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

Highlights

• Anopheles subpictus in Thailand is genetically and/or morphologically distinct from An. subpictus species A, B, C and D described elsewhere.

• Crossing experiments between three Thai COI clades of An. subpictus revealed no genetic incompatibility, suggesting that they were conspecific.

• Anopheles subpictus from South Sulawesi is genetically distinct from those found in mainland Asia.

Abstract

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.

Introduction

Anopheles subpictus Grassi of the Pyretophorus Series of subgenus Cellia Theobald was first described from a single specimen collected in India (Christophers, 1933). It has a very wide distribution, ranging eastward from Saudi Arabia and Iran across southern Asia (Pakistan, India, Sri Lanka, Bangladesh, Myanmar, Thailand), along southern coastal areas of Cambodia and Vietnam, and coastal areas of Malaysia, Indonesia and Papua New Guinea, and as far east as the Solomon Islands (Sinka et al., 2011). The taxon is a complex of species, the Subpictus Complex, consisting of four species in India, which are informally designated as species A, B, C and D (Suguna et al., 1994). However, current evidence is insufficient to determine which of the four species is conspecific with the nominotypical form. Identification of members of the complex has been based primarily on inversions in the X-arm of polytene chromosomes and morphological characters that include the number of float ridges of eggs, the number of branches of seta 4-M of larvae and seta 7-I of pupae, and pale and dark bands on the maxillary palpi of females. According to Suguna et al., species A, C and D generally occur in freshwater habitats of inland areas, whereas species B is restricted to coastal brackish water habitats. However, recent studies in Sri Lanka revealed that identification based on polytene chromosome inversions and morphological characteristics of the sibling species in India do not correspond to molecular identifications based on ITS2 and COI sequences, by which only two linages corresponding to species A and B were detected in the country, and species B is not restricted to coastal areas (Abhayawardana et al., 1996; Surendran et al., 2013). Similarly, morphological assessment and molecular identification in India using ITS2 and COI sequences (Kumar et al., 2007; Kaura et al., 2010; Chhilar and Chaudhry, 2012) suggested the presence of only species A and B. Additionally, sequences for species C and D are not present in GenBank. Therefore, further investigation is needed to determine whether differences in polytene chromosomes and morphological characters described for species C and D may be variations of either species A or B.

Phylogenetic analyses of the Subpictus Complex have revealed that species A and B do not form a monophyletic clade, but share genetic similarity with closely related species of the Pyretophorus Series: species A is closely related to An. vagus Dönitz whereas species B is closely related to An. epiroticus Linton & Harbach and An. sundaicus (Rodenwaldt) of the Sundaicus Complex (Surendran et al., 2013). The distributions of species A and B are well documented in India and Sri Lanka, but little is known about these forms elsewhere. Species A appears to be confined to India and Sri Lanka whereas species B may have a wider distribution (Surendran et al., 2010, 2013). In general, An. subpictus is primarily zoophilic and exophagic, but indices of anthropophilic behavior may be high (up to 20% human feeding) in some areas (reviewed by Chandra et al., 2010). The role the taxon plays in the transmission of human malarial parasites is well documented in coastal areas of India and Sri Lanka (Abhayawardana et al., 1996; Panicker et al., 1981). In the Indonesian Archipelago, An. subpictus is widely distributed from Sumatra to the Maluku Islands, but the role it plays in the transmission of malarial parasites is only well documented in Java, Sulawesi, the Lesser Sundas and Lombok Islands (reviewed by Elyazar et al., 2013). Whether differences in vectorial capacity reflect genetic distinctions is not known and requires further study.

In Thailand, An. subpictus is distributed throughout the country, including coastal areas in southern provinces, but it is not regarded as a vector of malarial plasmodia due to its zoophilic behavior (Rattanarithikul et al., 2006). The specific identity of An. subpictus in Thailand and elsewhere in Southeast Asia is not clear. Baimai et al. (1996) reported the finding of three karyotypic forms of An. subpictus in Thailand and two forms in Indonesia and the Philippines, but whether these forms reflect interspecific difference is not known. In the present study, we conducted a morphological and molecular study of specimens of An. subpictus collected in inland and coastal areas of Thailand, and in South Sulawesi, Indonesia, and compared ITS2 and COI sequences to sequences available in GenBank for specimens from other Asian countries in order to determine the genetic diversity of the taxon. We also conducted crossing experiments to evaluate the genetic compatibility of three COI clades that were found in specimens collected in Thailand.