Phylogeny of Anophelinae using mitochondrial protein coding genes

Abstract

Malaria is a vector-borne disease that is a great burden on the poorest and most marginalized communities of the tropical and subtropical world. Approximately 41 species of Anopheline mosquitoes can effectively spread species of Plasmodium parasites that cause human malaria. Proposing a natural classification for the subfamily Anophelinae has been a continuous effort, addressed using both morphology and DNA sequence data. The monophyly of the genus Anopheles, and phylogenetic placement of the genus Bironella, subgenera Kerteszia, Lophopodomyia and Stethomyia within the subfamily Anophelinae, remain in question. To understand the classification of Anophelinae, we inferred the phylogeny of all three genera (Anopheles, Bironella, Chagasia) and major subgenera by analysing the amino acid sequences of the 13 protein coding genes of 150 newly sequenced mitochondrial genomes of Anophelinae and 18 newly sequenced Culex species as outgroup taxa, supplemented with 23 mitogenomes from GenBank. Our analyses generally place genus Bironella within the genus Anopheles, which implies that the latter as it is currently defined is not monophyletic. With some inconsistencies, Bironella was placed within the major clade that includes Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia, which were found to be monophyletic groups within Anophelinae. Our findings provided robust evidence for elevating the monophyletic groupings Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia to genus level; genus Anopheles to include subgenera Anopheles, Baimaia, Cellia and Christya; Anopheles parvus to be placed into a new genus; Nyssorhynchus to be elevated to genus level; the genus Nyssorhynchus to include subgenera Myzorhynchella and Nyssorhynchus; Anopheles atacamensis and Anopheles pictipennis to be transferred from subgenus Nyssorhynchus to subgenus Myzorhynchella; and subgenus Nyssorhynchus to encompass the remaining species of Argyritarsis and Albimanus Sections.

Keywords: Anopheles; mitochondrial genomes; phylogenetics.

Figure 1.

Scheme of amplifications performed in this study. Blue bars show amplified fragments sequenced by Illumina technology, while remaining colours show Sanger-sequenced fragments. (a,b) The two methods of amplification of the mitochondrial genome used in this study. In both (a) and (b), the complete mitochondrial DNA was amplified in two steps. In (a), fragments of about 15.058 kbp and about 655 bp were amplified and sequenced. In (b), the fragments about 11.857 kbp and about 4.785 kbp were amplified and sequenced in both directions using the same set of primers employed for PCR amplification. (c) The problematic regions after assembly of the mitochondrial genome. Some samples showed low coverage in these regions (green and orange) and then new amplifications and sequencing were carried out to complete the genome sequence. The green region in Anopheles evansae and Anopheles eiseni was amplified employing species specific primers F.

Figure 2.

p-Distances between pairs of aligned, concatenated protein sequences, length 3735 aa. Empty bars show all p-distances except between pairs of sequences from the same species (the smallest distance in this set is 0.0005, representing two differences over the sequence pair), and filled bars show distances between taxa from different genera or subgenera (the smallest distance in this set is 0.064, representing 238 differences over the sequence pair).

Figure 3.

Most analyses described in this study place Bironella within genus Anopheles.

Figure 4.

Anophelinae with reduced taxa, rooted by Chagasia. Mitochondrial protein sequences, slow sites only using TIGER, analysed with PhyloBayes using the CAT-GTR model.