The phylogenetic analysis of concatenated ND5 and COI genes showed the existence of three distinct lineages for the five investigated species, i.e. 1) An. rivulorum, 2) An. funestus-like clade I and An. parensis clade II, and 3) An. funestus clades I and II, An. funestus-like clade II, An. parensis clade I and An. vaneedeni clades I and II. The results presented in this study support the hypothesis that there are at least two main divisions within An. funestus which is consistent with the results of Michel et al. as well as the An. funestus-like study .
Previous studies of the ITS2 and D3 regions of the rDNA and COI and Cytochrome Oxidase subunit II (COII) mtDNA genes, for African and Asian Anopheles groups [32, 33] showed that the An. rivulorum subgroup was clearly distinguished from members of the An. funestus subgroup (An. funestus, An. parensis and An. vaneedeni). Although there were slight differences in distances between members of the An. funestus subgroup reported in the two studies, the results reinforce the conclusion of distinct distance between An. funestus and An. rivulorum subgroups. Garros et al. suggested that An. rivulorum, which is assigned to its own subgroup within the broader An. funestus group  and the An. funestus subgroup might have evolved from a common ancestor based on the phylogenetic trees of ITS2, D3 and COI. It should be mentioned that An. rivulorum larvae are morphologically distinct from the members of the An. funestus subgroup  so it is not surprising that the molecular data should reflect this separation.
The phylogenetic data for An. funestus-like in this study suggest that it is a distinct lineage from the other species in the group. These results support Spillings et al. showing this to be a new member of the An. funestus group. However, the phylogenetic trees from the concatenated ND5 and COI both showed two clades for An. funestus-like. Four An. funestus-like specimens were separated into their own lineage with the rest falling into the An. funestus subgroup lineage. Furthermore, two An. parensis specimens were separated from the main An. funestus subgroup lineage and grouped with the An. funestus-like lineage. Further molecular investigations are needed to test the hypothesis of new species in the group and determine the relationship between An. funestus-like and the two An. parensis individuals.
In the study by Michel et al., samples from East Africa had significantly lower average heterozygosity (0.455) and allelic richness (3.9) across all microsatellite loci, and lower mean mtDNA haplotype diversity (0.773) compared with the rest of Africa (0.606, 6.0 and 0.924 respectively). Other studies [16, 35] have also reported two different subdivisions within An. funestus from the analysis of ND5. However, these proposed subdivisions were not correlated with the clades of Michel et al.. Analysis of the mtDNA cytochrome b gene and the ITS2 region in the rDNA  did not find any subdivisions within An. funestus. Previous studies using the RFLP method [17, 18] that included samples of An. funestus from 16 African countries, found evidence for five genetic subdivisions on the ITS2 and D3 regions in the rDNA but again these were not correlated with the Michel et al.  clades.
In the phylogenetic trees in this study, the results did not show clear phylogeny between An. funestus clade II, An. parensis clade I and An. vaneedeni in the An. funestus subgroup even though these three were separated clearly from An. funestus clade I. This undistinguishing relationship between the three species may reflect a selective sweep or non-discrimination due to recent divergence that is known to occur in the mtDNA .
Surprisingly, An. parensis from South Africa shared two of the haplotypes, one with An. funestus from Mozambique and one with An. vaneedeni from South Africa. Donnelly et al.  reported that shared haplotypes between species in the An. gambiae complex might reflect non-contemporary processes such as incomplete lineage sorting between species or historical introgression events. So, although no natural hybridization between An. funestus clade II, An. parensis and An. vaneedeni has been reported, mitochondrial introgression may have happened through a recent event. However, Green and Hunt  reported that cross-mating experiments between An. vaneedeni and An. funestus resulted in sterile male hybrids and asynapsis of the giant polytene chromosomes, two phenomena that occur regularly in crosses between species of Anopheles[11, 19, 40]. A more likely explanation, therefore, is that these shared haplotypes are ancestral.
Although there were apparent subdivisions in the species studied here, these divergences may only be limited to mtDNA. The nature of mtDNA, i.e. haploid, maternal heritage and non-recombination, could retain both distinct mtDNA clades in admixed populations, while their nuclear genome would become homogenized. Michel et al.  reported that there was no corresponding nuclear divergence in spite of deep mtDNA divergence between clades I and II within An. funestus. They suggested that the subdivisions may result from historical introgression either among previously isolated and divergent populations or with a related species . Additional research from other genomic regions is required to determine what these results mean in terms of specific status and relevance in epidemiology and to investigate their roles in malaria transmission in order to better understand the ecological aspects of this important vector group.