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-74203 Thonon les Bains cedex, France coll. J.M. Frigerio, P. Chaumeil & A. Franc INRA, UMR BioGeCo, 69, route d'arcachon ; Pierroton ; 33612 Cestas Cedex, France Essen Cost DNAquanet march 2017
R-syst French network of systematic: A. Franc Virus Bacteria Fungi Plants Insects Nematods Microalgae > diatoms R-Syst::diatom database.02
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 2016.03
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R-syst Open access: www.rsyst.inra.fr/ Quality indices Life-forms Functional groups Sizes, biovolumes, cellule shapes.08
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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
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
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
R-syst: curation Curation >Important genus for ecological indication: >Shallow turbulent lentic ecosystem Small unattached filaments, resist to turbulence.015
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
R-syst: completness? Estimation: 100.000 of extant species (Mann & Vanormelingen 2013) About 15.000 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
R-syst: completness? About 15.000 diatom species described Estimation: 100.000 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
R-syst: completness? About 15.000 diatom species described Estimation: 100.000 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
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
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. KP229547 Amphora.commutata KM084978 Amphora.ovalis MANN, A. PIUZ, R. TROBAJO, K. TAPOLCZAI, V. VASSELON, J. ZIMMERMANN. Fottea 92 KM084927 Amphora.ovalis KM084928 Amphora.ovalis KM084993 Amphora.ovalis 100 KM084926 Amphora.ovalis 48 KC954577 Amphora.ovalis AM710511 Amphora.sp. AM710425 Amphora.libyca KM084932 Amphora.cf..pediculus 86 62 93 52 TCC702-Rbcl-1 Amphora.pediculus KC954575 Amphora.pediculus 91 HQ912403 Amphora.pediculus KM084934 Amphora.cf..pediculus KM084933 Amphora.pediculus 36 76 18 AM710426 Amphora.pediculus AM710489 Amphora.fogediana 82 KM084979 Amphora.aff..atomoides KP229546 Amphora.aliformis KM084982 Amphora.berolinensis 100 KJ463471 Amphora.semperpalorum 26 100 KJ463462 Amphora.hyalina HQ912466 Halamphora.coffeaeformis 97 KJ463478 Halamphora.coffeaeformis 100 KP400296 Halamphora.sp. 43 79 KP400297 Halamphora.sp. KJ463458 Amphora.caribaea TCC477-Rbcl-1 Halamphora.montana 28 42 100 TCC479-Rbcl-1 Halamphora.montana KT023600 Amphora.subtropica 100 KJ463475 Amphora.subtropica 42 68 AM710424 Halamphora.normanii FJ002103 Halamphora.coffeaeformis 81 Otu00046 Halamphora.ganensis 37 Otu00047 Halamphora.ganensis KJ463481 Halamphora.oligotraphenta 95 100 KJ463480 Halamphora.coloradiana KJ463483 Halamphora.cf..veneta KJ463482 Halamphora.veneta 0.01.021
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
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
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 2014-2015 (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
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