Evaluating freshwater macroinvertebrates from eDNA metabarcoding: A river Nalón case study

Phylogenetic relationships among asteroids remain to be extremely controversial in spite of many morphological and molecular studies have been applied to this issue. In the present study, especially focusing on resolving the relationship of Asterina and Solasteridae, we reconstructed the molecular phylogenetic tree of asteroids using nuclear 18S rDNA. A close relationship between Asterina and Solasteridae, which has been suggested from analyses of mitochondrial 12S rDNA and 16S rDNA, is supported here by the nuclear 18S rDNA dataset. The support is even stronger when the sequences of mitochondrial rDNAs and nuclear 18S rDNA are combined as a total dataset. The independent support from both nuclear 18S rDNA and mitochondrial rDNAs strongly argues for a close relationship between the Asterina and Solasteridae.

In Taiwan, the authorities have spent years working on remedying polluted rivers. Generally, the remediation planning works are divided into two phases. During the first phase, the allowed pollution discharge quantity and abatement quantity of each drainage zone, including the assimilative capacity, are generated based on the total river basin. In the second phase, the abatement action plans for each pollution source in each drainage zone are respectively devised by the related organizations based on the strategies generated during the first phase. However, the effectiveness of linking the two phases is usually poor. Highly integrated performances are not always achieved because the separate two-phase method does not take system and management thinking into consideration in the planning stage. This study pioneers the use of the Managing for Results (MFR) method in planning strategies and action plans for river water quality management. A sustainable management framework is proposed based on the concept and method of MFR, Management Thinking, and System Analysis. The framework, consisting of planning, implementation, and controlling stages, systematically considers the relationships and interactions among four factors: environment, society, economy, and institution, based on the principles of sustainable development. Based on the framework, the Modified Bounded Implicit Enumeration algorithm, which is used as a solving method, is combined with Visual Basic software and MS Excel to develop a computer system for strategy planning. The Shetzu River, located in northern Taiwan, is applied as a case study. According to the theoretical, practical, and regulatory considerations, the result-oriented objectives are defined to first improve the pollution length of the Shetzu River in specific remediation periods to finally meet regulated water quality standards. The objectives are then addressed as some of the constraints for the strategy planning model. The model objective is to pursue the maximum assimilative capacity (environmental phase) subjected to the constraints of water quality standards (institutional phase), social equity (social phase), and proper available technology (economic phase). The pollution quantity abatement and allocation, which are named the top strategies, of each drainage zone for different scenarios can be obtained based on each water quality standard. The middle as well as lower strategies and action plans, which consist of pollution quantity abatement and allocation of each class (domestic, industrial, livestock, and non-point pollution sources) and their individual pollution sources in each drainage zone, are then generated based on the top strategies. The performance indicators and measure plans are proposed based on the action plans to promote the comprehensive effectiveness of river water quality management. The authorities have begun to develop a budget based on the strategies and action plans developed in this study. The analytical results indicate that the objectives, strategies, and action plans developed based on the sustainable management framework and strategy planning system can effectively help the related authorities to fulfill the tasks of water quality management for a river basin.

European native land snails, Trochulus hispidus (Linnaeus, 1758), were collected from Brighton, Ontario, Canada, over a 20-month period beginning in April 2020. Polysporocystic oocysts consistent with Klossia spp. (Apicomplexa: Adeleidae) were observed in macerated snail kidney tissues by light microscopy. Sporulated oocysts measured 103 ± 5.8 µm × 99 ± 7.4 µm with indistinct or absent oocyst residuum; oocysts contained more than 125 tetrazoic sporocysts. Sporocysts measured 12 ± 0.66 µm × 11 ± 0.76 µm with a centrally placed sporocyst residuum and a few irregularly dispersed granules. DNA was extracted from all kidney tissues and prevalence of the Klossia-infection determined by Klossia-specific PCR was 46% (19 of 41). Nuclear 18S rDNA (1800 bp) and complete mitochondrial genome (6757 bp) sequences differed sufficiently from available Klossia spp. sequences to justify description of a new Klossia species, Klossia vaderbriani n. sp. Phylogenetic analysis using combined nuclear (18S rDNA) and mitochondrial (cytochrome c oxidase subunit I—COI) sequences strongly support the monophyly of the genus Klossia but not of the family Adeleidae to which these parasites belong. These observations and a detailed literature review supported a wider discussion on the taxonomy of the genera in the family Adeleidae.

This study introduces a novel genus Robiniigena, with its type R.hyalinospora. The specimen was collected on dead aerial branches of Robiniapseudoacacia in Italy. Based on the examination of morphology and the results of phylogenetic analyses involving nuclear 18S rDNA (SSU), nuclear 28S rDNA (LSU), nuclear rDNA ITS1-5.8S-ITS2 (ITS), translation elongation factor 1-alpha (tef1-α) and RNA polymerase II second largest subunit (rpb2) sequences, Robiniigena is referred to the family Pleomonodictydaceae (Pleosporales). It is characterized by immersed to erumpent, ostiolate ascomata, filiform, septate and cellular pseudoparaphyses, bitunicate, clavate to cylindric-clavate asci and fusiform, hyaline ascospores surrounded by a mucilaginous sheath. This research also establishes the taxonomic placement of the previously unclassified Inflatispora (Pleosporales genus incertae sedis) within the Pleomonodictydaceae. The sexual morph of Ampelomycesquisqualis (Phaeosphaeriaceae) is described for the first time and it is characterized by immersed, perithecial ascomata, a peridium comprising two layers, branched, septate and filiform pseudoparaphyses, short-pedicellate, bitunicate asci with an ocular chamber and sub-hyaline, fusiform, septate ascospores. This species, previously known only in its asexual morph, has been found as a saprobe on Sonchus sp. in Italy. Our identification of the sexual morph was based on LSU rDNA and ITS rDNA sequence data. Melomastiamaolanensis (Pleurotremataceae) is reported for the first time in Thailand, collected from Chromolaenaodorata, while M.oleae is documented as a new record from Durantaerecta in Thailand.

In this study, Aporrectodea trapezoides were collected from three locations in the north, central and south of Baghdad (Rashdiyah, Jadriyah and Zafaraniyah). The species was initially identified using morphological characteristics, and then genetic links among the researched species in the three locations were analyzed using molecular techniques (DNA barcoding). In order to validate the species of earthworms examined, their nuclear 18S rDNA gene sequences were analyzed and a phylogenetic tree was constructed. The nucleotide sequences were compared to those in GenBank by using the online Basic Local Alignment Search Tool (Blast). The DNA barcoding of the nuclear 18S rDNA has been shown to be an effective tool for species identification of earthworms, and it provides a powerful complement to traditional morphological identification as shown by a Blast analysis showing 99-100% similarity with sequence annotated in GenBank at 18S region. DNA sequence from one of the species under study has been submitted in GenBank with an accession number.

Microbial community analysis based on the 16S rRNA-gene is used to investigate both beneficial and harmful microorganisms in various fields and environments. Recently, the next-generation sequencing (NGS) technology has enabled rapid and accurate microbial community analysis. Despite these advantages of NGS based metagenomics study, sample transport, storage conditions, amplification, library preparation kits, sequencing, and bioinformatics procedures can bias microbial community analysis results. In this study, eight mock communities were pooled from genomic DNA of Lactobacillus acidophilus KCTC 3164T, Limosilactobacillus fermentum KCTC 3112T, Lactobacillus gasseri KCTC 3163T, Lacticaseibacillus paracasei subsp. paracasei KCTC 3510T, Limosilactobacillus reuteri KCTC 3594T, Lactococcus lactis subsp. lactis KCTC 3769T, Bifidobacterium animalis subsp. lactis KCTC 5854T, and Bifidobacterium breve KCTC 3220T. The genomic DNAs were quantified by droplet digital PCR (ddPCR) and were mixed as mock communities. The mock communities were amplified with various 16S rRNA gene universal primer pairs and sequenced by MiSeq, IonTorrent, MGIseq-2000, Sequel II, and MinION NGS platforms. In a comparison of primer-dependent bias, the microbial profiles of V1-V2 and V3 regions were similar to the original ratio of the mock communities, while the microbial profiles of the V1-V3 region were relatively biased. In a comparison of platform-dependent bias, the sequence read from short-read platforms (MiSeq, IonTorrent, and MGIseq-2000) showed lower bias than that of long-read platforms (Sequel II and MinION). Meanwhile, the sequences read from Sequel II and MinION platforms were relatively biased in some mock communities. In the data of all NGS platforms and regions, L. acidophilus was greatly underrepresented while Lactococcus lactis subsp. lactis was generally overrepresented. In all samples of this study, the bias index (BI) was calculated and PCA was performed for comparison. The samples with biased relative abundance showed high BI values and were separated in the PCA results. In particular, analysis of regions rich in AT and GC poses problems for genome assembly, which can lead to sequencing bias. According to this comparative analysis, the development of reference material (RM) material has been proposed to calibrate the bias in microbiome analysis.

The benthic environment refers to the region defined by the interface between a body of water and the bottom substrate, including the upper part of the sediments, regardless of the depth and geographical location. Hence, benthic environments, their organisms, and their ecosystems are highly variable as they encompass the full depth range of the oceans with associated changes in physical and chemical properties as well as differences linked to latitudinal and geographical variation. The effects of ocean acidification on the full range of different benthic organisms and ecosystems are poorly known and difficult to ascertain. Nevertheless, by integrating our current knowledge on the effects of ocean acidification on major benthic biogeochemical processes, individual benthic organisms, and observed characteristics of benthic environments as a function of seawater carbonate chemistry, it is possible to draw conclusions regarding the response of benthic organisms and ecosystems to a world of increasingly higher atmospheric CO2 levels. The fact that there are large-scale geographical and spatial differences in seawater carbonate system chemistry (see Chapter 3), owing to both natural and anthropogenic processes, provides a powerful means to evaluate the effect of ocean acidification on marine benthic systems. In addition, there are local and regional environments that experience high-CO2 and low-pH conditions owing to special circumstances such as, for example, volcanic vents (Hall- Spencer et al . 2008 ; Martin et al . 2008 ; Rodolfo-Metalpa et al . 2010), seasonal stratification (Andersson et al . 2007), and upwelling (Feely et al . 2008 ; Manzello et al . 2008 ) that may provide important clues to the impacts of ocean acidification on benthic processes, organisms, and ecosystems. The objective of this chapter is to provide an overview of the potential consequences of ocean acidification on marine benthic organisms, communities, and ecosystems, and the major biogeochemical processes governing the cycling of carbon in the marine benthic environment, including primary production, respiration, calcification, and CaCO3 dissolution. The depth of the euphotic zone, i.e. the depth of water exposed to sufficient sunlight to support photosynthesis, varies depending on a range of factors affecting the clarity of seawater, including river input and run-off to the coastal ocean, upwelling, mixing, and planktonic production.

High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common "benchtop" sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.

BackgroundDiatoms are present in all waters and are highly sensitive to pollution gradients. Therefore, they are ideal bioindicators for water quality assessment. Current indices used in these applications are based on identifying diatom species and counting their abundances using traditional light microscopy. Several molecular techniques have been developed to help automate different steps of this process, but obtaining reliable estimates of diatom community composition and species abundance remains challenging.ResultsHere, we evaluated a recently developed quantification method based on Genotyping by Sequencing (GBS) for the first time in diatoms to estimate the relative abundances within a species complex. For this purpose, a reference database comprised of thousands of genomic DNA clusters was generated from cultures of Nitzschia palea. The sequencing reads from calibration and mock samples were mapped against this database for parallel quantification. We sequenced 25 mock diatom communities containing up to five taxa per sample in different abundances. Taxon abundances in these communities were also quantified by a diatom expert using manual counting of cells on light microscopic slides. The relative abundances of strains across mock samples were over- or under-estimated by the manual counting method, and a majority of mock samples had stronger correlations using GBS. Moreover, one previously recognized putative hybrid had the largest number of false positive detections demonstrating the limitation of the manual counting method when morphologically similar and/or phylogenetically close taxa are analyzed.ConclusionsOur results suggest that GBS is a reliable method to estimate the relative abundances of the N. palea taxa analyzed in this study and outperformed traditional light microscopy in terms of accuracy. GBS provides increased taxonomic resolution compared to currently available quantitative molecular approaches, and it is more scalable in the number of species that can be analyzed in a single run. Hence, this is a significant step forward in developing automated, high-throughput molecular methods specifically designed for the quantification of [diatom] communities for freshwater quality assessments.

The present work, included in the European Project BIOWAT, aims to develop and validate the use of genomic tools or metabarcoding for its utilization as a routine method for river biomonitoring in different European Biogeographical regions. The project included sampling points in three biogeographic regions, Mediterranean (Spain), Continental (Germany) and Boreal (Finland). The current development of the study was designed using mock communities obtained from the three mentioned areas and different aspects were tested: DNA extraction methods, selection of informative region (16S vs COI), design and performance of primers, bioinformatic pipeline, etc…Although the use of COI has become very popular, and its barcode database is more complete, the use of mitochondrial 16S as taxonomic marker can provide similar or even better results when accompanied by a rich local barcode database Elbrecht 2016. In this presentation, the results and conclusions obtained for the biomonitoring of nine rivers (3 for each of the biogeographic regions) using 16S as DNA marker and a local barcode database are shown. The results of ecological status assignment using 16S marker were promising, showing a good correlation between morphological determinations and metabarcoding data for most of the studied rivers. However, in some cases, the metabarcoding data showed a jump in the ecological status class (to better or worst status), highlighting the existence of some problems related with primers (for some taxonomic groups) or missing taxa in the barcode database that still need to be solved prior the utilization of this method on a routine basis.Additionally, for the studied Mediterranean rivers, a complementary analysis using COI as marker was made, using the universal primers developed by Elbrecht and Leese 2017. In general, this marker showed better results in the identification for some taxa, whereas other included in the mock communities were not identified showing important problems that could be related with primers (sometimes not well covering characteristic taxa present in other biogeographic regions) or the lack of a complete COI macroinvertebrate barcode databases in the Iberian Peninsula for the case of Spain Múrria 2020.Our results show that both markers have the potential to produce a good identification of benthic macroinvertebrates, showing an acceptable correlation between morphology and metabarcoding approaches. However, none of them is able to amplify all of the present groups, so the parallel use of both markers (mitochondrial 16S and COI) in a multimarker approach could solve some of the problems, giving a more complete profile of the macroinvertebrate community. This approach has already been proposed and can lead the future of macroinvertebrate community assessment Ficetola 2020, Martins 2020.

Efforts to resolve Darwin's "abominable mystery"-the origin of angiosperms-have led to the conclusion that Gnetales and various fossil groups are sister to angiosperms, forming the "anthophytes." Morphological homologies, however, are difficult to interpret, and molecular data have not provided clear resolution of relationships among major groups of seed plants. We introduce two sequence data sets from slowly evolving mitochondrial genes, cox1 and atpA, which unambiguously reject the anthophyte hypothesis, favoring instead a close relationship between Gnetales and conifers. Parsimony- and likelihood-based analyses of plastid rbcL and nuclear 18S rDNA alone and with cox1 and atpA also strongly support a gnetophyte-conifer grouping. Surprisingly, three of four genes (all but nuclear rDNA) and combined three-genome analyses also suggest or strongly support Gnetales as derived conifers, sister to Pinaceae. Analyses with outgroups screened to avoid long branches consistently identify all gymnosperms as a monophyletic sister group to angiosperms. Combined three- and four-gene rooted analyses resolve the branching order for the remaining major groups-cycads separate from other gymnosperms first, followed by Ginkgo and then (Gnetales + Pinaceae) sister to a monophyletic group with all other conifer families. The molecular phylogeny strongly conflicts with current interpretations of seed plant morphology, and implies that many similarities between gnetophytes and angiosperms, such as "flower-like" reproductive structures and double fertilization, were independently derived, whereas other characters could emerge as synapomorphies for an expanded conifer group including Gnetales. An initial angiosperm-gymnosperm split implies a long stem lineage preceding the explosive Mesozoic radiation of flowering plants and suggests that angiosperm origins and homologies should be sought among extinct seed plant groups.

BackgroundSample storage conditions, extraction methods, PCR primers, and parameters are major factors that affect metagenomics analysis based on microbial 16S rRNA gene sequencing. Most published studies were limited to the comparison of only one or two types of these factors. Systematic multi-factor explorations are needed to evaluate the conditions that may impact validity of a microbiome analysis. This study was aimed to improve methodological options to facilitate the best technical approaches in the design of a microbiome study. Three readily available mock bacterial community materials and two commercial extraction techniques, Qiagen DNeasy and MO BIO PowerSoil DNA purification methods, were used to assess procedures for 16S ribosomal DNA amplification and pyrosequencing-based analysis. Primers were chosen for 16S rDNA quantitative PCR and amplification of region V3 to V1. Swabs spiked with mock bacterial community cells and clinical oropharyngeal swabs were incubated at respective temperatures of -80°C, -20°C, 4°C, and 37°C for 4 weeks, then extracted with the two methods, and subjected to pyrosequencing and taxonomic and statistical analyses to investigate microbiome profile stability.ResultsThe bacterial compositions for the mock community DNA samples determined in this study were consistent with the projected levels and agreed with the literature. The quantitation accuracy of abundances for several genera was improved with changes made to the standard Human Microbiome Project (HMP) procedure. The data for the samples purified with DNeasy and PowerSoil methods were statistically distinct; however, both results were reproducible and in good agreement with each other. The temperature effect on storage stability was investigated by using mock community cells and showed that the microbial community profiles were altered with the increase in incubation temperature. However, this phenomenon was not detected when clinical oropharyngeal swabs were used in the experiment.ConclusionsMock community materials originated from the HMP study are valuable controls in developing 16S metagenomics analysis procedures. Long-term exposure to a high temperature may introduce variation into analysis for oropharyngeal swabs, suggestive of storage at 4°C or lower. The observed variations due to sample storage temperature are in a similar range as the intrapersonal variability among different clinical oropharyngeal swab samples.

Molecular characterization of enteric coccidia from domestic ferrets (Mustela putorius furo).

Four species of myxomycetes (Arcyria pseudodenudata, Diderma europaeum, Lycogala irregulare, and Trichia armillata) new to China were observed via light microscope and scanning electron microscope, and detailed descriptions and illustrations are provided, along with comparisons with related species. Among them, A. pseudodenudata was discovered for the first time outside of the type locality, D. europaeum was discovered for the first time outside of Europe, and L. irregulare and T. armillata were reported again after being named. Phylogenetic analyses based on nuclear 18S rDNA and elongation factor-1 alpha sequences or nuclear 18S rDNA and cytochrome oxidase subunit I sequences was performed to provide a molecular basis for morphological identification. These specimens were deposited in the Herbarium of Fungi of Nanjing Normal University.

In the present study, the DNA barcoding of the nematode parasite infecting Ompok bimaculatus and Nemacheilus anguilla fish species was carried out in Barvi Reservoir, Maharashtra. To ascertain the taxonomic status of these nematode parasites, an 18S gene marker was used. Accurate identification of fish parasites is essential to formulate preventive strategies and to study host–environment relations. The present study did barcoding of the nematode parasites of the fishes caught from the Barvi Reservoir using the nuclear 18S rDNA (SSU) sequence. The nuclear 18S rDNA (SSU) was amplified into two overlapping amplicons and sequenced to identify the species based on the sequence similarity with the NCBI GenBank database. The present study sequences (both fragments) showed 98% similarity with the species of Eustrongylides. The average genetic distance value between the present study sample and species of Eustrongylides was 0.003. In the phylogenetic tree also, the sequence was clustered with the species of Eustrongylides with significant bootstrap values. The present study identified the nematode parasite of the fish caught from the Barvi Reservoir, as species of Eustrongylides. The species‐level identification could not be possible due to the insufficient/lack of reference sequences in the database. It indicates the knowledge gap concerning the species‐specific molecular markers for nematode parasites of the fish.