Genetic diversity in two sibling species of the Anopheles punctulatus group of mosquitoes on Guadalcanal in the Solomon Islands

Arif U Hasan 2 Setsuo Suguri 2 Chigusa Fujimoto 1 2 Rodney L Itaki 2 Masakazu Harada - 2 Masato Kawabata 0 Hugo Bugoro 3 Bobogare Albino 3 0 International Center for Medical Research, School of Medicine, Kobe University , Kusunoki, Chuo, Kobe, 650-0017 , Japan 1 Department of Medical Technology, Faculty of Health Sciences, Kagawa Prefectural College of Health Sciences , Hara, Mure, Takamatsu, Kagawa, 761-0123 , Japan 2 Department of International Medical Zoology, Faculty of Medicine, Kagawa University , 1750-1, Ikenobe, Miki, Kita, Kagawa, 761-0793 , Japan 3 Solomon Islands Medical Training and Research Institute , Honiara, the Solomon Islands Background: The mosquito Anopheles irenicus, a member of the Anopheles punctulatus group, is geographically restricted to Guadalcanal in the Solomon Islands. It shows remarkable morphological similarities to one of its sibling species, An. farauti sensu stricto (An. farauti s.s.), but is dissimilar in host and habitat preferences. To infer the genetic variations between these two species, we have analyzed mitochondrial cytochrome oxidase subunit II (COII) and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences from Guadalcanal and from one of its nearest neighbours, Malaita, in the Solomon Islands. Results: An. farauti s.s. was collected mostly from brackish water and by the human bait method on both islands, whereas An. irenicus was only collected from fresh water bodies on Guadalcanal Island. An. irenicus is distributed evenly with An. farauti s.s. (SC = 0.033, 0.38%) and its range overlaps in three of the seven sampling sites. However, there is a significant population genetic structure between the species (CT = 0.863, P < 0.01; ST = 0.865, P < 0.01 and FST = 0.878, P < 0.01). Phylogenetic analyses suggest that An. irenicus is a monophyletic species, not a hybrid, and is closely related to the An. farauti s.s. on Guadalcanal. The time estimator suggests that An. irenicus diverged from the ancestral An. farauti s.s. on Guadalcanal within 29,000 years before present (BP). An. farauti s.s. expanded much earlier on Malaita (texp = 24,600 BP) than the populations on Guadalcanal (texp = 16,800 BP for An. farauti s.s. and 14,000 BP for An. irenicus). Conclusion: These findings suggest that An. irenicus and An. farauti s.s. are monophyletic sister species living in sympatry, and their populations on Guadalcanal have recently expanded. Consequently, the findings further suggest that An. irenicus diverged from the ancestral An. farauti s.s. on Guadalcanal. - Background Extensive sampling and genetic studies have suggested that an endemic mosquito named Anopheles irenicus resides exclusively in the northern part of Guadalcanal Island (one of the Solomon Islands), along with An. farauti sensu stricto (An. farauti s.s.), An. hinesorum, An. punctulatus and An. koliensis [1,2]. All these mosquitoes are members of the An. punctulatus group, which was originally considered to comprise four closely related species, An. farauti Laveran, An. punctulatus Donitz, An. koliensis Owen and An. clowi Rozeboom & Knight ([3] and references therein). Further studies with cross-mating experiments, allozyme analysis and DNA probes finally revealed 12 sibling species within this An. punctulatus group: An. farauti s.s. Laveran (formerly An. farauti No. 1), An. hinesorum Schmidt (formerly An. farauti No. 2), An. torresiensis Schmidt (formerly An. farauti No. 3), An. farauti Nos. 4, 5, 6, An. irenicus Schmidt (formerly An. farauti No. 7), An. punctulatus Donitz, An. sp. near punctulatus, An. koliensis Owen, An. rennellensis Taylor & Maffi and An. clowi Rozeboom & Knight [3,4]. Nevertheless, the origin and population structure of the group remain obscure because of the involvement of these complex cryptic species [2,5]. A few striking differences between the endemic An. irenicus mosquito on Guadalcanal and one of its sibling species, An. farauti s.s., provide an excellent opportunity for investigating their genetic relationship. As mentioned earlier, An. irenicus is only found on Guadalcanal [1,2,4], but An. farauti s.s. is distributed from the east through New Guinea, the Bismarck Archipelago and the Solomon Islands to Vanuatu and southward into northern Australia [5,6]. Although these two species generally breed in similar types of water bodies such as small ground pools, margins of creeks, streams and even road ruts [1], they do not readily share their breeding sites. An. farauti s.s. almost always breeds in brackish water, whereas even though An. irenicus shows potential tolerance of brackish water, it is always found in breeding sites containing fresh water [7]. The adult mosquitoes also have distinct patterns of host dependence: An. farauti s.s. is anthropophilic but An. irenicus is zoophilic [1] and never bites humans [2]. Little is known about the reproduction of adult An. irenicus. In natural conditions adult An. farauti s.s. require a blood meal and oviposition usually occurs 48 to 52 hours later (Suguri et al., to be published elsewhere). Larval development in the laboratory is similarly irregular for both species, and delayed hatching of some eggs is common, resulting in the simultaneous occurrence of second instar larvae and pupae in the same rearing bowls (personal observation, [8]). An. farauti s.s. and An. irenicus are morphologically nearly indistinguishable (like members of the An. gambiae complex [9]), but Schmidt et al. [4] described some subtle though definitive differences in morphological characters including the number of proepisternal setae in adults (An. farauti s.s. = 4 or more, An. irenicus = 3 or fewer), the number of branches of seta 5-V and seta 5-VI in pupae (An. farauti s.s. = 17 or fewer, An. irenicus = 18 or more), and the number of branches of seta 2-III in fourth instar larvae (An. farauti s.s. = 9 or fewer, An. irenicus = 10 or more). Schmidt et al. [4] therefore differentiated them taxonomically. However, some morphological variations were noted in the An. farauti s.s. collected from Australia, Papua New Guinea and the Solomon Islands [4]. Therefore, molecular analysis is necessary to identify and study them. Allozyme electrophoresis and mitochondrial DNA (mtDNA) sequences reveal distinguishable differences [2,5]. Moreover, Beebe et al. [1] showed species-specific ribosomal DNA polymorphisms by polymerase chain reaction-repeated fragment length analysis (PCR-RFLP) of ribosomal internal transcribed spacer (rITS) regions. There have been previous studies of the distribution, habitat and morphology of An. irenicus in relation to An. farauti s.s., but less attention has been paid to their genetic variations on a finer scale. Therefore, we aimed to assess this issue using both mitochondrial (cytochrome oxidase subunit II; COII) and nuclear (ribosomal internal transcribed spacer 2; ITS2) markers. Mitochondrial DNA shows ample signatures of genomic events such as gene flow, migration, bottlenecks, speciati (...truncated)