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Freshwater metazoan biodiversity assessment using environmental DNA (eDNA) captured on filters offers new opportunities for water quality management. Filtering of water in the field is a logistical advantage compared to transport of water to the nearest lab, and thus, appropriate filter preservation becomes crucial for maximum DNA recovery. Here, the effect of four different filter preservation strategies, two filter types, and pre-filtration were evaluated by measuring metazoan diversity and community composition, using eDNA collected from a river and a lake ecosystem. The filters were preserved cold on ice, in ethanol, in lysis buffer and dry in silica gel. Our results show that filters preserved either dry or in lysis buffer give the most consistent community composition. In addition, mixed cellulose ester filters yield more consistent community composition than polyethersulfone filters, while the effect of pre-filtration remained ambiguous. Our study facilitates development of guidelines for aquatic community-level eDNA biomonitoring, and we advocate filtering in the field, using mixed cellulose ester filters and preserving the filters either dry or in lysis buffer.

BackgroundDespite their ubiquity and high diversity in eukaryotic genomes, DNA transposons are rarely encountered in ribosomal DNA (rDNA). In contrast, R-elements, a diverse group of non-LTR retrotransposons, specifically target rDNA. Pokey is a DNA transposon that targets a specific rDNA site, but also occurs in many other genomic locations, unlike R-elements. However, unlike most DNA transposons, Pokey has been a stable component of Daphnia genomes for over 100 million years. Here we use qPCR to estimate the number of 18S and 28S ribosomal RNA genes and Pokey elements in rDNA (rPokey), as well as other genomic locations (gPokey) in two species of Daphnia. Our goals are to estimate the correlation between (1) the number of 18S and 28S rRNA genes, (2) the number of 28S genes and rPokey, and (3) the number of rPokey and gPokey. In addition, we ask whether Pokey number and distribution in both genomic compartments are affected by differences in life history between D. pulex and D. pulicaria.ResultsWe found differences in 18S and 28S gene number within isolates that are too large to be explained by experimental variation. In general, Pokey number within isolates is modest (< 20), and most are gPokey. There is no correlation between the number of rRNA genes and rPokey, or between rPokey and gPokey. However, we identified three isolates with unusually high numbers of both rPokey and gPokey, which we infer is a consequence of recent transposition. We also detected other rDNA insertions (rInserts) that could be degraded Pokey elements, R- elements or the divergent PokeyB lineage recently detected in the Daphnia genome sequence. Unlike rPokey, rInserts are positively correlated with rRNA genes, suggesting that they are amplified by the same mechanisms that amplify rDNA units even though rPokey is not. Overall, Pokey frequency and distribution are similar in D. pulex and D. pulicaria suggesting that differences in life history have no impact on Pokey.ConclusionsThe possibility that many rDNA units do not contain a copy of both 18S and 28S genes suggests that rDNA is much more complicated than once thought, and warrants further study. In addition, the lack of correlation between rPokey, gPokey and rDNA unit numbers suggests that Pokey transposition rate is generally very low, and that recombination, in combination with natural selection, eliminates rPokey much faster than gPokey. Our results suggest that further research to determine the mechanisms by which Pokey has escaped complete inactivation by its host (the usual fate of DNA transposons), would provide important insights into transposon biology.

Characterisation of freshwater benthic biodiversity using DNA metabarcoding may allow more cost-effective environmental assessments than the current morphological-based assessment methods. DNA metabarcoding methods where sorting or pre-sorting of samples are avoided altogether are especially interesting, since the time between sampling and taxonomic identification is reduced. Due to the presence of non-target material like plants and sediments in crude samples, DNA extraction protocols become important for maximising DNA recovery and sample replicability. We sampled freshwater invertebrates from six river and lake sites and extracted DNA from homogenised bulk samples in quadruplicate subsamples, using a published method and two commercially available kits: HotSHOT approach, Qiagen DNeasy Blood &amp; Tissue Kit and Qiagen DNeasy PowerPlant Pro Kit. The performance of the selected extraction methods was evaluated by measuring DNA yield and applying DNA metabarcoding to see if the choice of DNA extraction method affects DNA yield and metazoan diversity results. The PowerPlant Kit extractions resulted in the highest DNA yield and a strong significant correlation between sample weight and DNA yield, while the DNA yields of the Blood &amp; Tissue Kit and HotSHOT method did not correlate with the sample weights. Metazoan diversity measures were more repeatable in samples extracted with the PowerPlant Kit compared to those extracted with the HotSHOT method or the Blood &amp; Tissue Kit. Subsampling using Blood &amp; Tissue Kit and HotSHOT extraction failed to describe the same community in the lake samples. Our study exemplifies that the choice of DNA extraction protocol influences the DNA yield as well as the subsequent community analysis. Based on our results, low specimen abundance samples will likely provide more stable results if specimens are sorted prior to DNA extraction and DNA metabarcoding, but the repeatability of the DNA extraction and DNA metabarcoding results was close to ideal in high specimen abundance samples.

BackgroundTransposable elements (TEs) play a major role in genome evolution. Their capacity to move and/or multiply in the genome of their host may have profound impacts on phenotypes and dramatic consequences on genome structure. The population dynamics and distribution of TEs are influenced by their mode of transposition, the availability of niches in host genomes, and host population dynamics. Theories predict an increase in the number of TE insertions following hybridization or polyploidization. Evolution of TEs in hybrids and polyploids has mostly been studied in plants; few studies have examined the impacts of hybridization and/or polyploidization on TEs in animals. Hybrids and polyploids have arisen multiple times in the Daphnia pulex complex and are thought to reproduce by obligate parthenogenesis. Our study examines the effects of ploidy level on polymorphism and number of Pokey element insertions in diploid and polyploid hybrid isolates from the Daphnia pulex complex.ResultsThe polymorphism of Pokey insertion sites did not depend solely on either the ploidy level or the genetic background of their host; therefore, it may be the result of interactions between these parameters and other parameters such as Pokey activity, selection and/or drift. No significant effect of ploidy level was found on the number of Pokey insertions using TE display and qPCR. However, the load of Pokey insertion sites and the number of unique insertion sites were slightly (but not significantly) higher in polyploids than in diploids.ConclusionsThese results suggest a lack of increase in the number of Pokey insertions following polyploidization but higher availability of Pokey insertion sites in polyploids than in diploids. Compared to previous TE display and qPCR results, the load of Pokey insertions in hybrid diploids was higher than in non-hybrid sexual and asexual diploids, which suggests an increase in the density of Pokey insertions following hybridization.