DNA-barcoding is a rapidly developing method for efficiently identifying samples to species level by means of short standard DNA sequences. However, reliable species assignment requires the availability of a comprehensive DNA barcode reference library, and hence numerous initiatives aim at generating such barcode databases for particular taxa or geographic regions. Historical museum collections represent a potentially invaluable source for the DNA-barcoding of many taxa. This is particularly true for birds and mammals, for which collecting fresh (voucher) material is often very difficult to (nearly) impossible due to the special animal welfare and conservation regulations that apply to vertebrates in general, and birds and mammals in particular. Moreover, even great efforts might not guarantee sufficiently complete sampling of fresh material in a short period of time. DNA extracted from historical samples is usually degraded, such that only short fragments can be amplified, rendering the recovery of the barcoding region as a single fragment impossible. Here, we present a new set of primers that allows the efficient amplification and sequencing of the entire barcoding region in most higher taxa of Central European birds and mammals in six overlapping fragments, thus greatly increasing the value of historical museum collections for generating DNA barcode reference libraries. Applying our new primer set in recently established NGS protocols promises to further increase the efficiency of barcoding old bird and mammal specimens.

Background: The application of an appropriate extraction method is a relevant factor for the success of all molecular studies.Methods: Seven different DNA extraction methods suitable for high-throughput DNA sequencing with very small arthropods were compared by applying nine different protocols: three silica gel based spin methods, two cetyltrimethyl ammonium bromide (CTAB) based ones (one with an additional silica membrane), a protein precipitation method and a method based on a chelating resin (applying different protocols). The quantity (concentration) and quality (degradation, contamination, polymerase chain reaction (PCR) and sequencing success) of the extracted DNA as well as the costs, preparation times, user friendliness, and required supplies were compared across these methods. To assess the DNA quantity, two different DNA concentration measurements were applied. Additionally, the effect of varying amounts of starting material (different body sizes), variable lysis temperatures and mixing during DNA extraction was evaluated.Results: Although low DNA concentrations were measured for all methods, the results showed that—with the exception of two methods—the PCR success was 100%. However, other parameters show vast differences. The time taken to perform DNA extraction varied from 20 min to 2.5 h (Chelex vs. CTAB) and the costs from 0.02 to 3.46 € (Chelex vs. QIAamp kit) per sample. High quality genomic DNA was only gained from four methods. Results of DNA quantity measurements further indicated that some devices cannot deal with small amounts of DNA and show variant results.Discussion: In conclusion, using Chelex (chelating resin) turned out as a rapid, low-cost method which can provide high quality DNA for different kinds of molecular investigations.

Bilaterian mitochondrial (mt) genomes are circular molecules that typically contain 37 genes. To date, only a single complete mitogenome sequence is available for the species-rich sarcoptiform mite order Oribatida. We sequenced the mitogenome of Paraleius leontonychus, another species of this suborder. It is 14,186 bp long and contains 35 genes, including only 20 tRNAs, lacking tRNA ^Gly and tRNA ^Tyr . Re-annotation of the mitogenome of Steganacarus magnus increased the number of mt tRNAs for this species to 12. As typical for acariform mites, many tRNAs are highly truncated in both oribatid species. The total number of tRNAs and the number of tRNAs with a complete cloverleaf-like structure in P. leontonychus, however, clearly exceeds the numbers previously reported for Sarcoptiformes. This indicates, contrary to what has been previously assumed, that reduction of tRNAs is not a general characteristic for sarcoptiform mites. Compared to other Sarcoptiformes, the two oribatid species have the least rearranged mt genome with respect to the pattern observed in Limulus polyphemus, a basal arachnid species. Phylogenetic analysis of the newly sequenced mt genome and previously published data on other acariform mites confirms paraphyly of the Oribatida and an origin of the Astigmata within the Oribatida.

The use of molecular genetic data in phylogenetic systematics has revolutionized this field of research in that several taxonomic groupings defined by traditional taxonomic approaches have been rejected by molecular data. The taxonomic classification of the oribatid mite group Circumdehiscentiae ("Higher Oribatida") is largely based on morphological characters and several different classification schemes, all based upon the validity of diagnostic morphological characters, have been proposed by various authors. The aims of this study were to test the appropriateness of the current taxonomic classification schemes for the Circumdehiscentiae and to trace the evolution of the main diagnostic traits (the four nymphal traits scalps, centrodorsal setae, sclerits and wrinkled cuticle plus octotaxic system and pteromorphs both in adults) on the basis of a molecular phylogenetic hypothesis by means of parsimony, likelihood and Bayesian approaches.