| Mosquito is a kind of important medical insect. The research of its taxonomy has been studied for more than 200 years, and there has been a systematic framework about it. But the cryptic species and complex of mosquitoes consisted of some individuals shared with morphological characters make their identifying more difficult by traditional methods. Finding the suitable characters and methods becomes important for mosquitoes classifier in new century. With molecular biology developing, taxonomy has been into a new period since the 1980s. The taxonomy with molecular technology based on DNA showed more sensitive and stable than morphological characters as soon as it was used in molecular taxonomy. Therefore, the methods were accepted by most of biologist.The Anopheles (Cellia) maculatus complex includes at least eight species, five of which were found in China. The taxonomy of this complex has been investigated using a variety of morphological and genetic characters. However, because chromosomal preparations require considerable skill and morphological separation of these species is unreliable, the identification of this complex is confused and little understanding of relationship is obtained. In this paper, we applied molecular markers to identify and analyse interspecific variation among the five members of An. maculatus complex from China, and reconstructed phylogentic trees.The ribosomal DNA 28S-D3 region and mtDNA-COII gene were amplified by PCR with specific primers. The PCR-SSCP of 28S-D3 region was used to identify the five members. Whereafter, the 28S-D3 and mtDNA-COII were sequenced and analyzed. The phylogentic trees were obtained using Neighbor-joining, UPGMA, Minimum Evolution, Maximum parsimony and Maximum likelihood methods in PCGENE, PHYLIP and MEGA2. The results is as follows:1. The PCR-SSCP result showed that the five members of An. maculatus complex can be visually distinguished by the specific bands in nondenaturing polyacylamide gel. They were stable and objective after many repeats. The results demonstrated the taxonomy by chromosome karyotype, and rectified the identification by themorphologic characters. PCR-SSCP was a perfect molecular identification method.2. The length of 28S-D3 region of the five members in An. maculatus complex was from 388bp to 394bp, the GC contents were from 58.12% to 59.79%. The sequences were very conserved and had no intraspecific variation except one site mutation (T C) in An. dravidicus between Yunnan and Sichuan samples. However, large interspecific variation was found, the average was 4.59%. The largest variation was 6.55% between An. dravidicus and An. pseudowillmori, the smallest was 2.58% between An. dravidicus and An. maculates. The second structure of 28S-D3 were divided two types by the numbers of loops on the stems S2, which An. dravidicus and An. maculates have one loop and others have two. The length of mtDNA-COII gene of An. maculatus complex was 684bp, the GC contents ranged from 24.85% to 26.02%. Intraspecific variation was found in An. maculates, An. willmori and An. dravidicus, which ranged from 0.15% to 0.58%, and presented 3, 2, and 2 haplotypes, respectively. The interspecific variation among five members in DNA sequences ranged from 3.66% to 9.63%. But the deduced amino acid sequences were very conserved, An. willmori, An. sawadwongporni, An. dravidicus and An. maculates (Macu3-5) were the same, An. maculates (Macul-2) and An. pseudowillmori had one or six sites mutation.3. The phylogenetic trees obtained using several methods by 28S-D3 and mtDNA-COII gene. The trees were different from different molecule markers and methods. The results suggested that the rDNA may give strong phylogenetic information.4. As the first time, we analysed the evolution of the An. maculatus complex in subgenus Cellia based on predecessor's achievement acquired from GenBank. The result shows the complex evolved lonely after it co-originated with the ancestor of Myzomyia Series and Neomyzomyia Series at Series lever.
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