Decision‐making and best practices for taxonomy‐free environmental DNA metabarcoding in biomonitoring using Hill numbers

Environmental DNA (eDNA) metabarcoding is raising expectations for biomonitoring of organisms that have hitherto been neglected. To bypass current limitations in taxonomic assignments due to incomplete or erroneous reference databases, taxonomy-free approaches are proposed for biomonitoring at the level of operational taxonomic units (OTUs). This is challenging, because OTUs cannot be annotated and directly compared against classically derived taxonomic data. The application of good stringency treatments to infer the validity of OTUs and clear understanding of the consequences of such treatments is especially relevant for biodiversity assessments. We investigated how common practices of stringency filtering affect eDNA diversity estimates in the statistical framework of Hill numbers. We collected water eDNA samples at 61 sites across a 740-km2 river catchment, reflecting a spatially realistic scenario in biomonitoring. After bioinformatic processing of the data, we studied how different stringency treatments affect conclusions with respect to biodiversity at the catchment and site levels. The applied stringency treatments were based on the consistent appearance of OTUs across filter replicates, a relative abundance cut-off and rarefaction. We detected large differences in diversity estimates when accounting for presence/absence only, such that detected diversity at the catchment scale differed by an order of magnitude between the treatments. These differences disappeared when using stringency treatments with increasing weighting of the OTU abundances. Our study demonstrated the usefulness of Hill numbers for biodiversity analyses and comparisons of eDNA data sets that strongly differ in diversity. We recommend best practice for data stringency filtering for biomonitoring using eDNA

Heat-Shock Promoters: Targets for Evolution by <i>P</i> Transposable Elements in <i>Drosophila</i>

Transposable elements are potent agents of genomic change during evolution, but require access to chromatin for insertion—and not all genes provide equivalent access. To test whether the regulatory features of heat-shock genes render their proximal promoters especially susceptible to the insertion of transposable elements in nature, we conducted an unbiased screen of the proximal promoters of 18 heat-shock genes in 48 natural populations of Drosophila. More than 200 distinctive transposable elements had inserted into these promoters; greater than 96% are P elements. By contrast, few or no P element insertions segregate in natural populations in a “negative control” set of proximal promoters lacking the distinctive regulatory features of heat-shock genes. P element transpositions into these same genes during laboratory mutagenesis recapitulate these findings. The natural P element insertions cluster in specific sites in the promoters, with up to eight populations exhibiting P element insertions at the same position; laboratory insertions are into similar sites. By contrast, a “positive control” set of promoters resembling heat-shock promoters in regulatory features harbors few P element insertions in nature, but many insertions after experimental transposition in the laboratory. We conclude that the distinctive regulatory features that typify heat-shock genes (in Drosophila) are especially prone to mutagenesis via P elements in nature. Thus in nature, P elements create significant and distinctive variation in heat-shock genes, upon which evolutionary processes may act.</p

Heat-Shock Promoters: Targets for Evolution by P Transposable Elements in Drosophila

Transposable elements are potent agents of genomic change during evolution, but require access to chromatin for insertion—and not all genes provide equivalent access. To test whether the regulatory features of heat-shock genes render their proximal promoters especially susceptible to the insertion of transposable elements in nature, we conducted an unbiased screen of the proximal promoters of 18 heat-shock genes in 48 natural populations of Drosophila. More than 200 distinctive transposable elements had inserted into these promoters; greater than 96% are P elements. By contrast, few or no P element insertions segregate in natural populations in a “negative control” set of proximal promoters lacking the distinctive regulatory features of heat-shock genes. P element transpositions into these same genes during laboratory mutagenesis recapitulate these findings. The natural P element insertions cluster in specific sites in the promoters, with up to eight populations exhibiting P element insertions at the same position; laboratory insertions are into similar sites. By contrast, a “positive control” set of promoters resembling heat-shock promoters in regulatory features harbors few P element insertions in nature, but many insertions after experimental transposition in the laboratory. We conclude that the distinctive regulatory features that typify heat-shock genes (in Drosophila) are especially prone to mutagenesis via P elements in nature. Thus in nature, P elements create significant and distinctive variation in heat-shock genes, upon which evolutionary processes may act

Evaluation of primer pairs for eDNA-based assessment of Ephemeroptera, Plecoptera, and Trichoptera across a biogeographically diverse region

Macroinvertebrates serve as key indicators in ecological assessments of aquatic ecosystems, where the composition and richness of their communities are indicative of environmental and anthropogenic change. Established monitoring of macroinvertebrates is expensive and time-consuming, and relies on expert taxonomic knowledge. In contrast, biomonitoring based on molecular tools can support faster characterization of aquatic communities but needs validation for the target taxonomic groups and the study region. Here, we used data from a biomonitoring program covering a large biogeographic gradient to compare the routine kick-net method with eDNA metabarcoding. We used two primer pairs targeting COI, one targeting a broad metazoan spectrum (mICOIintF/jgHCO2198) and another more recently developed primer pair optimized for the detection of freshwater invertebrates (fwhF2/EPTDr2n). We used the data of the macroinvertebrate monitoring with a focus on the orders of Ephemeroptera, Plecoptera, and Trichoptera across 92 rivers in Switzerland, covering four continental drainage basins and an elevational range from 198 to 1650 m a.s.l. Across all sample sites, the kick-net detected more distinct taxa than either of the metabarcoding approaches. At a site level, however, both primer pairs detected on average more species. Comparing both primer pairs, the fwhF2/EPTDr2n primer pair captured more species assigned to the indicator groups Ephemeroptera, Plecoptera, and Trichoptera, and showed a significantly larger overlap with the kick-net method. However, the community composition still varied significantly among the different metabarcoding approaches. Fewer Trichoptera species were recovered by eDNA, whereas the fwhF2/EPTDr2n primer pair detected more Plecopterans than the other two approaches. This study highlights the importance of the optimization and validation of novel molecular approaches under consideration of the target organismal group and the study area

Temperature, phytoplankton density and bacteria diversity drive the biotransformation of micropollutants in a lake ecosystem

For most micropollutants (MPs) present in surface waters, such as pesticides and pharmaceuticals, the contribution of biotransformation to their overall removal from lake ecosystems is largely unknown. This study aims at empirically determining the biotransformation rate constants for 35 MPs at different periods of the year and depths of a meso-eutrophic lake. We then tested statistically the association of environmental parameters and microbial community composition with the biotransformation rate constants obtained. Biotransformation was observed for 14 out of 35 studied MPs for at least one sampling time. Large variations in biotransformation rate constants were observed over the seasons and between compounds. Overall, the transformation of MPs was mostly influenced by the lake's temperature, phytoplankton density and bacterial diversity. However, some individual MPs were not following the general trend or association with microorganism biomass. The antidepressant mianserin, for instance, was transformed in all experiments and depths, but did not show any relationship with measured environmental parameters, suggesting the importance of specific microorganisms in its transformation. The results presented here contribute to our understanding of the fate of MPs in surface waters and thus support improved risk assessment of contaminants in the environment. Keywords: Biotransformation; Microbial community; Pesticides; Pharmaceuticals; Phytoplankton; Random fores

RNA sequencing of early round goby embryos reveals that maternal experiences can shape the maternal RNA contribution in a wild vertebrate

It has been proposed that non-genetic inheritance could promote species fitness. Non-genetic inheritance could allow offspring to benefit from the experience of their parents, and could advocate pre-adaptation to prevailing and potentially selective conditions. Indeed, adaptive parental effects have been modeled and observed, but the molecular mechanisms behind them are far from understood. In the present study, we investigated whether maternal RNA can carry information about environmental conditions experienced by the mother in a wild vertebrate. Maternal RNA directs the development of the early embryo in many non-mammalian vertebrates and invertebrates. However, it is not known whether vertebrate maternal RNA integrates information about the parental environment. We sequenced the maternal RNA contribution from a model that we expected to rely on parental effects: the invasive benthic fish species Neogobius melanostomus (Round Goby). We found that maternal RNA expression levels correlated with the water temperature experienced by the mother before oviposition, and identified temperature-responsive gene groups such as core nucleosome components or the microtubule cytoskeleton. Our findings suggest that the maternal RNA contribution may incorporate environmental information. Maternal RNA should therefore be considered a potentially relevant pathway for non-genetic inheritance. Also, the ability of a species to integrate environmental information in the maternal RNA contribution could potentially contribute to species fitness and may also play a role in extraordinary adaptive success stories of invasive species such as the round goby

Microbial residence time is a controlling parameter of the taxonomic composition and functional profile of microbial communities.

A remaining challenge within microbial ecology is to understand the determinants of richness and diversity observed in environmental microbial communities. In a range of systems, including activated sludge bioreactors, the microbial residence time (MRT) has been previously shown to shape the microbial community composition. However, the physiological and ecological mechanisms driving this influence have remained unclear. Here, this relationship is explored by analyzing an activated sludge system fed with municipal wastewater. Using a model designed in this study based on Monod-growth kinetics, longer MRTs were shown to increase the range of growth parameters that enable persistence, resulting in increased richness and diversity in the modeled community. In laboratory experiments, six sequencing batch reactors treating domestic wastewater were operated in parallel at MRTs between 1 and 15 days. The communities were characterized using both 16S ribosomal RNA and non-target messenger RNA sequencing (metatranscriptomic analysis), and model-predicted monotonic increases in richness were confirmed in both profiles. Accordingly, taxonomic Shannon diversity also increased with MRT. In contrast, the diversity in enzyme class annotations resulting from the metatranscriptomic analysis displayed a non-monotonic trend over the MRT gradient. Disproportionately high abundances of transcripts encoding for rarer enzymes occur at longer MRTs and lead to the disconnect between taxonomic and functional diversity profiles

Spatio-temporal patterns of multi-trophic biodiversity and food-web characteristics uncovered across a river catchment using environmental DNA

Accurate characterisation of ecological communities with respect to their biodiversity and food-web structure is essential for conservation. However, combined empirical study of biodiversity and multi-trophic food webs at a large spatial and temporal resolution has been prohibited by the lack of appropriate access to such data from natural systems. Here, we assessed biodiversity and food-web characteristics across a 700 km$^{2}$ riverine network over seasons using environmental DNA. We found contrasting biodiversity patterns between major taxonomic groups. Local richness showed statistically significant, season-dependent increases and decreases towards downstream location within the catchment for fish and bacteria, respectively. Meanwhile, invertebrate richness remained spatially unchanged but varied across seasons. The structure of local food webs, such as link density and nestedness, also varied across space and time. However, these patterns did not necessarily mirror those observed for biodiversity and functional feeding characteristics. Our results suggest that biodiversity patterns and food-web dynamics are not directly scalable to each other even at the same spatial and temporal scales. In order to conserve species diversity as well as the functional trophic integrity of communities, patterns of biodiversity and food-web characteristics must thus be jointly studied

A Photoreceptor Contributes to the Natural Variation of Diapause Induction in Daphnia magna

Diapause is an adaptation that allows organisms to survive harsh environmental conditions. In species occurring over broad habitat ranges, both the timing and the intensity of diapause induction can vary across populations, revealing patterns of local adaptation. Understanding the genetic architecture of this fitness-related trait would help clarify how populations adapt to their local environments. In the cyclical parthenogenetic crustacean Daphnia magna, diapause induction is a phenotypic plastic life history trait linked to sexual reproduction, as asexual females have the ability to switch to sexual reproduction and produce resting stages, their sole strategy for surviving habitat deterioration. We have previously shown that the induction of resting stage production correlates with changes in photoperiod that indicate the imminence of habitat deterioration and have identified a Quantitative Trait Locus (QTL) responsible for some of the variation in the induction of resting stages. Here, new data allows us to anchor the QTL to a large scaffold and then, using a combination of a new mapping panel, targeted association mapping and selection analysis in natural populations, to identify candidate genes within the QTL. Our results show that variation in a rhodopsin photoreceptor gene plays a significant role in the variation observed in resting stage induction. This finding provides a mechanistic explanation for the link between diapause and day-length perception that has been suggested in diverse arthropod taxa

Monitoring invasive alien macroinvertebrate species with environmental DNA

Regular monitoring of ecosystems can be used for the early detection of invasive alien species (IAS), and provide information for management and preventing them from becoming established or advancing into new areas. Current methods of monitoring freshwater systems for IAS can be both financially costly and time-consuming, with routine monitoring often carried out at low intensity and at only a small number of sites. In this study, we evaluate how environmental DNA (eDNA) metabarcoding for monitoring freshwater macroinvertebrate IAS compares to traditional kick-net sampling as part of a national (Switzerland) and a catchment monitoring programme. Kick-net sampling was more fruitful for the detection of several well-known target macroinvertebrate IAS. However, eDNA samples proved complementary for the detection of IAS that belong to species often being unnoticed by traditional sampling due to methodological or taxonomic reasons. Specifically, the invasive jellyfish Craspedacusta sowerbii, hardly detectable using classic kick-net sampling, was found to be widespread in both the national and the catchment-scale monitoring with the eDNA method only. Our study shows that IAS detection using eDNA is easily implemented in both national- and catchment-scale monitoring campaigns. However, successful detection of target IAS is still highly dependent on primer choice, species' biology, and availability of adequate markers. Specifically, multiple markers should be considered for detecting IAS from several different taxonomic groups, such as those under the ‘freshwater macroinvertebrate’ umbrella term. While eDNA is still developing in terms of these fundamental methodological requirements, surveillance for both target and non-target IAS using eDNA is likely to increase efficiency in early detection of IAS in freshwater systems