R-Syst::diatom a reference library for diatoms: overview, uses and perspectives

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1 R-Syst::diatom a reference library for diatoms: overview, uses and perspectives F. Rimet, V. Vasselon, K. Tapolczai, A. Bouchez INRA, UMR Carrtel, 75 av. de Corzent, BP 511, F Thonon les Bains cedex, France coll. J.M. Frigerio, P. Chaumeil & A. Franc INRA, UMR BioGeCo, 69, route d'arcachon ; Pierroton ; Cestas Cedex, France Essen Cost DNAquanet march 2017

2 R-syst French network of systematic: A. Franc Virus Bacteria Fungi Plants Insects Nematods Microalgae > diatoms R-Syst::diatom database.02

3 R-syst R-Syst::diatom database Website: P. Chaumeil (coll. A. Franc) Objectives: Link: DNA-barcodes species names metadata Two markers: 18s, rbcl (Zimmermann et al. 2011, Kermarrec et al. 2013) Curation of the data Open access Maintained since 2012 Published in

4 R-syst Open access:

5 R-syst Open access:

6 R-syst Open access:

7 R-syst Open access:

8 R-syst Open access: Quality indices Life-forms Functional groups Sizes, biovolumes, cellule shapes.08

9 R-syst Open access:

10 R-syst Open access: Download.010

11 R-syst Open access:

12 R-syst: curation Where do the data come from? Collection: TCC (Thonon Culture Collection) Every 6 months rbcl + 18s NCBI Innovative algorithms (A. Franc) Curation procedure Phylogenetic tools R-Syst::diatom database 5617 barcodes (18s, rbcl).012

13 R-syst: curation Curation? Homogenization of taxonomic identifications for a given clade Why? Identification skills of authors (who deposited in NCBI) of the barcodes are different Taxonomy evolve from a year to another Length and quality of sequences can be not adapted for correct taxonomic affiliation.013

14 R-syst: curation Curation > Formerly one genus > Splitted into 7 different genera > Based on morphological criteria visible in SEM > Paraphyletic genera Impossible to detect this genus in metabarcoding.014

15 R-syst: curation Curation >Important genus for ecological indication: >Shallow turbulent lentic ecosystem Small unattached filaments, resist to turbulence.015

16 R-syst: curation Curation: Important step to have libraries usable for metabarcoding: algorithms need monophyletic taxa Cf. Bold is not correctly curated taxonomically (for diatoms) = NCBI + metadata Curation must be supervised by a few experienced people.016

17 R-syst: completness? Estimation: of extant species (Mann & Vanormelingen 2013) About diatom species described R-Syst::diatom: 1250 species Chiers in Longlaville Polluted river Microscope 8% 1 92% 12 Barcoding 10 L. Kermarrec et al. Metamatch + R-Syst::diatom.017

18 R-syst: completness? About diatom species described Estimation: of extant species (Mann & Vanormelingen 2013) R-Syst::diatom: 1250 species Lake Bourget littoral sampling 2015 Microscope 78% 94 22% 26 Barcoding 46 2 km Very good Good Moderate S. Riviera, V. Vasselon Mothur + R-Syst::diatom.018

19 R-syst: completness? About diatom species described Estimation: of extant species (Mann & Vanormelingen 2013) R-Syst::diatom: 1250 species Shell scraping of sea turtle Chelonia mydas Microscope 95% 54 5% 3 Barcoding 23 Mayotte Island V. Vasselon, F. Rimet Mothur + R-Syst::diatom.019

20 R-syst: how can we complete the library? Cell isolation, cultures: Classical method until now, Tedious Single cell PCR (Hamilton et al. 2015): Identification of living cell Many mistakes Cloning-sequencing of natural samples (Khan-Bureau et al. 2016) Long sequences of good quality Expensive Can be done on samples with low diversities.020

21 R-syst: how can we complete the library? Use of HTS sequences Select samples with target species never sequenced Criteria: - sequences must be abundant - rbcl (coding): no indel, no stop codon - Phylogenetic checkout F. RIMET, N. ABARCA, A. BOUCHEZ, R. JAHN, M. KAHLERT, F. KECK, M.G. KELLY, D.G. KP Amphora.commutata KM Amphora.ovalis MANN, A. PIUZ, R. TROBAJO, K. TAPOLCZAI, V. VASSELON, J. ZIMMERMANN. Fottea 92 KM Amphora.ovalis KM Amphora.ovalis KM Amphora.ovalis 100 KM Amphora.ovalis 48 KC Amphora.ovalis AM Amphora.sp. AM Amphora.libyca KM Amphora.cf..pediculus TCC702-Rbcl-1 Amphora.pediculus KC Amphora.pediculus 91 HQ Amphora.pediculus KM Amphora.cf..pediculus KM Amphora.pediculus AM Amphora.pediculus AM Amphora.fogediana 82 KM Amphora.aff..atomoides KP Amphora.aliformis KM Amphora.berolinensis 100 KJ Amphora.semperpalorum KJ Amphora.hyalina HQ Halamphora.coffeaeformis 97 KJ Halamphora.coffeaeformis 100 KP Halamphora.sp KP Halamphora.sp. KJ Amphora.caribaea TCC477-Rbcl-1 Halamphora.montana TCC479-Rbcl-1 Halamphora.montana KT Amphora.subtropica 100 KJ Amphora.subtropica AM Halamphora.normanii FJ Halamphora.coffeaeformis 81 Otu00046 Halamphora.ganensis 37 Otu00047 Halamphora.ganensis KJ Halamphora.oligotraphenta KJ Halamphora.coloradiana KJ Halamphora.cf..veneta KJ Halamphora.veneta

22 R-syst: how can we complete the library? Use of HTS sequences Easy and fast method Summer 2016: large scale sampling Coll. Regional Env. Agencies, Water Agencies, Private officies Definition of important species never sequenced 2017: retrieving of the HTS sequences.022

23 Perspectives? Curation Curation is sometimes complicated for some taxa because of discrepancies between morphological criteria used for species delimitations and DNA Needs: Integrative approach needed (DNA, morphology, ecology, biogeography) Collaborative work Nitzschia palea (UK, SP, F) Nitzschia inconspicua s.l. (U.K., SP) Fragilaria - Maria Kahlert (S, UK, F).023

24 Perspectives? On going standardization (original idea: M. Kelly) Collaborative work: France: A Bouchez, F Rimet, Germany: R Jahn, N Enke, J Zimmermann, Hungary: E Acs, Czech Republic: P Dvorak, A. Poulickova, Spain: R Trobajo, UK: D Mann, Dr. M Kelly, Belgium: K. Sabbe, Sweden: M Kahlert 2012 (Paris): 1st CEN meeting at TC230 (Water quality) Presentation of DNA metabarcoding 2013 (Vilnius): 3 preliminary work-items registered (Oslo, Prague): Circulation of document between experts Presentation to CEN 2016 (Delft): Nomination of experts by the national standardization offices 2 technical reports accepted: TR for the management of diatom barcodes for ecological status assessment TR for the routine sampling of benthic diatoms for metabarcoding analyses.024

25 Perspectives? Share the data! R-Syst::diatom = French initiative Algaterra = German initiative UK initiative Others? Not necessary to duplicate this work in several countries Need for a curated European database Discussion in WG1 DNA Barcode References? Thank you for your attention.025

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