DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

Cryptic diversity and population structure at small scales: the freshwater snail

Anthropogenic impacts like intensified land use and climate change are severe threats to freshwater biodiversity and effective biodiversity monitoring is therefore one of the most urgent tasks. This is, however, often hampered by the lack of knowledge regarding the number and ecology of species. Molecular tools have shown many freshwater taxa to comprise morphologically cryptic species, which often occur in sympatry on a small geographic scale. Here, we studied the freshwater snail Ancylus fluviatilis (Müller, 1774) species complex in the Iberian Montseny Mountains. We hypothesized that (1) several species of A. fluviatilis sensu lato occur in the Montseny and (2) the different species seldom co-occur in syntopy due to different ecological demands or interspecific competition. We barcoded 180 specimens from 36 sites in the Montseny for the cytochrome c oxidase subunit I (COI) barcoding gene and molecularly identified two Ancylus species. These species seldom occurred in syntopy and a species distribution modelling approach showed differing bioclimatic preferences of the species. One species occurs mainly in cooler, higher altitude streams while the second species occurs in lower-altitude areas with higher temperatures. Tests of population structure showed that both species possibly do not disperse well in the study area and that populations within species might be adapted to certain bioclimatic conditions in different regions of the Montseny. Our results highlight the need to incorporate molecular techniques into routine monitoring programmes.The freshwater limpet Ancylus fluviatilis is a known species complex. We applied COI barcoding and found two molecular clades on a small geographic scale. Their occurrence corresponds to bioclimatic conditions. We propose implementing molecular methods into diversity assessments on a regular basis

Exploring Pandora's Box: potential and pitfalls of low coverage genome surveys for evolutionary biology

High throughput sequencing technologies are revolutionizing genetic research. With this ‘‘rise of the machines’’, genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02–25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers

Characteristics of contigs with homology to viral protein sequences in the libraries.

<p>Characteristics of contigs with homology to viral protein sequences in the libraries.</p

Hits of nuclear genes against KEGG BRITE Ontology database using the KAAS pipeline for the 16 genomic libraries.

<p>The number of hits is listed below the species name. Colours assigned according to the highest level of KEGG Orthology hierarchy (different organismal/cellular pathway groups/ecosystem processes).</p

Correlation between genomic library size (y-axis) and total length of mitochondrial genome recovered (x-axis).

<p>A significantly positive linear correlation (Pearson r = 0.6049, P = 0.0219) between the number of base pairs sequenced and the proportion of the mitochondrial genome recovered was found.</p

Bacterial hits found in four genomic libraries.

<p>Hits for the bacterial species are displayed next to the chart pie for species with ≥4% of the hits. For the three pycnogonid species, Gammaproteobacteria are predominant, whereas for the vent limpet <i>Lepetodrilus</i> sp. nov. different bacterial groups were detected. The colours of the charts relate to the phyla/classes of Bacteria (see legend).</p

Characteristics of contigs with homology to known transposable elements in the libraries.

<p>Characteristics of contigs with homology to known transposable elements in the libraries.</p

Coverage estimations for the sequenced genomic libraries based upon genome size information of closely related taxa found in the Animal Genome Size database.

*<p>Information on genome size of <i>Euphausia superba</i> is based upon the flow-cytometry estimates listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049202#pone.0049202-Jeffery1" target="_blank">[46]</a>.</p