Polymerase chain reaction (PCR) products corresponding to 803 bp of the cytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNA COI-II) were deduced to consist of multiple haplotypes in three Sitobion species. We investigated the molecular basis of these observations. PCR products were cloned, and six clones from one individual per species were sequenced. In each individual, one sequence was found commonly, but also two or three divergent sequences were seen. The divergent sequences were shown to be nonmitochondrial by sequencing from purified mtDNA and Southern blotting experiments. All seven nonmitochondrial clones sequenced to completion were unique. Nonmitochondrial sequences have a high proportion of unique sites, and very few characters are shared between nonmitochondrial clones to the exclusion of mtDNA. From these data, we infer that fragments of mtDNA have been transposed separately (probably into aphid chromosomes), at a frequency only known to be equalled in humans. The transposition phenomenon appears to occur infrequently or not at all in closely related genera and other aphids investigated. Patterns of nucleotide substitution in mtDNA inferred over a parsimony tree are very different from those in transposed sequences. Compared with mtDNA, nonmitochondrial sequences have less codon position bias, more even exchanges between A, G, C and T, and a higher proportion of nonsynonymous replacements. Although these data are consistent with the transposed sequences being under less constraint than mtDNA, changes in the nonmitochondrial sequences are not random: there remains significant position bias, and probable excesses of synonymous replacements and of conservative inferred amino acid replacements. We conclude that a proportion of the inferred change in the nonmitochondrial sequences occurred before transposition. We believe that Sitobion aphids (and other species exhibiting mtDNA transposition) may be important for studying the molecular evolution of mtDNA and pseudogenes. However, our data highlight the need to establish the true evolutionary relationships between sequences in comparative investigations.

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae).

Population genetics has come of age. Three important components have come together: efficient techniques to examine informative segments of DNA, statistics to analyse DNA data and the availability of easy-to-use computer packages. Single-locus genetic markers and those that produce gene genealogies yield information that is truly comparable among studies. These markers answer biological questions most efficiently and also contribute to much broader investigations of evolutionary, population and conservation biology. For these reasons, single-locus and genealogical markers should be the focus of the intensive genetic data collection that has begun owing to the power of genetics in population biology.

/pdf/efficient-genetic-markers-for-population-biology-3iwlxszk91.pdf

Efficient genetic markers for population biology.

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-294X.2011.05403.x

Who is eating what: diet assessment using next generation sequencing.

Nuclear mitochondrial pseudogenes (numts) are nonfunctional copies of mtDNA in the nucleus that have been found in major clades of eukaryotic organisms. They can be easily coamplified with orthologous mtDNA by using conserved universal primers; however, this is especially problematic for DNA barcoding, which attempts to characterize all living organisms by using a short fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Here, we study the effect of numts on DNA barcoding based on phylogenetic and barcoding analyses of numt and mtDNA sequences in two divergent lineages of arthropods: grasshoppers and crayfish. Single individuals from both organisms have numts of the COI gene, many of which are highly divergent from orthologous mtDNA sequences, and DNA barcoding analysis incorrectly overestimates the number of unique species based on the standard metric of 3% sequence divergence. Removal of numts based on a careful examination of sequence characteristics, including indels, in-frame stop codons, and nucleotide composition, drastically reduces the incorrect inferences of the number of unique species, but even such rigorous quality control measures fail to identify certain numts. We also show that the distribution of numts is lineage-specific and the presence of numts cannot be known a priori. Whereas DNA barcoding strives for rapid and inexpensive generation of molecular species tags, we demonstrate that the presence of COI numts makes this goal difficult to achieve when numts are prevalent and can introduce serious ambiguity into DNA barcoding.

https://www.pnas.org/content/pnas/105/36/13486.full.pdf

Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified

Primers for a PCR-based approach to mitochondrial genome sequencing in birds and other vertebrates.

DNA-sequence data from the mitochondrial genome are being used with increasing frequency to estimate phylogenetic relationships among animal taxa. The advantage to using DNA-sequence data is that many of the processes governing the evolution and inheritance of DNA are already understood. DNA data, however, do not guarantee the correct phylogenetic tree because of problems associated with shared ancestral polymorphisms and multiple substitutions at single nucleotide sites. Knowledge of evolutionary processes can be used to improve estimates of patterns of relationships and can help to assess the phylogenetic usefulness of individual genes and nucleotides. This article reviews molecular processes, discusses the correction of genetic distances and the weighting of DNA data, and provides an assessment of the phylogenetic usefulness of specific mitochondrial genes. The Appendix presents a compilation of conserved polymerase chain reaction primers that can be used to amplify virtually any gene in the mitochondrial genome. DNA data sets vary tremendously in degree of phylogenetic usefulness. Correction or weighting (or both) of DNA-sequence data based on level of variability can improve results in some cases. Gene choice is of critical importance. For studies of relationships among closely related species, the use of ribosomal genes can be problematic, whereas unconstrained sites in protein coding genes appear to have fewer problems. In addition, information from studies of amino acid substitutions in rapidly evolving genes may help to decipher close relationships. For intermediate levels of divergence where silent sites contain many multiple hits, amino acid changes can be useful for construction phylogenetic relationships. For deep levels of divergence, protein coding genes may be saturated at the amino acid level and highly conserved regions of ribosomal RNA and transfer RNA genes may be useful. Because of the arbitrariness of taxonomic categories, no sweeping generalizations can be made about the taxonomic rank at which particular genes are useful. As more DNA-sequence data accumulate, we will be able to gain an even better understanding of the way in which genes and species evolve.

Paul Flook

Papers

Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers