Environmental DNA Sequencing Research Articles

Taxonomic revision of fleshy species of Hydnellum, Neosarcodon, and Sarcodon (Thelephorales) from Australasia

ABSTRACT Stipitate Thelephorales are basidiomycetous, mostly hydnoid, ectomycorrhizal fungi. Some species have declined considerably, and some are threat-listed as vulnerable or endangered. These ecological concerns require a well-resolved taxonomy to understand diversity in this group of fungi and facilitate conservation. However, phylogenetic studies have mostly neglected Southern Hemisphere representatives. This study examines the fleshy species of stipitate Thelephorales from native forests in Australia and New Zealand, using morphological analyses and phylogenetic analyses of nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) and D1–D2 domains at the 5′ end of nuc 28S rDNA (28S) sequences amplified from DNA isolated from fungarium collections and environmental DNA (eDNA) sequences from the Australian Microbiome initiative. Five new species, Sarcodon austrofibulatus, Hydnellum gatesiae, H. nothofagacearum, H. pseudoioeides, and H. variisporum, are described, Sarcodon carbonarius is transferred to Neosarcodon, and a key is provided for the six named species in the region. Boletopsis and Neosarcodon are reported from Australia for the first time based on detections from eDNA in soil samples taken from native forests. The Australasian species of Hydnellum occupy a highly derived position with the phylogeny of the genus, the members of which are otherwise all from the Northern Hemisphere, suggestive of a long-distance dispersal origin for the Australasian species.

Microbial life in preferential flow paths in subsurface clayey till revealed by metataxonomy and metagenomics

BackgroundSubsurface microorganisms contribute to important ecosystem services, yet little is known about how the composition of these communities is affected by small scale heterogeneity such as in preferential flow paths including biopores and fractures. This study aimed to provide a more complete characterization of microbial communities from preferential flow paths and matrix sediments of a clayey till to a depth of 400 cm by using 16S rRNA gene and fungal ITS2 amplicon sequencing of environmental DNA. Moreover, shotgun metagenomics was applied to samples from fractures located 150 cm below ground surface (bgs) to investigate the bacterial genomic adaptations resulting from fluctuating exposure to nutrients, oxygen and water.ResultsThe microbial communities changed significantly with depth. In addition, the bacterial/archaeal communities in preferential flow paths were significantly different from those in the adjacent matrix sediments, which was not the case for fungal communities. Preferential flow paths contained higher abundances of 16S rRNA and ITS gene copies than the corresponding matrix sediments and more aerobic bacterial taxa than adjacent matrix sediments at 75 and 150 cm bgs. These findings were linked to higher organic carbon and the connectivity of the flow paths to the topsoil as demonstrated by previous dye tracer experiments. Moreover, bacteria, which were differentially more abundant in the fractures than in the matrix sediment at 150 cm bgs, had higher abundances of carbohydrate active enzymes, and a greater potential for mixotrophic growth.ConclusionsOur results demonstrate that the preferential flow paths in the subsurface are unique niches that are closely connected to water flow and the fluctuating ground water table. Although no difference in fungal communities were observed between these two niches, hydraulically active flow paths contained a significantly higher abundance in fungal, archaeal and bacterial taxa. Metagenomic analysis suggests that bacteria in tectonic fractures have the genetic potential to respond to fluctuating oxygen levels and can degrade organic carbon, which should result in their increased participation in subsurface carbon cycling. This increased microbial abundance and activity needs to be considered in future research and modelling efforts of the soil subsurface.

Phylogenetic and morphological re-evaluation of Camptophora.

The genetic variety and habitats of Camptophora species, generally known as black yeast, have not been clarified. In this study, we re-evaluated Camptophora based on morphological observations and phylogenetic analyses. Because prior investigations on Camptophora only included a few strains/specimens, 24 Camptophora-related strains were newly obtained from 13 leaf samples of various plant species to redefine the genetic and species concepts of Camptophora. Their molecular phylogenetic relationships were examined using small subunit nuclear ribosomal DNA (nSSU, 18S rDNA), the internal transcribed spacer (ITS) rDNA operon, the large subunit nuclear ribosomal DNA (LSU, 28S rDNA), β-tubulin, the second largest subunit of RNA polymerase II (rpb2), and mitochondrial small subunit DNA (mtSSU). Single- and multi-locus analyses using nSSU-ITS-LSU-rpb2-mtSSU revealed a robust phylogenetic relationship among Camptophora species within Chaetothyriaceae. Camptophora species could be distinguished from other chaetothyriaceous genera by their snake-shaped conidia with microcyclic conidiation and loosely interwoven mycelial masses. Based on the results of phylogenetic analyses, two undescribed lineages were recognized, and Ca. schimae was excluded from the genus. ITS sequence comparison with environmental DNA sequences revealed that the distribution of the genus is restricted to the Asia-Pacific region. Camptophora has been isolated or detected from abrupt sources, and this was attributed to its microcycle. The mechanisms driving genetic diversity within species are discussed with respect to their phyllosphere habitats.

Phylogeny and ultrastructure of a non-toxigenic dinoflagellate Amphidoma fulgens sp. nov. (Amphidomataceae, Dinophyceae), with a wide distribution across Asian Pacific

Amphidoma languida, a marine thecate dinoflagellate that produces the lipophilic toxin azaspiracids (AZAs), is primarily found in the Atlantic. Although this species has not been recorded in the Asian Pacific, environmental DNAs related to Am. languida have been widely detected in the region by metabarcoding analysis. Their morphology and AZA production remain unclear. In this study, the morphology, ultrastructure, phylogeny, and AZA production of nine Amphidoma strains isolated from Japan, Malaysia, and Philippines were investigated. Phylogenetic trees inferred from rDNAs (SSU, ITS, and LSU rDNA) showed monophyly of the nine Pacific strains and were sister to the Am. languida clade, including the toxigenic strains from the Atlantic. Cells were ellipsoid, 8.7–16.7 µm in length and 7.4–14.0 µm in width, with a conspicuous apical pore complex. A large nucleus in the hyposome, parietal chloroplast with a spherical pyrenoid in the episome, and refractile bodies were observed. Thecal tabulation was typical of Amphidoma, Po, cp, X, 6ʹ, 6ʹʹ, 6C, 5S, 6ʹʹʹ, 2ʹʹʹʹ. A ventral pore was located on the anterior of 1ʹ plate, beside the suture to 6ʹ plate. The presence of a ventral depression, on the anterior of anterior sulcal plate, was different from Am. languida. A large antapical pore, containing approximately 10 small pores, was observed. Cells were apparently smaller than Am. trioculata, a species possessing three pores (ventral pore, ventral depression, and antapical pore). TEM showed the presence of crystalline structures, resembling guanine crystals, and cytoplasmic invaginations into the pyrenoid matrix. Flagellar apparatus lacking the striated root connective is similar to peridinioids and related dinoflagellates. AZAs were not detected from the Pacific strains by LC-MS/MS. This non-toxigenic Amphidoma species, here we propose as Amphidoma fulgens sp. nov., is widely distributed in the Asian Pacific. Moreover, molecular comparison also suggested that most of the environmental DNA sequences previously reported as Am. languida or related sequences from the Asian Pacific were attributable to Am. fulgens.

Soil bacterial community diversity and life strategy during slope restoration on an uninhabited island: changes under the aggregate spray-seeding technique.

We investigated the effects of the aggregate spray-seeding (ASS) technique on soil bacterial community diversity, life strategies, and seasonal change. Soil from six plots with original vegetation (CK, n=6) was compared to soil from 15 plots with spray-seeding restoration (SR, n=15) using environmental DNA sequencing. The bacterial Shannon and Chao1 indices of SR soils were significantly greater (P<0.05) than those of CK soils. The Chao1 index for the SR soil bacterial community was significantly greater in summer (P<0.05) than in winter. The ratio of the relative abundance of bacterial K-strategists to r-strategists (K/r) and the DNA guanine-cytosine (GC) content in the SR soil were significantly lower (P<0.05) than those in the CK soil. Principal coordinate analysis revealed significant differences between the SR and CK bacterial communities. The GC content was positively correlated with the K/r ratio. Soil conductivity was negatively associated with the K/r ratio and GC content, indicating that ionic nutrients were closely related to bacterial life strategies. The ASS technique improved soil bacterial diversity, altered community composition, and favored bacterial r-strategists.

Response of Microbial Communities to Naturally Occurring Radioactive Material-Contaminated Sediments: A Microcosm-Based Study.

There is a growing need to understand the potential ecological impacts of contaminants in offshore oil and gas infrastructure, especially if that infrastructure is to be left in situ as a decommissioning option. Naturally occurring radioactive material (NORM) is one type of contaminant found in solid deposits on internal surfaces of infrastructure that poses potential ecological harm if released into the marine environment. Microbes are important components of marine sediment ecosystems because they provide ecosystem services, yet the impacts of NORM contamination to these communities are not well understood. The present study aimed to investigate the response of benthic microbial communities to NORM-contaminated scale, collected from an offshore oil and gas system, via controlled laboratory microcosm studies. Changes to microbial communities in natural sediment and sediments spiked with NORM at radium-226 activity concentrations ranging from 9.5 to 59.8 Bq/kg (in partial equilibria with progeny) over 7 and 28 days were investigated using high-throughput sequencing of environmental DNA extracted from experimental sediments. There were no significant differences in microbial community composition between control and scale-spiked sediments over 7 and 28 days. However, we observed a greater presence of Firmicutes in the scale-mixed treatment and Chloroflexi in the scale-surface treatments after 28 days. This could suggest selection for species with contaminant tolerance or potential resilience to radiation and metal toxicity. Further research is needed to explore microbial tolerance mechanisms and their potential as indicators of effects of radionuclide-contaminated sediments. The present study demonstrated that microcosm studies can provide valuable insights about the potential impacts of contamination from oil and gas infrastructure to sediment microbial communities. Environ Toxicol Chem 2024;43:1648-1661. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Restoration via the aggregate spray-seeding technique affected the soil proteobacteria on an Uninhabited Island: Community structure, metabolic function, nutritional type, and life strategy

This study aimed to explore the impact of aggregate spray-seeding (ASS) restoration measures on the soil proteobacterial community. Using environmental DNA sequencing, we analyzed the proteobacterial communities in the soils of 3 natural vegetation (NV) plots, 3 traditional afforestation (TA) plots, and 12 spray-seeding restoration (SR) plots located on Triangle Island in Zhuhai city, China, during both the summer and winter seasons. We estimated the metabolic function, nutritional type, and life strategy of Proteobacteria through the FAPROTAX and rrnDB databases. Our findings demonstrated that Proteobacteria was the predominant phylum (relative abundance = 40.1–48.4%) in the soil bacterial communities across all three treatments. The relative abundance of Alphaproteobacteria ranged from 28.5% to 38.1%, which was significantly greater (2.6–10.3 times, p < 0.05) than that of Betaproteobacteria or Gammaproteobacteria. Most (90%) proteobacterial genera and all rhizobial genera found in the NV and TA soils were also present in the SR soil, but there were distinct differences in the proteobacterial community structures between the SR soil and NV/TA soil. Across all seasons and treatments, the proteobacterial communities were related to functions such as ureolysis, nitrogen fixation, nitrate reduction, and hydrocarbon degradation. The relative abundance of Proteobacteria associated with chitinolysis was greater in the SR soil than in the NV and TA soils. Among the overall proteobacterial community, the chemoheterotrophic, chemoautotrophic, and phototrophic bacteria accounted for 65–77%, 19–31%, and less than 5%, respectively. Alphaproteobacteria tended to be K strategic, while Betaproteobacteria and Gammaproteobacteria tended to be r strategic. The soil pH, organic carbon content, and nitrogen content were significantly correlated with the metabolic function and nutritional type of the soil Proteobacteria according to the Mantel test results. In conclusion, the application of the ASS technique can effectively restore the biodiversity, metabolic function, and nutritional-type structure of the soil proteobacterial community. Additionally, this study highlights that certain metabolic functions of the proteobacterial community in SR soil undergo changes in response to the use of certain restoration materials. These findings suggest that the targeted addition of specific repair materials can modulate soil microorganism functionality and provide a valuable theoretical foundation for ecological restoration engineering practices.

Distribution and Genetic Lineages of the Craspedacusta sowerbii Species Complex (Cnidaria, Olindiidae) in Italy.

Olindiid freshwater jellyfishes of the genus Craspedacusta Lankester, 1880 are native to eastern Asia; however, some species within the genus have been introduced worldwide and are nowadays present in all continents except Antarctica. To date, there is no consensus regarding the taxonomy within the genus Craspedacusta due to the morphological plasticity of the medusa stages. The species Craspedacusta sowerbii Lankester, 1880 was first recorded in Italy in 1946, and until 2017, sightings of the jellyfish Craspedacusta were reported for 40 water bodies. Here, we shed new light on the presence of the freshwater jellyfishes belonging to the genus Craspedacusta across the Italian peninsula, Sardinia, and Sicily. First, we report 21 new observations of this non-native taxon, of which eighteen refer to medusae sightings, two to environmental DNA sequencing, and one to the finding of polyps. Then, we investigate the molecular diversity of collected Craspedacusta specimens, using a Bayesian analysis of sequences of the mitochondrial gene encoding for Cytochrome c Oxidase Subunit I (mtDNA COI). Our molecular analysis shows the presence of two distinctive genetic lineages: (i) a group that comprises sequences obtained from populations ranging from central to northern Italy; (ii) a group that comprises three populations from northern Italy-i.e., those from the Lake Levico, the Lake Santo of Monte Terlago, and the Lake Endine-and the single known Sicilian population. We also report for the first time a mtDNA COI sequence obtained from a Craspedacusta medusa collected in Spain.

Singleton-based species names and fungal rarity: Does the number really matter?

Fungi are among the least known organisms on earth, with an estimated number of species between 1.5 and 10 million. This number is expected to be refined, especially with increasing knowledge about microfungi in undersampled habitats and increasing amounts of data derived from environmental DNA sequencing. A significant proportion of newly generated sequences fail to match with already named species, and thus represent what has been referred to as fungal “dark taxa”. Due to the challenges associated with observing, identifying, and preserving sporophores, many macro- and microfungal species are only known from a single collection, specimen, isolate, and/or sequence—a singleton. Mycologists are consequently used to working with “rare” sequences and specimens. However, rarity and singleton phenomena lack consideration and valorization in fungal studies. In particular, the practice of publishing new fungal species names based on a single specimen remains a cause of debate. Here, we provide some elements of reflection on this issue in the light of the specificities of the fungal kingdom and global change context. If multiple independent sources of data support the existence of a new taxon, we encourage mycologists to proceed with formal description, irrespective of the number of specimens at hand. Although the description of singleton-based species may not be considered best practice, it does represent responsible science in the light of closing the Linnean biodiversity shortfall.

Fungal Spore Richness and Abundance of Allergenic Taxa: Comparing a Portable Impactor and Passive Trap Indoors and Outdoors in an Urban Setting

Fungal spores are common airborne allergens, and fungal richness has been implicated in allergic disease. Amplicon sequencing of environmental DNA from air samples is a promising method to estimate fungal spore richness with semi-quantification of hundreds of taxa and can be combined with quantitative PCR to derive abundance estimates. However, it remains unclear how the choice of air sampling method influences these estimates. This study compared active sampling with a portable impactor and passive sampling with a passive trap over different durations to estimate fungal spore richness and the abundance of allergenic taxa. Air sampling was conducted indoors and outdoors at 12 residences, including repeated measurements with a portable impactor and passive traps with 1-day and 7-day durations. ITS2 amplicon sequence data were transformed to spore equivalents estimated by quantitative PCR, repeated active samples were combined, and abundance-based rarefaction was performed to standardize sample coverage for estimation of genus-level richness and spore abundance. Rarefied fungal richness was similar between methods indoors but higher for passive traps with a 7-day duration outdoors. Rarefied abundance of allergenic genera was similar between methods but some genera had lower abundance for passive traps with a 1-day duration, which differed indoors and outdoors indicating stochasticity in the collection of spores on collocated samplers. This study found that similar estimates of fungal spore richness and abundance of allergenic taxa can be obtained using a portable impactor or a passive trap within one day and that increased passive sample duration provides limited additional information.

Revision of Seriphium plumosum (Gnaphalieae: Asteraceae) in southern Africa with description of new species in Seriphium

Seriphium plumosum L. from southern Africa has been shown to comprise at least seven distinct species based on analysis of morphological, environmental, and multilocus DNA sequence data. On that evidence, Stoebe burchellii Levyns, Stoebe virgata Thunb., and Stoebe vulgaris Levyns are reinstated in Seriphium, and four new species are described, viz., Seriphium alto-argillaceum Z.Shaik, Se. burrowsii Z.Shaik, Se. crypticum Z.Shaik, and Se. dunensis Z.Shaik. Four of these species are endemic to the Cape Floristic Region of South Africa, and one is so far known from a single high-elevation locality. We present a key to species in the Seriphium plumosum complex, and for each species provide a formal description with accompanying images, typification and synonymy, with distribution maps based on verified field-collected and herbarium specimens.

Non-invasive real-time genomic monitoring of the critically endangered kākāpō.

We used non-invasive real-time genomic approaches to monitor one of the last surviving populations of the critically endangered kākāpō (Strigops habroptilus). We first established an environmental DNA metabarcoding protocol to identify the distribution of kākāpō and other vertebrate species in a highly localized manner using soil samples. Harnessing real-time nanopore sequencing and the high-quality kākāpō reference genome, we then extracted species-specific DNA from soil. We combined long read-based haplotype phasing with known individual genomic variation in the kākāpō population to identify the presence of individuals, and confirmed these genomically informed predictions through detailed metadata on kākāpō distributions. This study shows that individual identification is feasible through nanopore sequencing of environmental DNA, with important implications for future efforts in the application of genomics to the conservation of rare species, potentially expanding the application of real-time environmental DNA research from monitoring species distribution to inferring fitness parameters such as genomic diversity and inbreeding.

Non-invasive real-time genomic monitoring of the critically endangered kākāpō

We used non-invasive real-time genomic approaches to monitor one of the last surviving populations of the critically endangered kākāpō (Strigops habroptilus). We first established an environmental DNA metabarcoding protocol to identify the distribution of kākāpō and other vertebrate species in a highly localized manner using soil samples. Harnessing real-time nanopore sequencing and the high-quality kākāpō reference genome, we then extracted species-specific DNA from soil. We combined long read-based haplotype phasing with known individual genomic variation in the kākāpō population to identify the presence of individuals, and confirmed these genomically informed predictions through detailed metadata on kākāpō distributions. This study shows that individual identification is feasible through nanopore sequencing of environmental DNA, with important implications for future efforts in the application of genomics to the conservation of rare species, potentially expanding the application of real-time environmental DNA research from monitoring species distribution to inferring fitness parameters such as genomic diversity and inbreeding.

Introduction to special issue: Advancing disease ecology through eDNA monitoring of infectious agents

AbstractThis special issue focuses on the applications of environmental DNA (eDNA) sequencing for the detection and monitoring of disease‐causing agents, including viruses, bacteria, protozoans, myxozoans, fungi, trematodes, and arthropods. We explore the impact of eDNA technologies, such as metabarcoding and qPCR, in understanding the dynamics of pathogens in various environments as well as their implications for conservation, biosecurity, and veterinary and agricultural health under the “One Health” framework. This issue addresses how molecular sequencing provides innovative solutions to the challenges faced by conventional parasite and pathogen detection methods, enabling a more comprehensive understanding of the spatiotemporal dynamics of disease agents. Finally, we discuss the challenges in eDNA applications, such as primer development and taxonomic resolution, and the opportunities for future research in advancing eDNA methodologies for infectious disease studies. This issue highlights the growing importance of eDNA surveillance in understanding and managing the health of ecosystems and at‐risk species.

Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection.

Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3-V4 region to investigate whether the RC-PCR reveals more of the 'unseen' diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3-V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject.

Soil as a transdisciplinary research catalyst: from bioprospecting to biorespecting.

The vast microbial biodiversity of soils is beginning to be observed and understood by applying modern DNA sequencing techniques. However, ensuring this potentially valuable information is used in a fair and equitable way remains a challenge. Here, we present a public engagement project that explores this topic through collaborative research of soil microbiomes at six urban locations using nanopore-based DNA sequencing. The project brought together researchers from the disciplines of synthetic biology, environmental humanities and microbial ecology, as well as school students aged 14-16 years old, to gain a broader understanding of views on the use of data from the environment. Discussions led to the transformation of 'bioprospecting', a metaphor with extractive connotations which is often used to frame environmental DNA sequencing studies, towards a more collaborative approach-'biorespecting'. This shift in terminology acknowledges that genetic information contained in soil arises as a result of entire ecosystems, including the people involved in its creation. Therefore, any use of sequence information should be accountable to the ecosystems from which it arose. As knowledge can arise from ecosystems and communities, science and technology should acknowledge this link and reciprocate with care and benefit-sharing to help improve the wellbeing of future generations.

Microbial biomarkers of tree water status for next-generation biomonitoring of forest ecosystems.

Next-generation biomonitoring proposes to combine machine-learning algorithms with environmental DNA data to automate the monitoring of the Earth's major ecosystems. In the present study, we searched for molecular biomarkers of tree water status to develop next-generation biomonitoring of forest ecosystems. Because phyllosphere microbial communities respond to both tree physiology and climate change, we investigated whether environmental DNA data from tree phyllosphere could be used as molecular biomarkers of tree water status in forest ecosystems. Using an amplicon sequencing approach, we analysed phyllosphere microbial communities of four tree species (Quercus ilex, Quercus robur, Pinus pinaster and Betula pendula) in a forest experiment composed of irrigated and non-irrigated plots. We used these microbial community data to train a machine-learning algorithm (Random Forest) to classify irrigated and non-irrigated trees. The Random Forest algorithm detected tree water status from phyllosphere microbial community composition with more than 90% accuracy for oak species, and more than 75% for pine and birch. Phyllosphere fungal communities were more informative than phyllosphere bacterial communities in all tree species. Seven fungal amplicon sequence variants were identified as candidates for the development of molecular biomarkers of water status in oak trees. Altogether, our results show that microbial community data from tree phyllosphere provides information on tree water status in forest ecosystems and could be included in next-generation biomonitoring programmes that would use in situ, real-time sequencing of environmental DNA to help monitor the health of European temperate forest ecosystems.

High‐throughput metabarcoding of SAR11 assemblages from the southwest Atlantic shelf and arid Patagonia: richness and associated rank abundance distributions

The SAR11 clade is likely the most abundant microbial lineage on earth. Here, we provide detailed analyses of the group’s richness and AD in poorly studied Southern microbiomes. This requires whole community composition analyses to be performed, which are also provided in conjunction with the SAR11‐specific studies. In this way, the work describes unknown aspects of the diversity of an outstanding bacterial lineage and novel data on remote and relatively little‐studied microbial communities.BackgroundMassively parallel sequencing of environmental DNA allows microbiological studies to be performed in greater detail than was possible with first‐generation sequencing. For example, it facilitates the use of approaches hitherto largely applied to flora and fauna, such as rank abundance distribution (RAD) analyses.MethodsHere, we set out to advance the knowledge on Ca. Pelagibacterales (SAR11) communities from southern South America using environmental sequences from the open ocean in the Argentine sea, the uncharted Engaño Bay, as well as a river and an oligohaline shallow lake from the Patagonian Steppe ecoregion. The structures of the SAR11 assemblages present in these ecosystems were dissected by direct and rarefaction‐based estimates of species richness, and evaluations of the corresponding abundance distributions (ADs), which was addressed by RAD analyses.ResultsMicrobial community composition analyses revealed that the studied SAR11 assemblages coexist with 27 bacterial phyla. SAR11 richness was in general very high, but ADs turned out to be highly uneven. The results were compatible with prior knowledge, and similar to that derived from point estimates of diversity. However, our comprehensive dissection allowed for more detailed quantitative comparisons to be made between the environments surveyed, and revealed differences regarding both richness and the underlying ADs.ConclusionsDespite SAR11 assemblages being extremely rich, their ADs are very uneven. Richness and ADs can vary, not only between fresh and salt water, but also between oceanic and coastal marine environments. The obtained results provide insights on general topics such as adaptation and the contrast between marine and freshwater radiations.

Response of protists to nitrogen addition, arbuscular mycorrhizal fungi manipulation, and mesofauna reduction in a tropical montane rainforest in southern Ecuador.

The tropical Andes are a species-rich and nitrogen-limited system, susceptible to increased nitrogen (N) inputs from the atmosphere. However, our understanding of the impacts of increased N input on belowground systems, in particular on protists and their role in nutrient cycling, remains limited. We explored how increased N affects protists in tropical montane rainforests in Ecuador using high-throughput sequencing (HTS) of environmental DNA from two litter layers. In addition, we manipulated the amount of arbuscular mycorrhizal fungi (AMF) and mesofauna, both playing a significant role in N cycling and interacting in complex ways with protist communities. We found that N strongly affected protist community composition in both layers, while mesofauna reduction had a stronger effect on the lower layer. Changes in concentration of the AMF marker lipid had little effect on protists. In both layers, the addition of N increased phagotrophs and animal parasites and decreased plant parasites, while mixotrophs decreased in the upper layer but increased in the lower layer. In the upper layer with higher AMF concentration, mixotrophs decreased, while in the lower layer, photoautotrophs increased and plant parasites decreased. With reduced mesofauna, phagotrophs increased and animal parasites decreased in both layers, while plant parasites increased only in the upper layer. The findings indicate that to understand the intricate response of protist communities to environmental changes, it is critical to thoroughly analyze these communities across litter and soil layers, and to include HTS.

Environmental DNA metabarcoding of Danish soil samples reveals new insight into the hidden diversity of eutardigrades in Denmark

Abstract Tardigrades are rarely included in large biodiversity surveys, mainly because of the impracticalities that follow larger sampling and identification of these microscopic animals. Consequently, there is a lack of data on their biogeographical distribution. Here, we analyse environmental DNA sequences of eutardigrades obtained with a metabarcoding protocol on Danish soil samples collected during a national biodiversity project (Biowide). Specifically, we aimed to investigate the applicability of the V4 region (~400 bp) of the 18S rRNA marker gene to assign taxonomy to 96 eutardigrade molecular operational taxonomic units (MOTUs), using three different methods (alignment-, tree- and phylogeny-based methods). Tardigrade reference libraries are currently suffering from insufficient taxon coverage, in some cases challenging the interpretation of data based on similarity searches. This can, to some extent, be accounted for by supplementing identification with algorithms that incorporate a backbone phylogeny and infer models of evolution. Together, the present findings suggest that the V4 region of the 18S rRNA gene offers a promising tool to identify unknown MOTUs of eutardigrades to genus or family level and can, in some cases, be used to assign to species level.