Ground-truthing Phylotype Assignments for Antarctic Invertebrates

AbstractBiodiversity information from Antarctic terrestrial habitats helps conservation efforts, but the distribution and diversity particularly of microinvertebrates remains poorly understood. Springtails, mites, tardigrades, nematodes and rotifers are difficult to identify using morphological features, hence DNA-based metabarcoding methods are well suited for their study. We compared taxonomy assignments of a high throughput sequencing metabarcoding approach using one ribosomal DNA (18S rDNA) and one mitochondrial DNA (cytochrome c oxidase subunit I - COI) marker with morphological reference data. Specifically, we compared metabarcoding or morphological taxonomic assignments on multiple taxonomic levels in an artificial DNA blend containing Australian invertebrates, and in seven extracts of Antarctic soils containing known micro-faunal taxa. Avoiding arbitrary application of metabarcoding analysis parameters, we calibrated those parameters with metabarcoding data from non-Antarctic soils. Metabarcoding approaches employing 18S rDNA and COI markers enabled detection of small and cryptic Antarctic invertebrates, and on low taxonomic ranks 18S data outperformed COI data in this respect. Morphological taxonomy determination did not outperform metabarcoding approaches. Our study demonstrates how barcoding markers can be tested prior to their application to specific taxonomic groups, and that taxonomy fidelity of markers needs to be validated in relation to environment, taxa, and available reference information.

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Genomic and Microscopic Analysis of Ballast Water in the Great Lakes Region

Applied Sciences ◽

10.3390/app9122441 ◽

2019 ◽

Vol 9 (12) ◽

pp. 2441 ◽

Cited By ~ 2

Author(s):

David A. Wright ◽

Carys L. Mitchelmore ◽

Allen Place ◽

Ernest Williams ◽

Celia Orano-Dawson

Keyword(s):

Invasive Species ◽

Great Lakes ◽

Ballast Water ◽

High Throughput Sequencing ◽

Microscopic Analysis ◽

Significant Advance ◽

Individual Organism ◽

Irreversible Effects ◽

Taxonomic Assignments ◽

Taxonomic Groups

Invasive aquatic species can have damaging effects on fisheries and aquaculture through significant, and irreversible, effects on biodiversity. Human health may also be affected. To combat this threat the International Maritime Organization (IMO) Convention for Ballast Water and Sediments (BWMC) came into force in September 2017. U.S. Federal and IMO ballast water standards for discharged organisms stipulate discharge limits for different size classes of organisms. Several studies including recent trials aboard Great Lakes freighters have shown that many phytoplankton found in ballast water do not fall into the regulated 10–50 µM size class. Such issues illustrate the need for new methods of assessing microorganism populations that will supersede laborious microscopy requiring rare technical expertise. Recent progress has been made in the use of DNA (deoxyribose nucleic acid)-based methods as a means of identifying the appearance of invasive species in aquatic environments. A significant advance has been the development of high throughput sequencing (HTS), which has expanded DNA barcoding, relating to an individual organism, into second generation sequencing (metabarcoding), capable of mapping whole populations of organisms in an environmental sample. Several recent studies of HTS in ships’ ballast water, have shown that the technique has the capacity for detecting potentially harmful taxonomic groups and is capable of differentiating among water from different sources. The current study was undertaken to investigate the suitability (or otherwise) of HTS as a tool for ballast water management. Possible applications include improved risk assessment relating to invasive species. Feasibility for indicative testing for ballast water treatment efficacy was also addressed. However, pending analysis of treated samples, the current study was confined to a comparison of HTS and microscope counts in untreated samples. A correlation of visual and molecular taxonomic assignments of microorganisms found in the ballast water from different ports and during different seasons indicated that such a comparison was best conducted at Family level, although Principal Components Analysis showed that the two methods differed qualitatively among major taxonomic groups.

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Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes

Applied and Environmental Microbiology ◽

10.1128/aem.00560-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4757-4766 ◽

Cited By ~ 55

Author(s):

Caterina R. Giner ◽

Irene Forn ◽

Sarah Romac ◽

Ramiro Logares ◽

Colomban de Vargas ◽

...

Keyword(s):

Microbial Diversity ◽

High Throughput ◽

Relative Abundance ◽

Ribosomal Dna ◽

18S Rdna ◽

High Throughput Sequencing ◽

454 Sequencing ◽

Epifluorescence Microscopy ◽

Environmental Sequencing

ABSTRACTHigh-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescencein situhybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances.IMPORTANCEDirect studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescencein situhybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed.

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Accurate Reconstruction of Microbial Strains from Metagenomic Sequencing Using Representative Reference Genomes

10.1101/215707 ◽

2017 ◽

Cited By ~ 2

Author(s):

Zhemin Zhou ◽

Nina Luhmann ◽

Nabil-Fareed Alikhan ◽

Christopher Quince ◽

Mark Achtman

Keyword(s):

Evaluation Studies ◽

Species Level ◽

Metagenomic Sequencing ◽

Sequencing Data ◽

Reference Databases ◽

Microbial Strains ◽

Taxonomic Assignments ◽

Taxonomic Groups ◽

Reference Genomes ◽

Recent Evaluation

AbstractExploring the genetic diversity of microbes within the environment through metagenomic sequencing first requires classifying these reads into taxonomic groups. Current methods compare these sequencing data with existing biased and limited reference databases. Several recent evaluation studies demonstrate that current methods either lack sufficient sensitivity for species-level assignments or suffer from false positives, overestimating the number of species in the metagenome. Both are especially problematic for the identification of low-abundance microbial species, e. g. detecting pathogens in ancient metagenomic samples. We present a new method, SPARSE, which improves taxonomic assignments of metagenomic reads. SPARSE balances existing biased reference databases by grouping reference genomes into similarity-based hierarchical clusters, implemented as an efficient incremental data structure. SPARSE assigns reads to these clusters using a probabilistic model, which specifically penalizes non-specific mappings of reads from unknown sources and hence reduces false-positive assignments. Our evaluation on simulated datasets from two recent evaluation studies demonstrated the improved precision of SPARSE in comparison to other methods for species-level classification. In a third simulation, our method successfully differentiated multiple co-existing Escherichia coli strains from the same sample. In real archaeological datasets, SPARSE identified ancient pathogens with ≤ 0.02% abundance, consistent with published findings that required additional sequencing data. In these datasets, other methods either missed targeted pathogens or reported non-existent ones. SPARSE and all evaluation scripts are available at https://github.com/zheminzhou/SPARSE.

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Contribution of Citizen Science to Biodiversity Data Mobilization in Russia

Biodiversity Information Science and Standards ◽

10.3897/biss.4.59197 ◽

2020 ◽

Vol 4 ◽

Author(s):

Natalya Ivanova ◽

Maxim Shashkov

Keyword(s):

New Species ◽

Protected Areas ◽

Citizen Science ◽

Biodiversity Data ◽

Breeding Bird ◽

Breeding Bird Atlas ◽

Research Grade ◽

Taxonomic Groups ◽

Species Occurrences ◽

Biodiversity Information

Currently Russia doesn't have a national biodiversity information system, and is still not a GBIF (Global Biodiversity Information Facility) member. Nevertheless, GBIF is the largest source of biodiversity data for Russia. As of August 2020, >5M species occurrences were available through the GBIF portal, of which 54% were published by Russian organisations. There are 107 institutions from Russia that have become GBIF publishers and 357 datasets have been published. The important trend of data mobilization in Russia is driven by the considerable contribution of citizen science. The most popular platform is iNaturalist. This year, the related GBIF dataset (Ueda 2020) became the largest one for Russia (793,049 species occurrences as of 2020-08-11). The first observation for Russia was posted in 2011, but iNaturalist started becoming popular in 2017. That year, 88 observers added >4500 observations that represented 1390 new species for Russia, 7- and 2-fold more respectively, than for the previous 6 years. Now we have nearly 12,000 observers, about 15,000 observed species and >1M research-grade observations. The ratio of observations for Tracheophyta, Chordata, and Arthropoda in Russia is different compared to the global scale. There are almost an equal amount of observations in the global iNaturalist GBIF dataset for these groups. At the same time in Russia, vascular plants make up 2/3rds of the observations. That is due to the "Flora of Russia" project, which attracted many professional botanists both as observers and experts. Thanks to their activity, Russia has a high proportion of research-grade observations in iNaturalist, 78% versus 60% globally. Another consequence of wide participation by professional researchers is the high rate of species accumulation. For some taxonomic groups conspicuous species were already revealed. There are about 850 bird species in Russia of which 398 species were observed in 2018, and only 83 new species in 2019. Currently, the number of new species recorded over time is decreasing despite the increase in observers and overall user activity. Russian iNaturalist observers have shared a lot of archive photos (taken during past years). In 2018, it was nearly 1/4 of the total number of observations and about 3/4 of new species for the year, with similar trends observed during 2019. Usually archive photos are posted from December until April, but the 2020 pandemic lockdown spurred a new wave of archive photo mobilisation in April and May. There are many iNaturalist projects for protected areas in Russia: 27 for strict nature reserves and national parks, and about 300 for others. About 100,000 observations (7.5% of all Russian observations) from the umbrella project "Protected areas of Russia" represent >34% of the species diversity observed in Russia. For some regions, e.g., Novosibirsk, Nizhniy Novgorod and Vladimir Oblasts, almost all protected areas are covered by iNaturalist projects, and are often their only source of available biodiversity data. There are also other popular citizen science platforms developed by Russian researchers. The first one is the Russian birdwatching network RU-BIRDS.RU. The related GBIF dataset (Ukolov et al. 2019) is the third largest dataset for Russia (>370,000 species occurrences). Another Russian citizen science system is wildlifemonitoring.ru, which includes thematic resources for different taxonomic groups of vertebrates. This is the crowd-sourced web-GIS maintained by the Siberian Environmental Center NGO in Novosibirsk. It is noteworthy that iNaturalist activities in Russia are developed more as a social network than as a way to attract volunteers to participate in scientific research. Of 746 citations in the iNaturalist dataset, only 18 articles include co-authors from Russia. iNaturalist data are used for the management of regional red lists (in the Republic of Bashkortostan, Novosibirsk Oblast and others), and as an additional information source for regional inventories. RU-BIRDS data were used in the European Russia Breeding Bird Atlas and the new edition of the European Breeding Bird Atlas. In Russia, citizen science activities significantly contribute to filling gaps in the global biodiversity map. However, Russian iNaturalist observations available through GBIF originate from the USA. It is not ideal, because the iNaturalist GBIF dataset is growing rapidly, and in the future it will represent more than all other datasets for Russia combined. In our opinion, iNaturalist data should be repatriated during the process of publishing through GBIF, as it is implemented for the eBird dataset (Levatich and Ligocki 2020).

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Metagenomic Characterization Reveals Pronounced Seasonality in the Diversity and Structure of the Phyllosphere Bacterial Community in a Mediterranean Ecosystem

Microorganisms ◽

10.3390/microorganisms7110518 ◽

2019 ◽

Vol 7 (11) ◽

pp. 518 ◽

Cited By ~ 2

Author(s):

Despoina Vokou ◽

Savvas Genitsaris ◽

Katerina Karamanoli ◽

Katerina Vareli ◽

Marina Zachari ◽

...

Keyword(s):

Bacterial Community ◽

High Throughput Sequencing ◽

Bacterial Colonization ◽

The Other ◽

Mediterranean Ecosystem ◽

Seasonal Differences ◽

Operational Taxonomic Units ◽

Seasonal Environment ◽

Habitat Specialists ◽

Taxonomic Groups

We explore how the phyllosphere microbial community responds to a very seasonal environment such as the Mediterranean. For this, we studied the epiphytic bacterial community of a Mediterranean ecosystem in summer and winter, expecting to detect seasonal differences at their maximum. With high-throughput sequencing (HTS), we detected the operational taxonomic units (OTUs) present in the phyllosphere and also in the surrounding air. The epiphytic community is approximately five orders of magnitude denser than the airborne one and is made almost exclusively by habitat specialists. The two communities differ considerably but Proteobacteria and Actinobacteria are dominant in both. Of the five most abundant phyllosphere OTUs, two were closely related to Sphingomonas strains, one to Methylobacterium and the other two to Rhizobiales and Burkholderiales. We found the epiphytic community to become much richer, more distinct, even and diverse, denser and more connected in summer. In contrast, there was no difference in the level of bacterial colonization of the phyllosphere between the two seasons, although there were seasonal differences for individual taxonomic groups: Firmicutes, Gemmatimonadetes and Chlroroflexi had a higher participation in summer, whereas the major Proteobacteria classes presented reverse patterns, with Betaproteobacteria increasing in summer at the expense of the prominent Alphaproteobacteria.

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Metabarcoding From Microbes to Mammals: Comprehensive Bioassessment on a Global Scale

Frontiers in Ecology and Evolution ◽

10.3389/fevo.2020.581835 ◽

2020 ◽

Vol 8 ◽

Cited By ~ 1

Author(s):

Zacchaeus G. Compson ◽

Beverly McClenaghan ◽

Gregory A. C. Singer ◽

Nicole A. Fahner ◽

Mehrdad Hajibabaei

Keyword(s):

High Throughput Sequencing ◽

Spatial Scales ◽

Individual Species ◽

Biodiversity Assessment ◽

Occupancy Models ◽

Widespread Adoption ◽

Sampling Protocols ◽

Dna Metabarcoding ◽

Global Biodiversity ◽

Taxonomic Groups

Global biodiversity loss is unprecedented, and threats to existing biodiversity are growing. Given pervasive global change, a major challenge facing resource managers is a lack of scalable tools to rapidly and consistently measure Earth's biodiversity. Environmental genomic tools provide some hope in the face of this crisis, and DNA metabarcoding, in particular, is a powerful approach for biodiversity assessment at large spatial scales. However, metabarcoding studies are variable in their taxonomic, temporal, or spatial scope, investigating individual species, specific taxonomic groups, or targeted communities at local or regional scales. With the advent of modern, ultra-high throughput sequencing platforms, conducting deep sequencing metabarcoding surveys with multiple DNA markers will enhance the breadth of biodiversity coverage, enabling comprehensive, rapid bioassessment of all the organisms in a sample. Here, we report on a systematic literature review of 1,563 articles published about DNA metabarcoding and summarize how this approach is rapidly revolutionizing global bioassessment efforts. Specifically, we quantify the stakeholders using DNA metabarcoding, the dominant applications of this technology, and the taxonomic groups assessed in these studies. We show that while DNA metabarcoding has reached global coverage, few studies deliver on its promise of near-comprehensive biodiversity assessment. We then outline how DNA metabarcoding can help us move toward real-time, global bioassessment, illustrating how different stakeholders could benefit from DNA metabarcoding. Next, we address barriers to widespread adoption of DNA metabarcoding, highlighting the need for standardized sampling protocols, experts and computational resources to handle the deluge of genomic data, and standardized, open-source bioinformatic pipelines. Finally, we explore how technological and scientific advances will realize the promise of total biodiversity assessment in a sample—from microbes to mammals—and unlock the rich information genomics exposes, opening new possibilities for merging whole-system DNA metabarcoding with (1) abundance and biomass quantification, (2) advanced modeling, such as species occupancy models, to improve species detection, (3) population genetics, (4) phylogenetics, and (5) food web and functional gene analysis. While many challenges need to be addressed to facilitate widespread adoption of environmental genomic approaches, concurrent scientific and technological advances will usher in methods to supplement existing bioassessment tools reliant on morphological and abiotic data. This expanded toolbox will help ensure that the best tool is used for the job and enable exciting integrative techniques that capitalize on multiple tools. Collectively, these new approaches will aid in addressing the global biodiversity crisis we now face.

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Comparison of Ileum Microflora of Pigs Fed Corn-, Wheat-, or Barley-Based Diets by Chaperonin-60 Sequencing and Quantitative PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.2.867-875.2005 ◽

2005 ◽

Vol 71 (2) ◽

pp. 867-875 ◽

Cited By ~ 71

Author(s):

Janet E. Hill ◽

Sean M. Hemmingsen ◽

Blair G. Goldade ◽

Tim J. Dumonceaux ◽

Jonathan Klassen ◽

...

Keyword(s):

Quantitative Pcr ◽

High Throughput Sequencing ◽

Pcr Analysis ◽

Reference Database ◽

Pcr Product ◽

Culture Independent ◽

Chaperonin 60 ◽

Small Intestinal ◽

Primer Sets ◽

Taxonomic Assignments

ABSTRACT We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.

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Consortia of anti-nematode fungi and bacteria in the rhizosphere of soybean plants attacked by root-knot nematodes

10.1101/332403 ◽

2018 ◽

Author(s):

Hirokazu Toju ◽

Yu Tanaka

Keyword(s):

Crop Production ◽

High Throughput Sequencing ◽

Infected Plant ◽

Agricultural Practices ◽

Root Knot Nematodes ◽

Indigenous Resident ◽

Agroecosystem Management ◽

Soybean Plants ◽

Bacteria And Fungi ◽

Taxonomic Groups

AbstractCyst and root-knot nematodes are major risk factors of agroecosystem management, often causing devastating impacts on crop production. The use of microbes that parasitize or prey on nematodes has been considered as a promising approach for suppressing phytopathogenic nematode populations. However, as effects and persistence of those biological control agents often vary substantially depending on regions, soil characteristics, and agricultural practices, more insights into microbial community processes are required to develop reproducible control of nematode populations. By performing high-throughput sequencing profiling of bacteria and fungi, we examined how root and soil microbiomes differ between benign and nematode-infected plant individuals in a soybean field in Japan. Results indicated that various taxonomic groups of bacteria and fungi occurred preferentially on the soybean individuals infected by root-knot nematodes. Based on a network analysis of potential microbe–microbe associations, we further found that several fungal taxa potentially preying on nematodes [Dactylellina (Orbiliales), Rhizophydium (Rhizophydiales), Clonostachys (Hypocreales), Pochonia (Hypocreales), and Purpureocillium (Hypocreales)] co-occurred in the soybean rhizosphere at a small spatial scale. Overall, this study suggests how “consortia” of anti-nematode microbes can derive from indigenous (resident) microbiomes, thereby providing basic information for managing anti-nematode microbial communities in agroecosystems.

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Biodiversity Information Services: A (not-so-) little knowledge that acts

Biodiversity Information Science and Standards ◽

10.3897/biss.2.25738 ◽

2018 ◽

Vol 2 ◽

pp. e25738 ◽

Cited By ~ 1

Author(s):

Arturo Ariño ◽

Daniel Noesgaard ◽

Angel Hjarding ◽

Dmitry Schigel

Keyword(s):

Critical Mass ◽

The Body ◽

Global Biodiversity Information Facility ◽

Entire List ◽

Darwin Core ◽

Biogeographical Regions ◽

Continuous Presence ◽

Set Up ◽

Taxonomic Groups ◽

Biodiversity Information

Standards set up by Biodiversity Information Standards-Taxonomic Databases Working Group (TDWG), initially developed as a way to share taxonomical data, greatly facilitated the establishment of the Global Biodiversity Information Facility (GBIF) as the largest index to digitally-accessible primary biodiversity information records (PBR) held by many institutions around the world. The level of detail and coverage of the body of standards that later became the Darwin Core terms enabled increasingly precise retrieval of relevant records useful for increased digitally-accessible knowledge (DAK) which, in turn, may have helped to solve ecologically-relevant questions. After more than a decade of data accrual and release, an increasing number of papers and reports are citing GBIF either as a source of data or as a pointer to the original datasets. GBIF has curated a list of over 5,000 citations that were examined for contents, and to which tags were applied describing such contents as additional keywords. The list now provides a window on what users want to accomplish using such DAK. We performed a preliminary word frequency analysis of this literature, starting at titles, which refers to GBIF as a resource. Through a standardization and mapping of terms, we examined how the facility-enabled data seem to have been used by scientists and other practitioners through time: what concepts/issues are pervasive, which taxon groups are mostly addressed, and whether data concentrate around specific geographical or biogeographical regions. We hoped to cast light on which types of ecological problems the community believes are amenable to study through the judicious use of this data commons and found that, indeed, a few themes were distinctly more frequently mentioned than others. Among those, generally-perceived issues such as climate change and its effect on biodiversity at global and regional scales seemed prevalent. The taxonomic groups were also unevenly mentioned, with birds and plants being the most frequently named. However, the entire list of potential subjects that might have used GBIF-enabled data is now quite wide, showing that the availability of well-structured data has spawned a widening spectrum of possible use cases. Among them, some enjoy early and continuous presence (e.g. species, biodiversity, climate) while others have started to show up only later, once a critical mass of data seemed to have been attained (e.g. ecosystems, suitability, endemism). Biodiversity information in the form of standards-compliant DAK may thus already have become a commodity enabling insight into an increasingly more complex and diverse body of science. Paraphrasing Tennyson, more things were wrought by data than TDWG dreamt of.

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Spatial diversity of planktonic protists in the Lagoon of Venice (LTER-Italy) based on 18S rDNA

Advances in Oceanography and Limnology ◽

10.4081/aiol.2020.8961 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Simona Armeli Minicante ◽

Roberta Piredda ◽

Stefania Finotto ◽

Fabrizio Bernardi Aubry ◽

Francesco Acri ◽

...

Keyword(s):

18S Rdna ◽

High Throughput Sequencing ◽

Sampling Period ◽

Spatial Diversity ◽

Lagoon Of Venice ◽

Space And Time ◽

Α Diversity ◽

Spatial Differences ◽

Diatom Blooms ◽

High Degree

Transitional waters are subject to a high degree of variability in space and time. In this study, protist plankton communities of the Lagoon of Venice were compared among four sites characterised by different environmental conditions with a metabarcoding approach. High throughput sequencing (HTS) of the V4-18S rDNA fragment in 32 samples collected on four dates, from April 2016 to February 2017, produced 1,137,113 reads, which were grouped into 4,058 OTUs at 97% similarity. Bacillariophyta and Ciliophora were the most abundant groups in the entire dataset in terms of read number (27.6% and 16.6%, respectively), followed by Dinophyta (10.9%), Cryptophyceae (9.7%), and Syndiniales (6.1%). The contribution of protist groups markedly varied across the seasons, but spatial differences were also recorded in the lagoon. In April, a higher contribution of Bacillariophyta characterized St1 and 5 (68.0% and 61.1%), whereas Sts2 and 3 showed a higher percentage of Ciliophora (18.6 and 23.4%, respectively) and dinoflagellates (10.3 and 7.7%). In July, diatom blooms occurred at Sts1, 2 and 3, with some differences in the dominant species. At St2 Dinophyta reached the highest contribution of the whole sampling period in the area (30.6%), while St5 was quite distinct, with a low contribution of diatoms and a dominance of Ciliophora (34.0%) and Trebouxiophyceae (36.4%). All the stations in November were characterized by relatively high abundance of Ciliophora (21.4-51.9%). In February, diatom contribution was relevant only at St5 (29.3%), Teleaulax acuta peaked at St3 (ca. 36%), Syndiniales at St2 (38.8%) and Dictyochophyceae at St1 (24.2%). The α-diversity indexes (observed OTUs, Shannon and Pielou evenness) showed a high variability over space and time. Overall, diversity and community composition were rather similar between the intermediate and deeper Sts2 and 3 on all sampling dates whereas they at time differed between the landward and shallow Sts1 and 5. While the most marked differences occurred over the temporal scale, the depth of the station and the relatedness with the external marine coastal environment appear to play a major role in the spatial distribution of protist communities within the lagoon.

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