Extracting the invisible: obtaining high quality DNA is a challenging task in small arthropods

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

Ancestral state reconstruction reveals multiple independent evolution of diagnostic morphological characters in the "Higher Oribatida" (Acari), conflicting with current classification schemes

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>The molecular phylogeny based on three nuclear markers (28S rDNA, <it>ef-1α</it>, <it>hsp82</it>) revealed considerable discrepancies to the traditional classification of the five "circumdehiscent" subdivisions, suggesting paraphyly of the three families Scutoverticidae, Ameronothridae, Cymbaeremaeidae and also of the genus <it>Achipteria</it>. Ancestral state reconstructions of six common diagnostic characters and statistical evaluation of alternative phylogenetic hypotheses also partially rejected the current morphology-based classification and suggested multiple convergent evolution (both gain and loss) of some traits, after a period of rapid cladogenesis, rendering several subgroups paraphyletic.</p> <p>Conclusions</p> <p>Phylogenetic studies revealed non-monophyly of three families and one genus as a result of a lack of adequate synapomorphic morphological characters, calling for further detailed investigations in a framework of integrative taxonomy. Character histories of six morphological traits indicate that their evolution followed a rather complex pattern of multiple independent gains (and losses). Thus, the observed pattern largely conflicts with current morphological classifications of the Circumdehiscentiae, suggesting that the current taxonomic classification schemes are not appropriate, apart from a recently proposed subdivision into 24 superfamilies.</p

Revisiting the Evolution of Arboreal Life in Oribatid Mites

Though mostly soil dwelling, oribatid mites are found in all kind of habitats, with several species exclusively living on trees. Using previously published DNA sequences and eco-morphological data available from the literature, we inferred the number of transitions between soil dwelling to a truly arboreal lifestyle in oribatid mites and the shape evolution of a particular morphological structure of a sense organ (bothridial seta (= sensillus) of a trichobothrium), the shape of which was previously reported to be associated with an arboreal lifestyle. Our data suggest that a truly arboreal lifestyle evolved several times independently in oribatid mites, but much less often than previously proposed in the past. Even though all truly arboreal species indeed seem to possess a capitate sensillus, this character is not exclusive for arboreal taxa. Nonetheless, since all truly arboreal species do have a capitate sensillus, this might be considered an important (pre-)adaptation to a life on trees. We further provide guidelines on how the term &ldquo;arboreal&rdquo; should be applied in future mite research and emphasize the importance of exact microhabitat characterization, as this will greatly facilitate comparisons across studies

Unexpected diversity in the host-generalist oribatid mite Paraleius leontonychus (Oribatida, Scheloribatidae) phoretic on Palearctic bark beetles

Bark beetles are feared as pests in forestry but they also support a large number of other taxa that exploit the beetles and their galleries. Among arthropods, mites are the largest taxon associated with bark beetles. Many of these mites are phoretic and often involved in complex interactions with the beetles and other organisms. Within the oribatid mite family Scheloribatidae, only two of the three nominal species of Paraleius have been frequently found in galleries of bark beetles and on the beetles themselves. One of the species, P. leontonychus, has a wide distribution range spanning over three ecozones of the world and is believed to be a host generalist, reported from numerous bark beetle and tree species. In the present study, phylogenetic analyses of one mitochondrial and two nuclear genes identified six well supported, fairly divergent clades within P. leontonychus which we consider to represent distinct species based on molecular species delimitation methods and largely congruent clustering in mitochondrial and nuclear gene trees. These species do not tend to be strictly host specific and might occur syntopically. Moreover, mito-nuclear discordance indicates a case of past hybridization/introgression among distinct Paraleius species, the first case of interspecific hybridization reported in mites other than ticks

Claw shape variation in oribatid mites of the genera Carabodes and Caleremaeus: exploring the interplay of habitat, ecology and phylogenetics

Background Claws are a commonly observed biological adaptation across a wide range of animal groups. They serve different functions and their link to evolution is challenging to analyze. While there are many studies on the comparative anatomy and morphology of claws in reptiles, birds and several arthropods, knowledge about claws of soil-living oribatid mites, is still limited. Recent research on intertidal oribatid mites has shown that claw shape is strongly correlated with microhabitat and is subject to ecological selective pressures. However, the selective constraints shaping claws in terrestrial oribatid mites are still unknown. Methods In this study, 300 specimens from 12 different species and two genera were examined. Geometric morphometrics were used to quantify claw length and curvature, and to analyze two-dimensional claw shape. In combination with molecular phylogenetic analyses of investigated populations phylogenetic signal was quantified within genera using Blomberg’s K and random replicates. Additionally, ecological information on the investigated species was gathered from previous studies and compiled into tables. Results The claw shapes of Carabodes species vary moderately, with the three species C. reticulatus, C. rugosior and C. tenuis deviating the most from the others. These three species are only found in a small number of habitats, which may require a more specialized claw shape. Our results show that there is a phylogenetic influence on claw shape in Carabodes but not in Caleremaeus. Additionally, habitat specificity and lifestyle were found to have ecological impact on claw shape in both genera. The present results demonstrate that characteristics of the claws of terrestrial oribatid mites are correlated with ecology, but this correlation is apparently weaker than in intertidal oribatid mites that are prone to strong external forces

Opening the treasure chest: A DNA-barcoding primer set for most higher taxa of Central European birds and mammals from museum collections

<div><p>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.</p></div

Stuffed and tanned hides tested with the new primer set, with information on voucher ID, preservation method, collection year, fragments of the COI barcoding region that could be successfully amplified (colors refer to the fragments in Fig 1) and GenBank accession number.

<p>Stuffed and tanned hides tested with the new primer set, with information on voucher ID, preservation method, collection year, fragments of the COI barcoding region that could be successfully amplified (colors refer to the fragments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174449#pone.0174449.g001" target="_blank">Fig 1</a>) and GenBank accession number.</p

Fresh, frozen or ethanol preserved samples of birds (representatives of 18 orders) and mammals (representatives of 6 orders) used to test the performance of the new primer sets, with information on voucher ID, fragments of the COI barcoding region that could be successfully amplified (colors refer to the fragments in Fig 1), and GenBank accession numbers.

<p>Fresh, frozen or ethanol preserved samples of birds (representatives of 18 orders) and mammals (representatives of 6 orders) used to test the performance of the new primer sets, with information on voucher ID, fragments of the COI barcoding region that could be successfully amplified (colors refer to the fragments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174449#pone.0174449.g001" target="_blank">Fig 1</a>), and GenBank accession numbers.</p