Integrated systematics of Anopheles subpictus (Diptera: Culicidae) in the Oriental Region, with emphasis on forms in Thailand and Sulawesi, Indonesia.

The Anopheles subpictus complex consists of four species informally designated, based on fixed inversions of polytene chromosomes and morphology, as species A, B, C and D in India. However, recent studies revealed the presence of only species A and B in Sri Lanka. Little is known about the specific identity of the taxon in other countries in Asia. This paper reports the results of a molecular and morphological study of An. subpictus in Thailand and South Sulawesi, Indonesia. The maxillary palpi of most females from Thailand have the apical pale band longer than the subapical dark band, seta 7-I of pupae branched and short, and eggs with 18-25 float ridges. These characters do not agree with those described for species A, B, C and D in India. The females of An. subpictus from South Sulawesi usually have the subapical dark band of the maxillary palpus equal in length to the apical pale band. Phylogenetic analyses of sequences of the internal transcribed spacer 2 (ITS2) region of rDNA and the cytochrome c oxidase subunit I (COI) gene of mtDNA of specimens from Thailand, and South Sulawesi, and from various localities in GenBank, were conducted. ITS2 sequences of specimens from all localities in Thailand were identical, except for a small divergence in specimen from Phang Nga Province. Three distinct COI clades were detected in specimens from Chiang Mai Province in northern Thailand. However, crossing experiments between the three clades revealed no genetic incompatibility, suggesting that they were conspecific. ITS2 and COI sequences of most specimens from Thailand fell in clades other than those of An. subpictus species A and B and An. subpictus from Indonesia (East Nusa Tenggara, Java, South Sulawesi) and the Philippines. ITS2 sequences from South Sulawesi and East Nusa Tenggara were very similar, and fell in a clade consisting of specimen from Phang Nga in southern Thailand and sequences of some specimens from Cambodia and Vietnam, but their COI sequences were distinct. DNA sequences and morphological differences suggest the presence of two species within An. subpictus in Thailand, and more than one species in Indonesia.

Molecular identification of mosquitoes of the Anopheles maculatus group of subgenus Cellia (Diptera: Culicidae) in the Indonesian Archipelago.

This study reports the molecular differentiation of females of Anopheles maculatus s.l. collected in eight localities on five islands in the Indonesian Archipelago: Hargowilis and Hargotirto villages of Central Java Province, North Kalimantan Province, Sabang off the northern tip of Sumatra Province, Sumba Island of East Nusa Tenggara Province and Sulawesi Province. Analyses based on rDNA (ITS2 and D3) and mtDNA (COII) sequences revealed the presence of An. greeni for the first time in North Kalimantan, and at least one novel (previously unrecognized) species of the Maculatus Group in Central Java (Hargowilis). Despite the similarity of rDNA markers of specimens of An. maculatus s.l. from Central Java and Sulawesi, their COII sequences are highly divergent (3.3%), which might indicate the presence of a further new species. Specimens of An. maculatus s.l. from the other localities had identical rDNA sequences to most An. maculatus s.s. from mainland Southeast Asia, but moderate divergence in their COII sequences (1.2-2.1%). The latter might indicate there are further novel species within the Maculatus Complex. However, as the divergence at COII may be the result of geographical structuring within species related to the historical biogeography of the region, further studies are needed to shed light on this possibility.

Genetic and morphological evidence for a new species of the Maculatus Group of Anopheles subgenus Cellia (Diptera: Culicidae) in Java, Indonesia.

BACKGROUND: Anopheles maculatus, a species of the Maculatus Group of subgenus Cellia (Diptera: Culicidae), is an important vector of human malarial protozoa in Java, Indonesia. However, the identity of this species in Indonesia has been questionable because published reports and records are based mainly on morphological identification, which is unreliable for distinguishing members of the Maculatus Group due to overlapping characters. METHODS: We performed morphological assessments, metaphase karyotype preparations, phylogenetic analyses of ITS2 and cox2 sequence data and cross-mating experiments to determine whether the Javanese form and An. maculatus (s.s.) from Thailand were conspecific. RESULTS: The adults of the Java strain are similar to those of An. maculatus (s.s.), but the larvae and pupae exhibit significant differences. The metaphase karyotype of Javanese specimens includes a long acrocentric X chromosome and a small telocentric Y chromosome, which are distinct from other members of the Maculatus Group. Cross-mating of the Java strain with An. maculatus (s.s.) revealed genetic incompatibility. Phylogenetic analysis of ITS2 and cox2 sequences revealed that the Java strain forms a single clade that is distinct from clades of other members of the group (Kimura 2-parameter, K2P, genetic distances 3.1-19.2% and 1.6-9.6%, respectively). CONCLUSIONS: This study provides evidence that the Javanese form of An. maculatus is not conspecific with An. maculatus (s.s.) and constitutes a previously unrecognized species of the Maculatus Group.