications). Primers can be combined in a single PCR reaction providing a rapid, sensitive and straightforward method of species identification. Only small quantities of DNA are required, leaving most of the mosquito to be used for other analyses.

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.

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.

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.

Geographical versus ecological isolation of closely related black flies (Diptera: Simuliidae) inferred from phylogeny, geography, and ecology

To investigate patterns of geographical and ecological separation among morphologically similar, closely related species of black flies, we integrated ecological, geographical, and phylogenetic information, based on multiple gene sequences, for 12 species in the subgenus Gomphostilbia in Thailand. Molecular characters supported the monophyly of the Simulium ceylonicum species group, but not of the Simulium batoense species group, suggesting that revisionary work is needed for the latter. Both ecological and geographical isolation of similar taxa were revealed. Stream velocity and altitude were among the principal ecological factors differing between closely related species. Most closely related species in the subgenus Gomphostilbia overlap geographically, suggesting the possibility of sympatric speciation driven by ecological divergence. Geographical isolation via dispersal also might have contributed to species divergence, while Pleistocene climate changes possibly influenced population genetic structure, demographic history, and speciation of some members of the subgenus.

Cytotaxonomy, Morphology and Ecology of the Simulium nobile Species Group (Diptera: Simuliidae) in Thailand

The polytene chromosomes of 512 larvae of the Simulium nobile species group collected from 16 stream sites in northern, central and southern Thailand were examined. Band-by- band comparisons relative to the established standard chromosome map for the subgenus Simulium distinguished the 2 species of this group, S. nobile and S. nodosum. The differences are mainly due to the position of the nucleolar organizer, pseudochromocenter, and fixed and polymorphic inversions. S. nobile is a monomorphic species, while S. nodosum is a polymorphic species. The morphology of these taxa is described based on the gill filaments and the color of the antennae and female genitalia. S. nobile is distributed in southern Thailand at low altitudes, whereas the distribution of S. nodosum is in the north at higher altitudes.