Mitochondrial genomes of Anopheles arabiensis, An. gambiae and An. coluzzii show no clear species division

Introduction

Historically, mtDNA sequence has been used in taxonomy as a source of species diagnostic markers (Cronin et al. (1991); De Barba et al. (2014); Pegg et al. (2006)) or in population genetics and evolutionary studies (Fu et al. (2013); Harrison (1989); Llamas et al. (2016)). One advantage of using mitochondrial over nuclear DNA for such studies is that the mutation rate of mtDNA is about 10 times faster than nuclear DNA (Brown et al. (1979); Haag-Liautard et al. (2008)), hence amplifying the evolutionary trajectory of populations and species. In addition, mtDNA is easy to amplify, because there are more copies of mitochondrial DNA relative to nuclear DNA. Also, universal primers can be applied to a wide range of species. Widely used universal primers target the cytochrome b and cytochrome oxidase 1 genes (Tahir et al. (2016)), because both have conserved and highly variable regions. In addition to these, other genes as described in De Mandal et al. (2014), can also be used as markers. However, phylogenetic trees based on mtDNA can deviate from the ones that are derived from nuclear DNA (Phillips et al. (2013); Shaw (2002); Sota & Vogler, 2001).

The Anopheles gambiae species complex consists of eight morphologically identical species that can only be distinguished with molecular markers (Scott et al. (1993)) or, for some of the species, by cytological examination of polytene chromosomes (Green, 1972; Pombi et al., 2008). The currently used molecular markers are located within genomic islands of divergence located proximal to the centromeres (Lee et al. (2014); Turner et al. (2005)). Monitoring additional species-specific markers on mitochondrial DNA (mtDNA) could increase the ease of application and accuracy of species detection assays. In addition, mtDNA markers could enhance our understanding of divergence times among taxa within the complex.

In this study we wished to identify species-specific markers within the mtDNA for Anopheles arabiensis, An. coluzzii and An. gambiae, including among the chromosomal forms currently subsumed under the designations An. gambiae and An. coluzzii, with the goal of adding these to our existing Anopheles species detection assay (Lee et al. (2014)). We sequenced the whole mitogenomes of 70 individual mosquito specimens collected throughout Sub-Saharan Africa. The raw Illumina sequencing reads were mapped to the AgamP4 reference sequence, which included both nuclear and mitochondrial sequences. We explore the relationship among An. arabiensis, An. coluzzii, An. gambiae and four of the sub-specific chromosomal form mitogenome sequences.

Methods

Results and Discussion

We identified a total of 783 single nucleotide polymorphisms (SNPs) over the entire mitogenome. The majority of these (58.7%) were singletons (found on one of the 70 mitogenomes). We did not identify any SNPs unique to the species or chromosomal forms (Table 2) and therefore conclude that mtDNA is not suitable for Anopheles gambiae complex species identification.