Environmental DNA Sequencing Research Articles

Soil Micro-eukaryotic Diversity Patterns Along Elevation Gradient Are Best Estimated by Increasing the Number of Elevation Steps Rather than Within Elevation Band Replication.

The development of high-throughput sequencing (HTS) of environmental DNA (eDNA) has stimulated the study of soil microbial diversity patterns and drivers at all scales. However, given the heterogeneity of soils, a challenge is to define effective and efficient sampling protocols that allow sound comparison with other records, especially vegetation. In studies of elevational diversity pattern, a trade-off is choosing between replication within elevation bands vs. sampling more elevation bands. We addressed this question for soil protists along an elevation gradient on Mt. Asahi, Hokkaido, Japan. We compared two sampling approaches: (1) the replicate strategy (five replicates at six elevational bands, total = 30) and (2) the transect strategy (one sample in each of 16 different elevational bands). Despite a nearly twofold lower sampling effort, the transect strategy yielded congruent results compared to the replicate strategy for the estimation of elevational alpha diversity pattern: the regression coefficients between diversity indices and elevation did not differ between the two options. Furthermore, for a given total number of samples, gamma diversity estimated across the entire transect was higher when sampling more elevational bands as compared to replication from fewer elevational bands. Beta diversity (community composition turnover) was lower within a given elevational band than between adjacent bands and increased with elevation distance. In redundancy analyses, soil organic matter-related variable (the first principal component of soil organic matter, water content, total organic carbon, and nitrogen by whom were highly correlated) and elevation best explained elevational beta diversity pattern for both sampling approaches. Taken together, our results suggest that sampling a single plot per elevation band will be sufficient to obtain a good estimate of soil micro-eukaryotic diversity patterns along elevation gradients. This study demonstrated the effectiveness of the transect strategy in estimating diversity patterns along elevation gradients which is instructive for future environmental or even experimental studies. While not advocating for completely replacing replication-based sampling practices, it is important to note that both replicate and transect strategies have their merits and can be employed based on specific research goals and resource limitations.

Phytophthora in Horticultural Nursery Green Waste—A Risk to Plant Health

Phytophthora is a genus of destructive plant pathogens. Certain species are damaging to native ecosystems, forestry, and the horticultural sector, and there is evidence of their dissemination in plant imports. Horticultural nurseries are central nodes of the plant trade and previous studies have found a high diversity of Phytophthora associated with plant nursery stock. It was subsequently hypothesized that green waste disposal sites in nurseries could harbour diverse Phytophthora communities and act as a pathogen reservoir and conduit, facilitating further Phytophthora infection of nursery stock and its spread into the wider environment. This project identified Phytophthora species associated with green waste at three Scottish nurseries by sampling material from waste piles, water run-off from piles, and roots from discarded plants. Species were identified using a baiting method and sequencing of environmental DNA. Plant nursery green waste was shown to harbour diverse and varied Phytophthora species assemblages, with differences among nurseries reflecting biosecurity management practices. Eighteen Phytophthora species were detected in the samples, including the highly destructive pathogens P. ramorum and P. austrocedri. Results suggest that the improved management of waste, for example through effective on-site composting, is essential to reduce the risk of Phytophthora pathogens spreading from nurseries into the wider environment.

Soil protist functional composition shifts with atmospheric nitrogen deposition in subtropical forests

Abstract Soil protists play a key role in driving ecological functions through predation and parasitism. However, little is known about how nitrogen (N) deposition and seasonal variation influence soil protist functions in forest soils. Here, we assessed first the impacts of N deposition (control, 50, 100 and 150 kg N ha−1 year−1) on the functional composition of the soil protist community in summer and winter, using amplicon sequencing of environmental DNA from a subtropical natural forest. We found that soil protists were dominated by consumers (42.6%–51.6%), followed by parasites (32.9%–40.9%) and phototrophs (3.2%–13.1%), implying a predominant role of consumers and potential top‐down effects on the other trophic groups in subtropical forest soils. The functional composition of soil protists was greatly influenced by N deposition, but these responses were dependent on seasonal variation. The diversity of phototrophs was lower in summer than in winter. Instead, an opposite pattern was observed for consumers, resulting in a significantly higher protist diversity in summer than in winter. Furthermore, low and high N deposition simplified the structural complexity of soil protist communities, suggesting a nonlinear response of protist structural stability to N deposition. Synthesis and applications. This study provides unprecedented evidence that seasonal variation plays an important role in regulating responses of soil protist functional composition to N deposition, and highlights the nonlinear effects of rising N deposition on the soil food web.

From river to ocean: Connectivity and heterogeneity of aquatic ecosystems depicted by planktonic microeukaryotes

Understanding how the diverse habitats incubated a high diverse biosphere is a core question for biodiversity conservation. Community assembly of organisms in connected but different habitats could provide insight into this issue. As an important group of plankton, microeukaryotes are widely distributed in aquatic habitats and are sensible to ecological conditions. However, how the distribution of planktonic microeukaryotes from river to ocean are shaped by habitat connectivity and heterogeneity remains unclear. In order to assess the connectivity and heterogeneity of aquatic habitats, environmental DNA sequencing was used to profile the planktonic microeukaryotic communities in typical habitats throughout the major rivers, estuaries, coastal waters, and ocean of China, covering a geographic distance of 3530 km. The hypothesized “freshwater species pool (FSP)” and “marine species pool (MSP)” of planktonic microeukaryotes were confirmed by their significant dissimilarities in community composition. The planktonic microeukaryotic communities completed turnover of FSP-pattern and MSP-pattern within salinity range of 5–15 in estuaries of the river-to-ocean continuum. In this scale covering fresh and salty waters, the community assembly of planktonic microeukaryotes was driven primarily by habitat filtering rather than salinity nor geographic distance. Four types of habitat, i.e., river, estuary, tidal zone, and ocean, were outlined by their planktonic microeukaryotic communities, which depicted the habitat heterogeneity in this continuum. As to the constructive species in these habitats, 98.1 % of them were shared between at least two types of habitat and 41.3 % of them shared among all habitats. These shared constructive species depicted the habitat connectivity of aquatic ecosystems from river to ocean. The findings suggest that the assessment of planktonic microorganism assemblages is a potential approach to indicate habitat diversity in connected aquatic ecosystems.

Comparing Microsporidia-targeting primers for environmental DNA sequencing.

Metabarcoding is a powerful tool to detect classical, and well-known "long-branch" Microsporidia in environmental samples. Several primer pairs were developed to target these unique microbial parasites, the majority of which remain undetected when using general metabarcoding primers. Most of these Microsporidia-targeting primer pairs amplify fragments of different length of the small subunit ribosomal RNA (SSU-rRNA) gene. However, we lack a broad comparison of the efficacy of those primers. Here, we conducted in silico PCRs with three short-read (which amplify a few-hundred base pairs) and two long-read (which amplify over a thousand base pairs) metabarcoding primer pairs on a variety of publicly available Microsporidia sensu lato SSU-rRNA gene sequences to test which primers capture most of the Microsporidia diversity. Our results indicate that the primer pairs do result in slight differences in inferred richness. Furthermore, some of the reverse primers are also able to bind to microsporidian subtaxa beyond the classical Microsporidia, which include the metchnikovellidan Amphiamblys spp., the chytridiopsid Chytridiopsis typographi and the "short-branch" microsporidian Mitosporidium daphniae.

Biodiversity in a forest ecosystem in India using environmental DNA sequence analysis of pathogen hostile eucalyptus trees

<p>Trees in the forest are an unprecedented cluster of organisms in ecological, monetary and social importance. With a wide distribution, mostly random spread over and a large population in terms of size, the majority of tree species show considerable variation in genetics both within and between populations. The genus Phytophthora, is a most destructive plant pathogen and attacks a wide range of tree hosts, including inexpensively its significant species. Many species of Phytophthora are known to be persistent and brought in through nurseries and commercial agriculture. Diseases from various eucalyptus tree species were first reported in India and the symptoms, incidence and leaf damage have been described. As an observation of a larger project to use genomic data for tree disease diagnosis, pathogen detection and surveillance, in this study the significant analysis of various DNA sequences of P.meadii 2 on eucalyptus tree species in Indian Forests. The identified outcomes observed that the high frequency of nucleotides and their combinations found in the organisms in trees threatening their lives may be observed and has to be condensed</p>

Antarctic biodiversity predictions through substrate qualities and environmental DNA

Antarctic conservation science is crucial for enhancing Antarctic policy and understanding alterations to terrestrial Antarctic biodiversity. Antarctic conservation will have limited long‐term impacts in the absence of large‐scale biodiversity data, but if such data were available, it is likely to improve environmental protection regimes. To enable the prediction of Antarctic biodiversity across continental spatial scales through proxy variables, in the absence of baseline surveys, we linked Antarctic substrate‐derived environmental DNA (eDNA) sequence data from the remote Antarctic Prince Charles Mountains to a selected range of concomitantly collected measurements of substrate properties. We achieved this through application of a statistical method commonly used in machine learning. Our analysis indicated that neutral substrate pH, low conductivity, and certain substrate minerals are important predictors of the presence of basidiomycetes, chlorophytes, ciliophorans, nematodes, and tardigrades. A bootstrapped regression revealed how variations in the identified substrate parameters influence probabilities of detecting eukaryote phyla across vast and remote areas of Antarctica. We believe that our work will improve future taxon distribution modeling and aid in developing more targeted surveys of biodiversity conducted under logistically challenging conditions.

Development of a machine-learning model to identify the impacts of pesticides characteristics on soil microbial communities from high-throughput sequencing data.

High-throughput sequencing (HTS) of soil environmental DNA provides an advanced insight into the effects of pesticides on soil microbial systems. However, the association between the properties of the pesticide and its ecological impact remains methodically challenging. Risks associated with pesticide use can be minimized if pesticides with optimal structural traits were applied. For this purpose, we merged the 20 independent HTS studies, to reveal that pesticides significantly reduced beneficial bacteria associated with soil and plant immunity, enhanced the human pathogen and weaken the soil's ecological stability. Through the machine-learning approach, correlating these impacts with the physicochemical properties of the pesticides yielded a random forest model with good predictive capabilities. The models revealed that physical pesticide properties such as the dissociation constant (pKa), the molecular weight and water solubility, determined the ecological impact of pesticides to a large extent. Moreover, this study identified that eco-friendly pesticides should possess a value of pKa > 5 and a molecular weight in the range of 200-300 g/mol, which were found to be conducive to bacteria related to plant immunity promotion and exerted the lowest fluctuation of human opportunistic pathogen and keystone species. This guides the design of pesticides for which the impacts on soil biota are minimized.

Comparative analysis of zooplankton diversity in freshwaters: What can we gain from metagenomic analysis?

AbstractMolecular genetic approaches applied to environmental DNA have great potential for biodiversity research and ecosystem monitoring. A metagenome is produced via shotgun sequencing of DNA collected directly from the environment and represents a sample of genetic information from all organisms captured in an environmental sample. Metagenomes have been primarily used to study bacteria and archaea, but promising reports focusing on metazoan diversity are emerging. However, methodological uncertainties remain, and studies are required to validate the power and the limitations of such an approach when applied to macro‐eukaryotes. Here, we analyzed water sample metagenomes to estimate zooplankton diversity in 22 freshwater lakes across eastern Canada. We tested the coherence of data based on field samples collected at the same time: 1) morphologically identified zooplankton specimens and 2) molecular genetic data derived from shotgun sequencing of environmental DNA for which we applied two different bioinformatic workflows: a whole metagenome mapping approach and a small subunit (SSU) rRNA gene prediction approach. We further evaluated diversity trends emerging from each dataset in relation to major environmental gradients. We found a significant correlation between the relative abundance of zooplankton families identified based on SSU rRNA gene prediction and morphology. However, differences in congruence between metagenomes and morphological identifications were detected when varied bioinformatic approaches were applied to the presence–absence data. This study presents one of the first diversity assessments of a group of aquatic metazoans using metagenomes and validates the coherence of the community composition derived from genomic and classical species surveys. Overall, our results suggest that metagenomics has the potential to be further developed to describe metazoan biodiversity in aquatic ecosystems, and to advance this area we provide key recommendations for workflow improvement.

Allogromia Arnoldi n. sp.: Distribution, Phylogenetic Placement, Culture Methods, and Fine Structure of a New Monothalamid Foraminiferan

ABSTRACT Allogromiids, organic-walled foraminifera, are common members of foraminiferal associations in reef and back-reef settings of the Florida Keys and other locales, and many live in cryptic or otherwise protected microhabitats associated with macroalgae and seagrasses. A new species of Allogromia was isolated from the alga Dasycladus vermicularis (Scopoli) collected from prop-roots of the red mangrove, Rhizophora mangle (Linneaus), in Zane Grey Creek, Long Key, Florida. This species, Allogromia arnoldi n. sp., genetically matches sequences of undescribed and uncharacterized allogromiids deposited in GenBank from Cyprus and Jamaica. Allogromia arnoldi is genetically and morphologically distinct from A. laticollaris Arnold, A. laticollaris strain CSH, A. sp. NF (Lee & Pierce, 1963), environmental DNA sequences of morphologically unknown allogromiids, a number of freshwater forms, and several undescribed marine allogromiids known in the literature by nicknames (e.g., “squatter,” “rubble dome,” “twinkle”). This new species of Allogromia belongs to Clade M of the monothalamid foraminifera. Allogromia arnoldi appears to be broadly distributed, and it is fairly easy to isolate and rear in culture. Individuals typically have bright orange cytoplasm, a variable number of apertures, and can assume a wide range of shapes. Transmission electron microscopy (TEM) following high-pressure freezing and freeze substitution shows that the test has a complex fine structure that varies in thickness. It consists of two layers: a thick inner layer with a “herringbone” fine structure, and a thinner, outer electron-opaque mesh-like layer that occurs in patches. The number of nuclei varies per individual, and the non-reproductive nucleus is morphologically similar to that of many other monothalamid foraminifera.

Temperature Controls eDNA Persistence across Physicochemical Conditions in Seawater.

Environmental DNA (eDNA) quantification and sequencing are emerging techniques for assessing biodiversity in marine ecosystems. Environmental DNA can be transported by ocean currents and may remain at detectable concentrations far from its source depending on how long it persist. Thus, predicting the persistence time of eDNA is crucial to defining the spatial context of the information derived from it. To investigate the physicochemical controls of eDNA persistence, we performed degradation experiments at temperature, pH, and oxygen conditions relevant to the open ocean and the deep sea. The eDNA degradation process was best explained by a model with two phases with different decay rate constants. During the initial phase, eDNA degraded rapidly, and the rate was independent of physicochemical factors. During the second phase, eDNA degraded slowly, and the rate was strongly controlled by temperature, weakly controlled by pH, and not controlled by dissolved oxygen concentration. We demonstrate that marine eDNA can persist at quantifiable concentrations for over 2 weeks at low temperatures (≤10 °C) but for a week or less at ≥20 °C. The relationship between temperature and eDNA persistence is independent of the source species. We propose a general temperature-dependent model to predict the maximum persistence time of eDNA detectable through single-species eDNA quantification methods.

Changes in the Understanding of Fungal Diversity Based on Research in the Tropics

Fungi are representative one of the world’s eukaryotic kingdoms and play an important role in various ecosystems. For example, they are responsible for breaking down organic matter and recycling nutrients (as decomposers), mutualistic interactions with plants (as mycorrhizae and endophytes) and causal agents of disease (as pathogens) [1–3]. The question of how many fungal species actually exist is indisputably important. Over the past two decades, a conservative estimate of 1.5 million species of fungi has been reported globally [4]. However, this estimation has been based on extrapolations derived from several independent sources of data and would still not include sequence data that have been derived from environmental DNA sequences. Notably, there is still no comprehensive database pertaining to fungal diversity in tropical areas. New fungal taxa have already been described and a number of recent studies have revealed that fungal diversity may actually exceed previous estimates. During the last decade, with the use of morphological and molecular approaches, new fungal species have been described. However, the presence of cryptic species or species complexes has frequently led to overestimated geographic distributions of certain taxa. Accordingly, new fungal species are usually discovered in hotspot areas or areas where most fungal taxonomists are presently working. Thus, global fungi species records have been revised to between 2.2 and 3.8 million species [5,6]. The rate of recovery for new fungal species has been reported in known hot spot areas to be more than 2,000 species per year [6]. A comparison of new fungal species described from each continent in 2017 [6] and 2019 [7] is presented in Figure 1. The majority of new fungal species has been recorded from Asia, followed by Europe, America, Oceania, Africa, and Antarctica. However, less than 10% of the fungal species in the world have been accurately described [5]. Remarkably, the increasing trend in newly recorded fungal species is expected to continue to rise in the future.

Environmental DNA Sequencing to Monitor Restoration Practices on Soil Bacterial and Archaeal Communities in Soils Under Desertification in the Brazilian Semiarid.

Soils from Brazilian semiarid regions are highly vulnerable to desertification due to their geology, climate, human actions, and intensive land use that contribute to desertification. Therefore, areas under desertification have increased in the Brazilian semiarid region and it has negatively changed the soil bacterial and archaeal communities and their functionality. On the other hand, although restoration strategies are expensive and there are few soils restoration programs, some practices have been applied to restore these soils under desertification. For instance, conservationist practices and grazing exclusion have been strategically implemented, and they created a new altered soil condition for soil microbial communities, boosting soil microbial diversity. Here, we discuss the potential of these restoration strategies to recover the richness and diversity of soil bacterial and archaeal communities that were described through environmental DNA (eDNA) sequencing of soil samples. eDNA sequencing results show that areas where restoration strategies have been applied in regions under desertification in the Brazilian semiarid have increased species richness, diversity, and structure of the bacterial and archaeal community. In addition, network connectivity and functionality of the soil microorganisms have been improved over time. Altogether, we show that management strategies for soil restoration have positive effects on soil microbial communities and these effects can be monitored using the eDNA sequencing approach.

Aphelidium parallelum, sp. nov., a new aphelid parasitic on selenastracean green algae

ABSTRACT Aphelids (phylum Aphelida = Aphelidiomycota) are intracellular parasitoids of algae and represent one of the early-diverging or sister lineages of the kingdom Fungi. Although aphelids are a small group comprising four genera and 17 species, molecular phylogenetic analyses revealed that numerous environmental DNA sequences represent undescribed lineages, indicating their hidden diversity. Here, we investigated a novel aphelid strain, KS114, that parasitizes selenastracean green algae. KS114 exhibited a life cycle typical of aphelids and produced posteriorly uniflagellate zoospores that resembled those of Aphelidium chlorococcorum f. majus in possessing a single apical filopodium but could be distinguished by ultrastructure features. In KS114, the kinetosome and nonflagellated centriole were aligned in parallel, a unique characteristic among the known aphelids. Kinetid-associated structures, such as fibrillar root and microtubules, were not found in the zoospores of KS114. In the molecular phylogeny of nuc 18S rDNA sequences, KS114 clustered with two environmental sequences and was distinct from all other sequenced species. Based on these results, we describe this aphelid as a new species, Aphelidium parallelum. http://www.zoobank.org/urn:lsid:zoobank.org:act:3CB658DB-1F12-41EF-A57D-2CBFCDE6A49A

DNA sequence and taxonomic gap analyses to quantify the coverage of aquatic cyanobacteria and eukaryotic microalgae in reference databases: Results of a survey in the Alpine region

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99–100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.

Machine Learning and Deep Learning Applications in Metagenomic Taxonomy and Functional Annotation.

Shotgun sequencing of environmental DNA (i.e., metagenomics) has revolutionized the field of environmental microbiology, allowing the characterization of all microorganisms in a sequencing experiment. To identify the microbes in terms of taxonomy and biological activity, the sequenced reads must necessarily be aligned on known microbial genomes/genes. However, current alignment methods are limited in terms of speed and can produce a significant number of false positives when detecting bacterial species or false negatives in specific cases (virus, plasmids, and gene detection). Moreover, recent advances in metagenomics have enabled the reconstruction of new genomes using de novo binning strategies, but these genomes, not yet fully characterized, are not used in classic approaches, whereas machine and deep learning methods can use them as models. In this article, we attempted to review the different methods and their efficiency to improve the annotation of metagenomic sequences. Deep learning models have reached the performance of the widely used k-mer alignment-based tools, with better accuracy in certain cases; however, they still must demonstrate their robustness across the variety of environmental samples and across the rapid expansion of accessible genomes in databases.

Lost in dead wood? Environmental DNA sequencing from dead wood shows little signs of saproxylic beetles

AbstracteDNA metabarcoding has become a standard method for assessing wood‐inhabiting fungi and bacteria, yet determination of dead‐wood‐inhabiting beetles still relies on time‐consuming collection of beetle specimens. We thus tested whether beetle species can be identified by eDNA sequencing of wood in a mesocosm experiment that manipulated species assemblages. Dead wood samples were taken at exit holes of beetles and DNA was extracted and analyzed using two comparative methods: (i) metabarcoding with standard arthropod primers (421 bp) and (ii) using short species‐specific primers (120–264 bp) with Sanger sequencing. Results showed that beetle DNA was amplified by each of the two approaches, however, with (i) we detected only one non‐target saproxylic beetle species. In addition, we identified 80 different OTUs with four non‐targeted species of arthropods. For (ii) we detected the targeted species in two fresh beetle exit holes out of 20 samples. We suggest that, in contrast to fungi and bacteria, this eDNA metabarcoding approach is not able to reliably detect saproxylic beetles from wood samples, likely due to rapid degradation of their target DNA. Adapting such an approach for large‐scale analyses thus requires a better knowledge of degradation processes affecting DNA quality and quantity in wood.

Benefits of merging paired-end reads before pre-processing environmental metagenomics data.

High throughput sequencing of environmental DNA has applications in biodiversity monitoring, taxa abundance estimation, understanding the dynamics of community ecology, and marine species studies and conservation. Environmental DNA, especially, marine eDNA, has a fast degradation rate. Aside from the good quality reads, the data could have a significant number of reads that fall slightly below the default PHRED quality threshold of 30 on sequencing. For quality control, trimming methods are employed, which generally precede the merging of the read pairs. However, in the case of eDNA, a significant percentage of reads within the acceptable quality score range are also dropped. To infer the ideal merge tool that is sensitive to eDNA, two Hiseq paired-end eDNA datasets were utilized to study the merging by the tools - FLASH (Fast Length Adjustment of SHort reads), PANDAseq, COPE, BBMerge, and VSEARCH without preprocessing. We assessed these tools on the following parameters: Time taken to process, the quality, and the number of merged reads. Trimmomatic, a widely-used preprocessing tool, was also assessed by preprocessing the datasets at different parameters for the two approaches of preprocessing: Sliding Window and Maximum Information. The preprocessed read pairs were then merged using the ideal merge tool identified earlier. FLASH is the most efficient merge tool balancing data conservation, quality of reads, and processing time. We compared Trimmomatic's two quality trimming options with increasing strictness with FLASH's direct merge. The raw reads processed with Trimmomatic then merged, yielded a significant drop in reads compared to the direct merge. An average of 29% of reads was dropped when directly merged with FLASH. Maximum Information option resulted in 30.7% to 68.05% read loss with lowest and highest stringency parameters, respectively. The Sliding Window approach conserves approximately 10% more reads at a PHRED score of 25 set as the threshold for a window of size 4. The lowered PHRED cut off conserves about 50% of the reads that could potentially be informative. We noted no significant reduction of data while optimizing the number of reads read in a window with the ideal quality (Q) score. Losing reads can negatively impact the downstream processing of the environmental data, especially for sequence alignment studies. The quality trim-first-merge-later approach can significantly decrease the number of reads conserved. However, direct merging of pair-end reads using FLASH conserved more than 60% of the reads. Therefore, direct merging of the paired-end reads can prevent potential removal of informative reads that do not comply by the trimming tool's strict checks. FLASH to be an efficient tool in conserving reads while carrying out quality trimming in moderation. Overall, our results show that merging paired-end reads of eDNA data before trimming can conserve more reads.

Mobile mud layer underneath the desiccated maar lake of Rincón de Parangueo and insights into its microbial fingerprints

We surveyed a subsurface layer of saturated mobile mud and its microbial fingerprints below the desiccated bottom of a maar lake at Rincón de Parangueo (RP), Mexico. A multi-scale approach was followed using geological fieldwork, coring of the sediments, ground penetrating radar survey, physico-chemical characterization including X-ray diffraction and scanning electron microscope, and high-throughput DNA sequencing methods. The mobile mud is an organic-rich silty clay, with high values of alkalinity, volumetric water content and conductivity. Mud mobility has been attributed to overpressure caused by disequilibrium compaction of sediments related to active subsidence and pore overpressure produced by an input of groundwater and gas content in the sediments and resulted in a diverse set of structures related to mud tectonics such as injection domes and fluid seeps through fractures. Extraction and sequencing of sedimentary environmental DNA in the mud layer were performed for Bacteria and Archaea. Despite the small number of samples obtained, the microbial fingerprint from the sedimentary environmental DNA at subsurface shares similarities with the microbial communities identified on the crater surface. Additionally, we identify the DNA of specific methanogenic microorganisms in the mud, such as Bathyarchaeia, Methanomassiliicoccales, and Methanobacteriales, and we speculate on their probable role in gas production and pore overpressure in the mud layer. The underground mud at Rincón de Parangueo represents a geologically dynamic environment with conditions that are favorable for the thriving of microbial communities.

Targeted next‐generation sequencing of environmental DNA improves detection of invasive European green crab (Carcinus maenas)

AbstractIn the northeast Pacific Ocean, there is high interest in developing eDNA‐based survey methods to aid management of invasive populations of European green crab (Carcinus maenas). Expected benefits are improved sensitivity for early detection of secondary spread and to assess the outcome of eradication efforts. A new eDNA‐based approach we term “targeted next‐generation sequencing (tNGS)” is introduced here and shown to improve detection relative to qPCR at sites with lower green crab CPUE values measured by trapping. DNA standards (gBlock) with starting molecule copies that were 10 to 100 times lower than the qPCR limit of detection returned significant numbers of sequencing reads, which in our field assessments translated to a 7%–10% increase in detection probability from tNGS relative to qPCR at sites with lower CPUE. We also found the number of sequencing reads from tNGS was significantly correlated with green crab CPUE whereas Ct values from qPCR were not. When sources of variation were partitioned for each assay, we found the difference between mean within‐site and mean between‐site variation was much larger and had non‐overlapping confidence intervals for tNGS relative to qPCR, suggesting the former may offer more power for detecting spatial variation in eDNA availability. Results presented here indicate this approach is suitable for species of known low abundances where a positive detection has high economic or environmental consequences, or for labs doing eDNA surveys for whom NGS‐based workflows are operationally more efficient than qPCR given its upward scalability. Any species with an existing qPCR assay can be easily tested with a tNGS assay using the approach presented here. We conclude with a discussion on the fitness for purpose of tNGS vs. qPCR for various applications and on how to best apply molecular surveys in management programs.