Molecular Identification of Birds: Performance of Distance-Based DNA Barcoding in Three Genes to Delimit Parapatric Species

Background

DNA barcoding based on the mitochondrial cytochrome oxidase subunit I gene (cox1 or COI) has been successful in species identification across a wide array of taxa but in some cases failed to delimit the species boundaries of closely allied allopatric species or of hybridising sister species.

Methodology/Principal Findings

In this study we extend the sample size of prior studies in birds for cox1 (2776 sequences, 756 species) and target especially species that are known to occur parapatrically, and/or are known to hybridise, on a Holarctic scale. In order to obtain a larger set of taxa (altogether 2719 species), we include also DNA sequences of two other mitochondrial genes: cytochrome b (cob) (4614 sequences, 2087 species) and 16S (708 sequences, 498 species). Our results confirm the existence of a wide gap between intra- and interspecies divergences for both cox1 and cob, and indicate that distance-based DNA barcoding provides sufficient information to identify and delineate bird species in 98% of all possible pairwise comparisons. This DNA barcoding gap was not statistically influenced by the number of individuals sequenced per species. However, most of the hybridising parapatric species pairs have average divergences intermediate between intraspecific and interspecific distances for both cox1 and cob.

Conclusions/Significance

DNA barcoding, if used as a tool for species discovery, would thus fail to identify hybridising parapatric species pairs. However, most of them can probably still assigned to known species by character-based approaches, although development of complementary nuclear markers will be necessary to account for mitochondrial introgression in hybridising species.     

The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome

Summary In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain ana ^r -14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.Abbreviations cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cytochrome - c oxidase - cob gene encoding apocytochrome b - cox1I1, cox1I2a, cox1I2b, cox1I3 introns in cox1 - cox1Ix ^+/– indicates the presence or absence of the intron either in the native gene or after intron DNA excision - cox1Ix is a deletion in the intron leading to respiratory deficiency     

Performance of distance-based DNA barcoding in the molecular identification of Primates

For comparative primatology proper recognition of basal taxa (i.e. species) is indispensable, and in this the choice of a suitable gene with high phylogenetic resolution is crucial. For the goals of species identification in animals, the cytochrome c oxidase subunit 1 (cox1) has been introduced as standard marker. Making use of the difference in intra- and interspecific genetic variation – the DNA barcoding gap – cox1 can be used as a fast and accurate marker for the identification of animal species. For the Order Primates we compare the performance of cox1 (166 sequences; 50 nominal species) in species-identification with that of two other mitochondrial markers, 16S ribosomal RNA (412 sequences, 92 species) and cytochrome b (cob: 547 sequences, 72 species). A wide gap exist between intra- and interspecific divergences for both cox1 and cob genes whereas this gap is less apparent for 16S, indicating that rRNA genes are less suitable for species delimitation in DNA barcoding. For those species where multiple sequences are available there are significant differences in the intraspecific genetic distances between different mitochondrial markers, without, however, showing a consistent pattern. We conclude that cox1 allows accurate differentiation of species and as such DNA barcoding may have an important role to play in comparative primatology.     

Complete mitochondrial exploration of Echinococcus multilocularis from French alveolar echinococcosis patients

Alveolar echinococcosis (AE) is a parasitosis that is expanding worldwide, including in Europe. The development of genotypic markers is essential to follow its spatiotemporal evolution. Sequencing of the commonly used mitochondrial genes cob, cox1, and nad2 shows low discriminatory power, and analysis of the microsatellite marker EmsB does not allow nucleotide sequence analysis. We aimed to develop a new method for the genotyping of Echinococcus multilocularis based on whole mitochondrial genome (mitogenome) sequencing, to determine the genetic diversity among 30 human visceral samples from French patients, and compare this method with those currently in use. Sequencing of the whole mitochondrial genome was carried out after amplification by PCR, using one uniplex and two multiplex reactions to cover the 13,738 bp of the mitogenome, combined with Illumina technology. Thirty complete mitogenome sequences were obtained from AE lesions. One showed strong identity with Asian genotypes (99.98% identity) in a patient who had travelled to China. The other 29 mitogenomes could be differentiated into 13 haplotypes, showing higher haplotype and nucleotide diversity than when using the cob, cox1, and nad2 gene sequences alone. The mitochondrial genotyping data and EmsB profiles did not overlap, probably because one method uses the mitochondrial genome and the other the nuclear genome. The pairwise fixation index (F_st) value between individuals living inside and those living outside the endemic area was high (F_st = 0.222, P = 0.002). This is consistent with the hypothesis of an expansion from historical endemic areas to peripheral regions.     

The complete mitochondrial genome of Tuta absoluta (Lepidoptera: Gelechiidae) and genetic variation in two newly invaded populations in China

Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is a devastating invasive pest worldwide, causing severe damage to tomatoes. Recently, it has been recorded in the northwestern and southwestern parts of China. Here, the mitogenomes and genetic variation of two newly invaded T. absoluta populations in Xinjiang and Yunnan, were determined. The results showed that the complete mitogenome size of T. absoluta is 15298 bp for the individual from Xinjiang and 15296 bp for the individual from Yunnan, which were both longer than the reported mitogenome from Spain (15290 bp). The mitogenome sequences of individuals collected from three locations showed high levels of sequence similarity, except for 8 polymorphic sites, which were in genes cox2 (1 site), cox3 (2 sites), cob (1 site), atp6 (1 site), nad1 (2 sites) and nad5 (1 site). Tuta absoluta mitogenomes share many features with other 6 Gelechiidae mitogenomes, except for several differences in the start and stop codons of protein-coding genes and the length of intergenic spacers. Seven partial mitochondrial genes (cox1, cox2, cox3, atp6, cob, nad1, and nad5) were used for genetic variation analysis, and significant population differentiation was found between the two populations based on cox2, atp6, nad1, and nad5. The complete mitogenomes and sensitive mitochondrial gene markers reported here provide useful data for further population genetics study of this pest.     

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.