An account of epiphytic diatoms in mediterranean wetlands: comparing morphological and molecular assessments

Benthic diatoms are well known bioindicators of water quality, used in many aquatic ecosystems. Since diatom-based monitoring of water quality is required by European legislation, the search for methods that facilitate this task has become more relevant. The aim of this study was to test the reliability of DNA metabarcoding combined with high-throughput sequencing (HTS) techniques in the bioassessment of 22 Mediterranean shallow ponds in Spain. For each pond, Trophic Diatom Index (TDI) was calculated from inventories obtained by using light microscopy, and then molecular (HTS) methods. Ponds were subsequently classified into five water quality classes. Our results showed a good correspondence between both methods, especially after applying a correction factor depending on the biovolume of the cells. This correspondence led to the assignment to the same quality class in 59% of the ponds. The determination and quantification of valves or DNA sequences was one of the main pitfalls, mainly those related to the variability in the relative abundances of some species. Accordingly, ponds with similar relative abundances for the dominant species were assigned to the same quality class. Moreover, other difficulties leading the discrepancies were the misidentification of some species due to the presence of semi-cryptic taxa, the incompleteness of the reference database and the bioinformatic protocol. Therefore, the validation of DNA-based methods for the identification of freshwater diatoms represents an important goal, as an alternative to traditional ones in Mediterranean shallow ponds.

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Epiphytic Diatom-Based Biomonitoring in Mediterranean Ponds: Traditional Microscopy versus Metabarcoding Approaches

Water ◽

10.3390/w13101351 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1351

Author(s):

María Borrego-Ramos ◽

Eloy Bécares ◽

Pedro García ◽

Alejandro Nistal ◽

Saúl Blanco

Keyword(s):

Water Quality ◽

Light Microscopy ◽

High Throughput ◽

Dna Sequences ◽

High Throughput Sequencing ◽

Reference Database ◽

Morphological Identification ◽

Quality Class ◽

Shallow Ponds ◽

Relative Abundances

Benthic diatoms have traditionally been used as bioindicators of aquatic ecosystems. Because diatom-based monitoring of water quality is required by European legislation, molecular-based methods had emerged as useful alternatives to classical methods based on morphological identification using light microscopy. The aim of this study was to test the reliability of DNA metabarcoding combined with High-Throughput Sequencing (HTS) techniques in the bioassessment of the trophic status of 22 Mediterranean shallow ponds in NW Spain. For each pond, the Trophic Diatom Index (TDI) was calculated from inventories obtained by identification using light microscopy (LM) followed by high-throughput sequencing (HTS) at the molecular level. Ponds were subsequently classified into five water quality classes. The results showed a good correspondence between both methods, especially after applying a correction factor that depended on the biovolume of the cells. This correspondence led to the assignment to the same quality class in 59% of the ponds. The determination and quantification of valves or DNA sequences was one of the main pitfalls, which mainly included those related to the variability in the relative abundances of some species. Accordingly, ponds with similar relative abundances for the dominant species were assigned to the same quality class. Moreover, other difficulties leading the discrepancies were the misidentification of some species due to the presence of semi-cryptic taxa, the incompleteness of the reference database and the bioinformatic protocol. Thus, the validation of DNA-based methods for the identification of freshwater diatoms represents an important goal, as an alternative to using traditional methods in Mediterranean shallow ponds.

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Field-based species identification in eukaryotes using real-time nanopore sequencing

10.1101/107656 ◽

2017 ◽

Cited By ~ 1

Author(s):

Joe Parker ◽

Andrew J. Helmstetter ◽

Dion Devey ◽

Alexander S.T. Papadopulos

Keyword(s):

Real Time ◽

Species Identification ◽

Dna Sequences ◽

High Throughput Sequencing ◽

De Novo ◽

Reference Database ◽

Nanopore Sequencing ◽

Technological Advances ◽

A Genome ◽

Hybrid Genome

Advances in DNA sequencing and informatics have revolutionised biology over the past four decades, but technological limitations have left many applications unexplored1,2. Recently, portable, real-time, nanopore sequencing (RTnS) has become available. This offers opportunities to rapidly collect and analyse genomic data anywhere3–5. However, the generation of datasets from large, complex genomes has been constrained to laboratories6,7. The portability and long DNA sequences of RTnS offer great potential for field-based species identification, but the feasibility and accuracy of these technologies for this purpose have not been assessed. Here, we show that a field-based RTnS analysis of closely-related plant species (Arabidopsis spp.)8 has many advantages over laboratory-based high-throughput sequencing (HTS) methods for species level identification-by-sequencing and de novo phylogenomics. Samples were collected and sequenced in a single day by RTnS using a portable, “al fresco” laboratory. Our analyses demonstrate that correctly identifying unknown reads from matches to a reference database with RTnS reads enables rapid and confident species identification. Individually annotated RTnS reads can be used to infer the evolutionary relationships of A. thaliana. Furthermore, hybrid genome assembly with RTnS and HTS reads substantially improved upon a genome assembled from HTS reads alone. Field-based RTnS makes real-time, rapid specimen identification and genome wide analyses possible. These technological advances are set to revolutionise research in the biological sciences9 and have broad implications for conservation, taxonomy, border agencies and citizen science.

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DNA metabarcoding and morphological methods show complementary patterns in the metacommunity organization of lentic epiphytic diatoms

ARPHA Conference Abstracts ◽

10.3897/aca.4.e63672 ◽

2021 ◽

Vol 4 ◽

Author(s):

Alejandro Nistal ◽

Pedro Garcia ◽

Jorge Garcia ◽

María Borrego ◽

Saúl Blanco ◽

...

Keyword(s):

Community Composition ◽

High Throughput Sequencing ◽

Freshwater Ecosystems ◽

Reference Database ◽

Compositional Variation ◽

Diatom Community ◽

Morphological Method ◽

Dna Metabarcoding ◽

Geographical Distances ◽

Diatom Community Composition

Diatoms are important organisms in freshwater ecosystems due to their position as primary producers and therefore, analyzing their communities provides relevant information on ecosystem functioning. Diatoms have historically been identified based on morphological traits, which is time-consuming and requires well-trained specialists. Nevertheless, DNA barcoding offers an alternative approach to overcome some limitations of the morphological method. Here, we assess if both approaches are comparable methods to study patterns and mechanisms (including environmental filtering and dispersal limitation) of epiphytic diatom metacommunities using a comprehensive dataset from 22 Mediterranean ponds at different taxonomic resolutions. We used a fragment of rbcL barcode gene combined with High-Throughput Sequencing to infer diatom community composition. The overall degree of correspondence between both approaches was assessed by Procrustean rotation analysis and Procrustean randomization tests, whereas the role of local environmental variables and geographical distances was studied using a comprehensive combination of BIOENV, Mantel tests and distance-based redundancy analysis. Our results showed a relatively poor correspondence in the compositional variation of diatom metacommunity between both approaches. We speculate that the incompleteness of the reference database and the bioinformatics processing are the biases most likely affecting the molecular approach, whereas the limited counting effort and the presence of cryptic species are presumably the major biases related with the morphological method. On the other hand, variation in diatom community composition detected with both approaches was strongly related to the environmental template, which may be related with the narrow ecological niche and the strong preferences for particular substrata of some diatom species. Nevertheless, we found no significant relationship between compositional variation and geographical distances at regional extent. Overall, our work highlights the importance of DNA metabarcoding to address empirical research questions of community ecology in freshwaters, especially once the reference databases include most genotypes of occurring taxa and bioinformatics biases are overcome.

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Can DNA-based ecosystem assessments quantify species abundance? Testing primer bias and biomass - sequence relationships with an innovative metabarcoding protocol

10.7287/peerj.preprints.1023v1 ◽

2015 ◽

Cited By ~ 1

Author(s):

Vasco Elbrecht ◽

Florian Leese

Keyword(s):

High Throughput ◽

Statistical Power ◽

High Throughput Sequencing ◽

Species Abundance ◽

Reference Database ◽

Dna Metabarcoding ◽

Coi Barcoding ◽

Species Abundances ◽

Species Specific ◽

Sequence Relationships

Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity should rely on presence-absence metrics.

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Issues of under-representation in quantitative DNA metabarcoding weaken the inference about diet of the tundra vole Microtus oeconomus

PeerJ ◽

10.7717/peerj.11936 ◽

2021 ◽

Vol 9 ◽

pp. e11936

Author(s):

Magne Neby ◽

Stefaniya Kamenova ◽

Olivier Devineau ◽

Rolf A. Ims ◽

Eeva M. Soininen

Keyword(s):

High Throughput ◽

Dna Sequences ◽

High Throughput Sequencing ◽

Plant Biomass ◽

Quantitative Relationship ◽

Food Plants ◽

Small Rodents ◽

Microtus Oeconomus ◽

Dna Metabarcoding ◽

Tundra Vole

During the last decade, methods based on high-throughput sequencing such as DNA metabarcoding have opened up for a range of new questions in animal dietary studies. One of the major advantages of dietary metabarcoding resides in the potential to infer a quantitative relationship between sequence read proportions and biomass of ingested food. However, this relationship’s robustness is highly dependent on the system under study, calling for case-specific assessments. Herbivorous small rodents often play important roles in the ecosystem, and the use of DNA metabarcoding for analyses of rodent diets is increasing. However, there has been no direct validation of the quantitative reliability of DNA metabarcoding for small rodents. Therefore, we used an experimental approach to assess the relationship between input plant biomass and sequence reads proportions from DNA metabarcoding in the tundra vole Microtus oeconomus. We found a weakly positive relationship between the number of high-throughput DNA sequences and the expected biomass proportions of food plants. The weak relationship was possibly caused by a systematic under-amplification of one of the three plant taxa fed. Generally, our results add to the growing evidence that case-specific validation studies are required to reliably make use of sequence read abundance as a proxy of relative food proportions in the diet.

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Can DNA-based ecosystem assessments quantify species abundance? Testing primer bias and biomass - sequence relationships with an innovative metabarcoding protocol

10.7287/peerj.preprints.1023 ◽

2015 ◽

Author(s):

Vasco Elbrecht ◽

Florian Leese

Keyword(s):

High Throughput ◽

Statistical Power ◽

High Throughput Sequencing ◽

Species Abundance ◽

Reference Database ◽

Dna Metabarcoding ◽

Coi Barcoding ◽

Species Abundances ◽

Species Specific ◽

Sequence Relationships

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Toward a global reference database of COI barcodes for marine zooplankton

Marine Biology ◽

10.1007/s00227-021-03887-y ◽

2021 ◽

Vol 168 (6) ◽

Author(s):

Ann Bucklin ◽

Katja T. C. A. Peijnenburg ◽

Ksenia N. Kosobokova ◽

Todd D. O’Brien ◽

Leocadio Blanco-Bercial ◽

...

Keyword(s):

Species Diversity ◽

Dna Sequences ◽

Reference Sequence ◽

Global Ocean ◽

Reference Database ◽

Data Repositories ◽

Marine Zooplankton ◽

The North ◽

Coi Sequences ◽

Taxonomic Groups

AbstractCharacterization of species diversity of zooplankton is key to understanding, assessing, and predicting the function and future of pelagic ecosystems throughout the global ocean. The marine zooplankton assemblage, including only metazoans, is highly diverse and taxonomically complex, with an estimated ~28,000 species of 41 major taxonomic groups. This review provides a comprehensive summary of DNA sequences for the barcode region of mitochondrial cytochrome oxidase I (COI) for identified specimens. The foundation of this summary is the MetaZooGene Barcode Atlas and Database (MZGdb), a new open-access data and metadata portal that is linked to NCBI GenBank and BOLD data repositories. The MZGdb provides enhanced quality control and tools for assembling COI reference sequence databases that are specific to selected taxonomic groups and/or ocean regions, with associated metadata (e.g., collection georeferencing, verification of species identification, molecular protocols), and tools for statistical analysis, mapping, and visualization. To date, over 150,000 COI sequences for ~ 5600 described species of marine metazoan plankton (including holo- and meroplankton) are available via the MZGdb portal. This review uses the MZGdb as a resource for summaries of COI barcode data and metadata for important taxonomic groups of marine zooplankton and selected regions, including the North Atlantic, Arctic, North Pacific, and Southern Oceans. The MZGdb is designed to provide a foundation for analysis of species diversity of marine zooplankton based on DNA barcoding and metabarcoding for assessment of marine ecosystems and rapid detection of the impacts of climate change.

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Gut Microbiome Changes in Captive Plateau Zokors (Eospalax baileyi)

Evolutionary Bioinformatics ◽

10.1177/1176934321996353 ◽

2021 ◽

Vol 17 ◽

pp. 117693432199635

Author(s):

Daoxin Liu ◽

Pengfei Song ◽

Jingyan Yan ◽

Haijing Wang ◽

Zhenyuan Cai ◽

...

Keyword(s):

Gut Microbiota ◽

Gut Microbiome ◽

High Throughput Sequencing ◽

Tibet Plateau ◽

Rrna Gene ◽

Diversity Analysis ◽

Bacterial Phyla ◽

In Captivity ◽

Relative Abundances ◽

Plateau Zokor

Wild-caught animals must cope with drastic lifestyle and dietary changes after being induced to captivity. How the gut microbiome structure of these animals will change in response receives increasing attention. The plateau zokor ( Eospalax baileyi), a typic subterranean rodent endemic to the Qinghai-Tibet plateau, spends almost the whole life underground and is well adapted to the environmental pressures of both plateau and underground. However, how the gut microbiome of the plateau zokor will change in response to captivity has not been reported to date. This study compared the microbial community structure and functions of 22 plateau zokors before (the WS group) and after being kept in captivity for 15 days (the LS group, fed on carrots) using the 16S rRNA gene via high-throughput sequencing technology. The results showed that the LS group retained 973 of the 977 operational taxonomic units (OTUs) in the WS group, and no new OTUs were found in the LS group. The dominant bacterial phyla were Bacteroides and Firmicutes in both groups. In alpha diversity analysis, the Shannon, Sobs, and ACE indexes of the LS group were significantly lower than those of the WS group. A remarkable difference ( P < 0.01) between groups was also detected in beta diversity analysis. The UPGMA clustering, NMDS, PCoA, and Anosim results all showed that the intergroup difference was significantly greater than the intragroup difference. And compared with the WS group, the intragroup difference of the gut microbiota in the LS group was much larger, which failed to support the assumption that similar diets should drive convergence of gut microbial communities. PICRUSt revealed that although some functional categories displayed significant differences between groups, the relative abundances of these categories were very close in both groups. Based on all the results, we conclude that as plateau zokors enter captivity for a short time, although the relative abundances of different gut microbiota categories shifted significantly, they can maintain almost all the OTUs and the functions of the gut microbiota in the wild. So, the use of wild-caught plateau zokors in gut microbial studies is acceptable if the time in captivity is short.

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Periphyton diversity in two different Antarctic lakes assessed using metabarcoding

Antarctic Science ◽

10.1017/s0954102021000316 ◽

2021 ◽

pp. 1-9

Author(s):

Paulo E.A.S. Câmara ◽

Láuren M.D. De Souza ◽

Otávio Henrique Bezerra Pinto ◽

Peter Convey ◽

Eduardo T. Amorim ◽

...

Keyword(s):

Dna Sequences ◽

High Throughput Sequencing ◽

Shannon Index ◽

South Shetland Islands ◽

King George Island ◽

Deception Island ◽

Maritime Antarctic ◽

Shetland Islands ◽

Antarctic Lakes ◽

Significant Difference

Abstract Antarctic lakes have generally simple periphyton communities when compared with those of lower latitudes. To date, assessment of microbial diversity in Antarctica has relied heavily on traditional direct observation and cultivation methods. In this study, sterilized cotton baits were left submerged for two years in two lakes on King George Island and Deception Island, South Shetland Islands (Maritime Antarctic), followed by assessment of diversity by metabarcoding using high-throughput sequencing. DNA sequences of 44 taxa belonging to four kingdoms and seven phyla were found. Thirty-six taxa were detected in Hennequin Lake on King George Island and 20 taxa were detected in Soto Lake on Deception Island. However, no significant difference in species composition was detected between the two assemblages (Shannon index). Our data suggest that metabarcoding provides a suitable method for the assessment of periphyton biodiversity in oligotrophic Antarctic lakes.

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The Relationship between the Community Structure and Function of Bacterioplankton and the Environmental Response in Qingcaosha Reservoir

Water ◽

10.3390/w13223155 ◽

2021 ◽

Vol 13 (22) ◽

pp. 3155

Author(s):

Shumin Liu ◽

Fengbin Zhao ◽

Xin Fang

Keyword(s):

Water Quality ◽

Community Structure ◽

High Throughput Sequencing ◽

Scientific Basis ◽

Vital Role ◽

Structure And Function ◽

Environmental Response ◽

Temporal And Spatial ◽

Density And Biomass ◽

And Function

Phytoplankton and bacterioplankton play a vital role in the structure and function of aquatic ecosystems, and their activity is closely linked to water eutrophication. However, few researchers have considered the temporal and spatial succession of phytoplankton and bacterioplankton, and their responses to environmental factors. The temporal and spatial succession of bacterioplankton and their ecological interaction with phytoplankton and water quality were analyzed using 16S rDNA high-throughput sequencing for their identification, and the functions of bacterioplankton were predicted. The results showed that the dominant classes of bacterioplankton in the Qingcaosha Reservoir were Gammaproteobacteria, Alphaproteobacteria, Actinomycetes, Acidimicrobiia, and Cyanobacteria. In addition, the Shannon diversity indexes were compared, and the results showed significant temporal differences based on monthly averaged value, although no significant spatial difference. The community structure was found to be mainly influenced by phytoplankton density and biomass, dissolved oxygen, and electrical conductivity. The presence of Pseudomonas and Legionella was positively correlated with that of Pseudanabaena sp., and Sphingomonas and Paragonimus with Melosira granulata. On the contrary, the presence of Planctomycetes was negatively correlated with Melosira granulata, as was Deinococcus-Thermus with Cyclotella sp. The relative abundance of denitrifying bacteria decreased from April to December, while the abundance of nitrogen-fixing bacteria increased. This study provides a scientific basis for understanding the ecological interactions between bacteria, algae, and water quality in reservoir ecosystems.

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