High‐throughput sequencing on preservative ethanol is effective at jointly examining infraspecific and taxonomic diversity,although bioinformatics pipelines do not perform equally

1. High‐throughput sequencing of amplicons (HTSA) has been proposed as an effective approach to evaluate taxonomic and genetic diversity at the same time. However, there are still uncertainties as to how the results produced by different bioinformatics treatments impact the conclusions drawn on biodiversity and population genetics indices.
2. We evaluated the ability of six bioinformatics pipelines to recover taxonomic and genetic diversity from HTSA data obtained from controlled assemblages. To that end, 20 assemblages were produced using 354 colonies of Botrylloides spp., sampled in the wild in ten marinas around Brittany (France). We used DNA extracted from preservative ethanol (ebDNA) after various time of storage (3, 6, and 12 months), and from a bulk of preserved specimens (bulkDNA). DNA was amplified with primers designed for targeting this ascidian genus. Results obtained from HTSA data were compared with Sanger sequencing on individual zooids (i.e., individual barcoding).
3. Species identification and relative abundance determined with HTSA data from either ebDNA or bulkDNA were similar to those obtained with traditional individual barcoding. However, after 12 months of storage, the correlation between HTSA and individual‐based data was lower than after shorter durations. The six bioinformatics pipelines were able to depict accurately the genetic diversity using standard population genetics indices (H_S and F_ST), despite producing false positives and missing rare haplotypes. However, they did not perform equally and dada2 was the only pipeline able to retrieve all expected haplotypes.
4. This study showed that ebDNA is a nondestructive alternative for both species identification and haplotype recovery, providing storage does not last more than 6 months before DNA extraction. Choosing the bioinformatics pipeline is a matter of compromise, aiming to retrieve all true haplotypes while avoiding false positives. We here recommend to process HTSA data using dada2, including a chimera‐removal step. Even if the possibility to use multiplexed primer sets deserves further investigation to expand the taxonomic coverage in future similar studies, we showed that primers targeting a particular genus allowed to reliably analyze this genus within a complex community.

     

Ecosystem type drives soil eukaryotic diversity and composition in Europe

Soil eukaryotes play a crucial role in maintaining ecosystem functions and services, yet the factors driving their diversity and distribution remain poorly understood. While many studies focus on some eukaryotic groups (mostly fungi), they are limited in their spatial scale. Here, we analyzed an unprecedented amount of observational data of soil eukaryomes at continental scale (787 sites across Europe) to gain further insights into the impact of a wide range of environmental conditions (climatic and edaphic) on their community composition and structure. We found that the diversity of fungi, protists, rotifers, tardigrades, nematodes, arthropods, and annelids was predominantly shaped by ecosystem type (annual and permanent croplands, managed and unmanaged grasslands, coniferous and broadleaved woodlands), and higher diversity of fungi, protists, nematodes, arthropods, and annelids was observed in croplands than in less intensively managed systems, such as coniferous and broadleaved woodlands. Also in croplands, we found more specialized eukaryotes, while the composition between croplands was more homogeneous compared to the composition of other ecosystems. The observed high proportion of overlapping taxa between ecosystems also indicates that DNA has accumulated from previous land uses, hence mimicking the land transformations occurring in Europe in the last decades. This strong ecosystem-type influence was linked to soil properties, and particularly, soil pH was driving the richness of fungi, rotifers, and annelids, while plant-available phosphorus drove the richness of protists, tardigrades, and nematodes. Furthermore, the soil organic carbon to total nitrogen ratio crucially explained the richness of fungi, protists, nematodes, and arthropods, possibly linked to decades of agricultural inputs. Our results highlighted the importance of long-term environmental variables rather than variables measured at the time of the sampling in shaping soil eukaryotic communities, which reinforces the need to include those variables in addition to ecosystem type in future monitoring programs and conservation efforts.     

Cobble community DNA as a tool to monitor patterns of biodiversity within kelp forest ecosystems

Kelp forest ecosystems dominate 150,000 km of global temperate coastline, rivalling the coastal occurrence of coral reefs. Despite the astounding biological diversity and productive ecological communities associated with kelp forests, patterns of species richness and composition are difficult to monitor and compare. Crustose coralline algae are a critically important substrate for propagule settlement for a range of kelp forest species. Coralline‐covered cobbles are home to hundreds of species of benthic animals and algae and form a replicable unit for ecological assays. Here, we use DNA metabarcoding of bulk DNA extracts sampled from cobbles to explore patterns of species diversity in kelp forests of the central California coast. The data from 97 cobbles within kelp forest ecosystems at three sites in Central California show the presence of 752 molecular operational taxonomic units (MOTUs) and 53 MOTUs assigned up to the species level with >95% similarity to current databases. We are able to detect spatial patterns of important management targets such as abalone recruits, and localized abundance of sea stars in 2012. Comparison of classic ecological surveys of these sites reveals large differences in species targets for these two approaches. In order to make such comparisons more quantitative, we use Presence/Absence Metabarcoding, using the fraction of replicate cobbles showing a species as a measure of its local abundance. This approach provides a fast and repeatable survey method that can be applied for biodiversity assessments across systems to shed light on the impact of different ecological disturbances and the role played by marine protected areas.     

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.     

Environmental factors and host genetic variation shape the fungal endophyte communities within needles of Scots pine (Pinus sylvestris)

To determine the role of environmental and host genetic factors in shaping fungal endophyte communities we used culturing and metabarcoding techniques to quantify fungal taxa within healthy Scots pine (Pinus sylvestris) needles in a 7-y old provenance-progeny trial replicated at three sites. Both methods revealed a community of ascomycete and basidiomycete taxa dominated by the needle pathogen Lophodermium seditiosum. Differences in fungal endophyte taxon composition and diversity indices were highly significant among trial sites. Within two sites, fungal endophyte communities varied significantly among provenances. Furthermore, the communities differed significantly among maternal families within provenances in 11/15 and 7/15 comparisons involving culture and metabarcoding data respectively. We conclude that both environmental and host genetic variation shape the fungal endophyte community of P. sylvestris needles.     

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring

Aquatic environmental DNA (eDNA) surveys are transforming how marine ecosystems are monitored. The time-consuming preprocessing step of active filtration, however, remains a bottleneck. Hence, new approaches that eliminate the need for active filtration are required. Filter-feeding invertebrates have been proven to collect eDNA, but side-by-side comparative studies to investigate the similarity between aquatic and filter-feeder eDNA signals are essential. Here, we investigated the differences among four eDNA sources (water; bivalve gill-tissue; sponges; and ethanol in which filter-feeding organisms were stored) along a vertically stratified transect in Doubtful Sound, New Zealand using three metabarcoding primer sets targeting fish and vertebrates. Combined, eDNA sources detected 59 vertebrates, while concurrent diver surveys observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was successfully extracted from the ethanol in which sponges were stored, although a reduced number of species were detected. Bivalve gill-tissue dissections, on the other hand, failed to reliably detect eDNA. Overall, our results show that vertebrate eDNA signals obtained from water samples and marine sponges are highly concordant. The strong similarity in eDNA signals demonstrates the potential of marine sponges as an additional tool for eDNA-based marine biodiversity surveys, by enabling the incorporation of larger sample numbers in eDNA surveys, reducing plastic waste, simplifying sample collection, and as a cost-efficient alternative. However, we note the importance to not detrimentally impact marine communities by, for example, nonlethal subsampling, specimen cloning, or using bycatch specimens.     

Integrating omics to characterize eco‐physiological adaptations: How moose diet and metabolism differ across biogeographic zones

1. With accelerated land conversion and global heating at northern latitudes, it becomes crucial to understand, how life histories of animals in extreme environments adapt to these changes. Animals may either adapt by adjusting foraging behavior or through physiological responses, including adjusting their energy metabolism or both. Until now, it has been difficult to study such adaptations in free‐ranging animals due to methodological constraints that prevent extensive spatiotemporal coverage of ecological and physiological data.
2. Through a novel approach of combining DNA‐metabarcoding and nuclear magnetic resonance (NMR)‐based metabolomics, we aim to elucidate the links between diets and metabolism in Scandinavian moose Alces alces over three biogeographic zones using a unique dataset of 265 marked individuals.
3. Based on 17 diet items, we identified four different classes of diet types that match browse species availability in respective ecoregions in northern Sweden. Individuals in the boreal zone consumed predominantly pine and had the least diverse diets, while individuals with highest diet diversity occurred in the coastal areas. Males exhibited lower average diet diversity than females.
4. We identified several molecular markers indicating metabolic constraints linked to diet constraints in terms of food availability during winter. While animals consuming pine had higher lipid, phospocholine, and glycerophosphocholine concentrations in their serum than other diet types, birch‐ and willow/aspen‐rich diets exhibit elevated concentrations of several amino acids. The individuals with highest diet diversity had increased levels of ketone bodies, indicating extensive periods of starvation for these individuals.
5. Our results show how the adaptive capacity of moose at the eco‐physiological level varies over a large eco‐geographic scale and how it responds to land use pressures. In light of extensive ongoing climate and land use changes, these findings pave the way for future scenario building for animal adaptive capacity.

     

Merging two eDNA metabarcoding approaches and citizen-science-based sampling to facilitate fish community monitoring along vast Sub-Saharan coastlines

The coastline of Sub-Saharan Africa hosts highly diverse fish communities of great conservation value, which are also key resources for local livelihoods. However, many costal ecosystems are threatened by overexploitation and their conservation state is frequently unknown due to their vast spatial extent and limited monitoring budgets. Here, we evaluated the potential of citizen science-based eDNA surveys to alleviate such chronic data deficiencies and assessed fish communities in Mozambique using two 12S metabarcoding primer sets. Samples were either collected by scientific personnel or trained community members and results from the two metabarcoding primers were combined using a new data merging approach. Irrespective of the background of sampling personnel, a high average fish species richness was recorded (38 ± 20 OTUs per sample). Individual sections of the coastline largely differed in the occurrence of threatened and commercially important species, highlighting the need for regionally differentiated management strategies. A detailed comparison of the two applied primer sets revealed an important trade-off in primer choice with MiFish primers amplifying a higher number of species but Riaz primers performing better in the detection of threatened fish species. This trade-off could be partly resolved by applying our new data-merging approach, which was especially designed to increase the robustness of multiprimer assessments in regions with poor reference libraries. Overall, our study provides encouraging results but also highlights that eDNA-based monitoring will require further improvements of, for example, reference databases and local analytical infrastructure to facilitate routine applications in Sub-Saharan Africa.     

Spatio-temporal patterns of eukaryotic biodiversity in shallow hard-bottom communities from the West Antarctic Peninsula revealed by DNA metabarcoding

Aim

We studied molecular eukaryotic biodiversity patterns in shallow hard-bottom Antarctic benthic communities using community DNA metabarcoding. Polar ecosystems are extremely exposed to climate change, and benthic macroinvertebrate communities have demonstrated rapid response to a range of natural and anthropogenic pressures. However, these rich and diverse ecosystems are poorly studied, revealing how little is known about the biodiversity of the Antarctic benthos associated with hard-bottom habitats.

Location

West Antarctic Peninsula and South Shetland Islands.

Methods

Using data collected in seven localities along the western Antarctic Peninsula, we calculated spatial patterns of alpha and beta diversities. Furthermore, we analysed temporal changes in benthic composition in one location (Deception Island) over 3 years. We calculated the temporal alpha and beta diversities to reveal changes in this community over time.

Results

We obtained a final list of 2057 molecular operational taxonomic units. We found significant differences in benthic community composition between localities and among years. Our dataset revealed a total of 10 different kingdom-level lineages and 34 different phyla in the samples. The most diverse phylum was Arthropoda, followed by Bacillariophyta, and Annelida, while the highest relative read abundances belonged to Annelida, Porifera and Echinodermata. Benthic community compositions changed between 2016 and 2018 in Deception Island, and decreasing species richness was the main component of temporal beta diversity.

Main Conclusions

Direct sampling methods are required for monitoring these complex communities. Informative biodiversity patterns can be retrieved even though most of the benthic biodiversity found in Antarctic habitats is yet to be taxonomically described and barcoded. Hard-bottom assemblages exhibit high spatial variability and heterogeneity, not related to depth, which represent a huge challenge for large-scale studies in the Southern Ocean. Local patchiness and structure within these communities are probably a consequence of a combination of several biotic and abiotic factors (i.e. ice disturbance, food supply and competition).