Novel DNA probes with low background and high hybridization-triggered fluorescence

Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5′-end and a non-fluorescent quencher at the 3′-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2–4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB–quencher interaction and concealment of the MGB moiety inside the minor groove

Unprecedented within-species chromosome number cline in the Wood White butterfly Leptidea sinapis and its significance for karyotype evolution and speciation

Background: Species generally have a fixed number of chromosomes in the cell nuclei while between-species differences are common and often pronounced. These differences could have evolved through multiple speciation events, each involving the fixation of a single chromosomal rearrangement. Alternatively, marked changes in the karyotype may be the consequence of within-species accumulation of multiple chromosomal fissions/fusions, resulting in highly polymorphic systems with the subsequent extinction of intermediate karyomorphs. Although this mechanism of chromosome number evolution is possible in theory, it has not been well documented. Results: We present the discovery of exceptional intraspecific variability in the karyotype of the widespread Eurasian butterfly Leptidea sinapis. We show that within this species the diploid chromosome number gradually decreases from 2n = 106 in Spain to 2n = 56 in eastern Kazakhstan, resulting in a 6000 km-wide cline that originated recently (8,500 to 31,000 years ago). Remarkably, intrapopulational chromosome number polymorphism exists, the chromosome number range overlaps between some populations separated by hundreds of kilometers, and chromosomal heterozygotes are abundant. We demonstrate that this karyotypic variability is intraspecific because in L. sinapis a broad geographical distribution is coupled with a homogenous morphological and genetic structure. Conclusions: The discovered system represents the first clearly documented case of explosive chromosome number evolution through intraspecific and intrapopulation accumulation of multiple chromosomal changes. Leptidea sinapis may be used as a model system for studying speciation by means of chromosomally-based suppressed recombination mechanisms, as well as clinal speciation, a process that is theoretically possible but difficult to document. The discovered cline seems to represent a narrow time-window of the very first steps of species formation linked to multiple chromosomal changes that have occurred explosively. This case offers a rare opportunity to study this process before drift, dispersal, selection, extinction and speciation erase the traces of microevolutionary events and just leave the final picture of a pronounced interspecific chromosomal difference

A novel endonuclease IV post-PCR genotyping system

Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5′ end and fluorophore attached to the 3′ end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3′ end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection

DNA Barcoding the Geometrid Fauna of Bavaria (Lepidoptera): Successes, Surprises, and Questions

BACKGROUND: The State of Bavaria is involved in a research program that will lead to the construction of a DNA barcode library for all animal species within its territorial boundaries. The present study provides a comprehensive DNA barcode library for the Geometridae, one of the most diverse of insect families. METHODOLOGY/PRINCIPAL FINDINGS: This study reports DNA barcodes for 400 Bavarian geometrid species, 98 per cent of the known fauna, and approximately one per cent of all Bavarian animal species. Although 98.5% of these species possess diagnostic barcode sequences in Bavaria, records from neighbouring countries suggest that species-level resolution may be compromised in up to 3.5% of cases. All taxa which apparently share barcodes are discussed in detail. One case of modest divergence (1.4%) revealed a species overlooked by the current taxonomic system: Eupithecia goossensiata Mabille, 1869 stat.n. is raised from synonymy with Eupithecia absinthiata (Clerck, 1759) to species rank. Deep intraspecific sequence divergences (>2%) were detected in 20 traditionally recognized species. CONCLUSIONS/SIGNIFICANCE: The study emphasizes the effectiveness of DNA barcoding as a tool for monitoring biodiversity. Open access is provided to a data set that includes records for 1,395 geometrid specimens (331 species) from Bavaria, with 69 additional species from neighbouring regions. Taxa with deep intraspecific sequence divergences are undergoing more detailed analysis to ascertain if they represent cases of cryptic diversity

DNA Barcoding for Community Ecology - How to Tackle a Hyperdiverse, Mostly Undescribed Melanesian Fauna

Trigonopterus weevils are widely distributed throughout Melanesia and hyperdiverse in New Guinea. They are a dominant feature in natural forests, with narrow altitudinal zonation. Their use in community ecology has been precluded by the "taxonomic impediment". We sampled >6,500 specimens from seven areas across New Guinea; 1,002 specimens assigned to 270 morphospecies were DNA sequenced. Objective clustering of a refined dataset (excluding nine cryptic species) at 3% threshold revealed 324 genetic clusters (DNA group count relative to number of morphospecies = 20.0% overestimation of species diversity, or 120.0% agreement) and 85.6% taxonomic accuracy (the proportion of DNA groups that "perfectly" agree with morphology-based species hypotheses). Agreement and accuracy were best at an 8% threshold. GMYC analysis revealed 328 entities (21.5% overestimation) with 227 perfect GMYC entities (84.1% taxonomic accuracy). Both methods outperform the parataxonomist (19% underestimation; 31.6% taxonomic accuracy). The number of species found in more than one sampling area was highest in the Eastern Highlands and Huon (Sørensen similarity index 0.07, 4 shared species); ⅓ of all areas had no species overlap. Success rates of DNA barcoding methods were lowest when species showed a pronounced geographical structure. In general, Trigonopterus show high α and β-diversity across New Guinea. DNA barcoding is an excellent tool for biodiversity surveys but success rates might drop when closer localities are included. Hyperdiverse Trigonopterus are a useful taxon for evaluating forest remnants in Melanesia, allowing finer-grained analyses than would be possible with vertebrate taxa commonly used to date. Our protocol should help establish other groups of hyperdiverse fauna as target taxa for community ecology. Sequencing delivers objective data on taxa of incredible diversity but mostly without a solid taxonomic foundation and should help pave the road for the eventual formal naming of new species

A Comprehensive DNA Barcode Library for the Looper Moths (Lepidoptera: Geometridae) of British Columbia, Canada

The construction of comprehensive reference libraries is essential to foster the development of DNA barcoding as a tool for monitoring biodiversity and detecting invasive species. The looper moths of British Columbia (BC), Canada present a challenging case for species discrimination via DNA barcoding due to their considerable diversity and limited taxonomic maturity.By analyzing specimens held in national and regional natural history collections, we assemble barcode records from representatives of 400 species from BC and surrounding provinces, territories and states. Sequence variation in the barcode region unambiguously discriminates over 93% of these 400 geometrid species. However, a final estimate of resolution success awaits detailed taxonomic analysis of 48 species where patterns of barcode variation suggest cases of cryptic species, unrecognized synonymy as well as young species.A catalog of these taxa meriting further taxonomic investigation is presented as well as the supplemental information needed to facilitate these investigations

A global phylogeny of butterflies reveals their evolutionary history, ancestral hosts and biogeographic origins

Butterflies are a diverse and charismatic insect group that are thought to have evolved with plants and dispersed throughout the world in response to key geological events. However, these hypotheses have not been extensively tested because a comprehensive phylogenetic framework and datasets for butterfly larval hosts and global distributions are lacking. We sequenced 391 genes from nearly 2,300 butterfly species, sampled from 90 countries and 28 specimen collections, to reconstruct a new phylogenomic tree of butterflies representing 92% of all genera. Our phylogeny has strong support for nearly all nodes and demonstrates that at least 36 butterfly tribes require reclassification. Divergence time analyses imply an origin similar to 100 million years ago for butterflies and indicate that all but one family were present before the K/Pg extinction event. We aggregated larval host datasets and global distribution records and found that butterflies are likely to have first fed on Fabaceae and originated in what is now the Americas. Soon after the Cretaceous Thermal Maximum, butterflies crossed Beringia and diversified in the Palaeotropics. Our results also reveal that most butterfly species are specialists that feed on only one larval host plant family. However, generalist butterflies that consume two or more plant families usually feed on closely related plants

Polyploidy in bisexual Lepidoptera species (Insecta: Lepidoptera): old hypotheses and new data

Volume: 48Start Page: 313End Page: 32

Some chromosomal aspects of Brenthis hecate (Denis & Schiffermuller), 1775 from south Altai, USSR (Lepidoptera, Nymphalidae)

Volume: 11Start Page: 234End Page: 23