Abstract
It is known that PCR amplification of highly homologous genes from complex DNA mixtures can generate a significant proportion of chimeric sequences. The 16S rRNA gene is not only widely used in estimating the species diversity of endosymbionts in aphids but also used to explore the co-diversification of aphids and their endosymbionts. Thus, chimeric sequences may lead to the discovery of non-existent endosymbiont species and mislead Buchnera-based phylogenetic analysis that lead to false conclusions. In this study, a high probability (6.49%) of chimeric sequence occurrence was found in the amplified 16S rRNA gene sequences of endosymbionts from aphid species in the subfamily Lachninae. These chimeras are hybrid products of multiple parent sequences from the dominant species of endosymbionts in each corresponding host. It is difficult to identify the chimeric sequences of a new or unidentified species due to the high variability of their main parent, Buchnera aphidicola, and because the chimeric sequences can confuse the phylogenetic analysis of 16S rRNA gene sequences. These chimeras present a challenge to Buchnera-based phylogenetic research in aphids. Thus, our study strongly suggests that using appropriate methods to detect chimeric 16S rRNA sequences may avoid some false conclusions in endosymbiont-based aphid research.
Highlights
Aphidina live in association with a diverse assemblage of heritable intra-cellular bacterial endosymbionts [1,2,3]
It is difficult to identify the chimeric sequences of a new or unidentified species due to the high variability of their main parent, Buchnera aphidicola, and because the chimeric sequences can confuse the phylogenetic analysis of 16S rRNA gene sequences
Most chimeric sequences were formed with the aphid primary symbiont Buchnera as well as the secondary symbiont Serratia symbiotica, which was found in many Lachninae [16,35] (Figure 1)
Summary
Aphidina live in association with a diverse assemblage of heritable intra-cellular bacterial endosymbionts [1,2,3]. Aphids can have a series of secondary endosymbionts, such as Regiella insecticola (Enterobacteriaceae), Hamiltonella defensa (Enterobacteriaceae), Serratia symbiotica (Enterobacteriaceae) [14], Wolbachia pipientis (Alphaproteobacteria: Rickettsiales) [15], and a Sodalis-like symbiont (Enterobacteriaceae) [16] These benefit their aphid hosts by providing protection against parasitoids, pathogens, or thermal stress, though they are generally not required for host development and reproduction [17]. The Lachninae species are known to have a high incidence of facultative symbiont infection [16], which makes Lachninae a perfect object to study the influences of chimeric sequences in endosymbiont-based aphid research. We evaluated the effect of these chimeras on the phylogenetic reconstruction of aphids and endosymbionts
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