Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcoding.

Pollen metabarcoding is emerging as a powerful tool for ecological research and offers unprecedented scale in citizen science projects for environmental monitoring via honey bees. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. While in metabarcoding studies pollen has been preserved at - 20 °C (FRZ), this is not the best method for citizen scientists. Herein, we compared this method with ethanol (EtOH), silica gel (SG) and room temperature (RT) for preservation of pollen collected from hives in Austria and Denmark. After ~ 4 months of storage, DNAs were extracted with a food kit, and their quality and concentration measured. Most DNA extracts exhibited 260/280 absorbance ratios close to the optimal 1.8, with RT samples from Austria performing slightly worse than FRZ and SG samples (P < 0.027). Statistical differences were also detected for DNA concentration, with EtOH samples producing lower yields than RT and FRZ samples in both countries and SG in Austria (P < 0.042). Yet, qualitative and quantitative assessments of floral composition obtained using high-throughput sequencing with the ITS2 barcode gave non-significant effects of preservation methods on richness, relative abundance and Shannon diversity, in both countries. While freezing and ethanol are commonly employed for archiving tissue for molecular applications, desiccation is cheaper and easier to use regarding both storage and transportation. Since SG is less dependent on ambient humidity and less prone to contamination than RT, we recommend SG for preserving pollen for metabarcoding. SG is straightforward for laymen to use and hence robust for widespread application in citizen science studies.

Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcoding.

One of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.

In vivo fertilizing ability of stallion spermatozoa processed by single layer centrifugation with Androcoll-E™.

A colloid with a species specific silane-coated, silica-based formulation, optimized for stallion (Androcoll-E™), enables a better sub-population of spermatozoa to be selected from stallion ejaculates. However, such a practice has not been critically evaluated in stallions with fertility problems. In this study we evaluate whether single-layer centrifugation (SLC) through Androcoll-E™ could be used to enhance fertility rates in a subfertile stallion. Ejaculates were obtained from two different stallions, one Lusitano (fertile) and one Sorraia (subfertile), with distinct sperm characteristics and fertility. Motility, morphology, plasma membrane structural (eosin-nigrosin) and functional integrity (HOS test), mitochondrial functionality (Δψm; JC-1) and longevity (motility after 72 h cooling) after centrifugation in Androcoll-E™, as well as pregnancy rates obtained after artificial insemination (AI), with and without (control group) SLC-treated sperm were assessed. The effect of SLC on sperm characteristics, and fertility results were evaluated by ANOVA and Fisher procedures, respectively. Our results showed that SLC-selected sperm did not differ from the raw semen in terms of viability, morphology, response to hypo-osmotic conditions (HOS test) and mitochondrial membrane potential (↑ΔΨmit; JC-1). Sperm motility in cooled samples was not improved by SLC treatment. Our data show that SLC through Androcoll-E™ has no effect on pregnancy rates in the stallions used in this trial.

New insights into the genetic composition and phylogenetic relationship of wolves and dogs in the Iberian Peninsula.

This study investigates the gene pool of Portuguese autochthonous dog breeds and their wild counterpart, the Iberian wolf subspecies (Canis lupus signatus), using standard molecular markers. A combination of paternal and maternal molecular markers was used to investigate the genetic composition, genetic differentiation and genetic relationship of native Portuguese dogs and the Iberian wolf. A total of 196 unrelated dogs, including breed and village dogs from Portugal, and other dogs from Spain and North Africa, and 56 Iberian wolves (wild and captive) were analyzed for nuclear markers, namely Y chromosome SNPs, Y chromosome STR loci, autosomal STR loci, and a mitochondrial fragment of the control region I. Our data reveal new variants for the molecular markers and confirm significant genetic differentiation between Iberian wolf and native domestic dogs from Portugal. Based on our sampling, no signs of recent introgression between the two subspecies were detected. Y chromosome data do not reveal genetic differentiation among the analyzed dog breeds, suggesting they share the same patrilineal origin. Moreover, the genetic distinctiveness of the Iberian wolf from other wolf populations is further confirmed with the description of new mtDNA variants for this endemism. Our research also discloses new molecular markers for wolf and dog subspecies assignment, which might become particularly relevant in the case of forensic or noninvasive genetic studies. The Iberian wolf represents a relic of the once widespread wolf population in Europe and our study reveals that it is a reservoir of unique genetic diversity of the grey wolf, Canis lupus. These results stress the need for conservation plans that will guarantee the sustainability of this threatened top predator in Iberia.