Unicellular opisthokonts diversity and distribution along the European coast analyzed using high-throughput sequencing
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Unicellular opisthokonts include relevant eukaryotic lineages from an evolutionary and an ecological point of view. In our study we analyzed the 18S rDNA and rRNA V4 region of the unicellular opisthokonts (excluding fungi). Sequences were obtained by high-throughput methods from the European coast in the context of the BioMarKs project. We described the abundance and the distributions of the studied organisms in different sampling points and size fractions. Furthermore the ratio RNA:DNA allowed us to determine the most active groups. By using pysosequencing we had access to the less abundant and rare unicellular opisthokonts of the community. Using a curated reference database we properly placed the sequences retrieved in the opisthokonts tree, describing new diversity and identifying some groups not previously reported in marine environments. High-throughput sequencing techniques successfully increase our knowledge on the diversity of unicellular opisthokonts, its geographical distribution and possible ecological role.
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Unicellular opisthokonts diversity and distribution along the European coast analyzed using high-throughput sequencing
- 1. Unicellular opisthokonts diversity and distribution along the European coast analyzed using high-throughput sequencing J del Campo, D Mallo, R Massana, C de Vargas and I Ruiz-Trillo ICOP XIV Vancouver
- 2. Our phylogenetic framework Collodictyonidae Phaeothamniophyceae Raphidophyceae Actinophryidae Phaeophycee Schizocladia Xanthophyceae Corallochytrium Acantharia Polycistinea Mantamonas Rigifilida Preaxostyla Fornicata Parabasalia Tsukubamonas Jakobida Euglenozoa Heterolobosea Palpitomonas Picozoa Cryptophyceae Glaucophyta Palmophyllales Prasinophytae Nephroselmis Mamiellophyceae Pedinophyceae Ulvophyceae Chlorophyceae Trebouxiophyceae Chlorodendrophyceae Charophyta Embriophyta Rhodophyceae Centrohelida Telonema Haptophyta Pinguiochrysidales Eustigmatales Synurales Chrysophyceae Dictyochophyceae Pelagophyceae Bolidomonas Diatomea Hyphochytriales Peronosporomycetes MAST 1 MAST 2 Blastocystis Opalinata MAST 12 Bicosoecida MAST 3 Placidida MAST 4 / 7 / 11 MAST 8 / 10 Labyrinthulomycetes Apicomplexa Chromerida Ciliophora Oxyrrhis Dinoflagellata Syndiniales Perkinsidae Colpodellida Chlorarachniophyta Fonticula Imbricatea Glissomonadida Cercomonadida Pansomonadida Thecofilosea Granofilosea Metromonadea Tremula Gromia Filoreta Ascetosporea Vampyrellida Phytomyxea Foraminifera Apusomonadida Aphelida Fungi Rozella Nuclearia Ichthyosporea Filasterea Choanomonada Metazoa Breviatea Myxogastria Schizoplasmodiida Gracilipodida Fractovitelliida Protosteliida Cavosteliida Protosporangiida Dictyostelia Multicilia Archamoebae Tubulinea Discosea Malawimonas Ancyromonadida Opisthokonta Amoebozoa Excavata Archaeplastida Rhizaria Alveolata Stramenopila Incertae sedis
- 3. Diversity and abundance Pedrós-Alió 2006 Trends in Microbiology Described sp.1 GenBank (97%)1 Cultures2 GOLD2 GOS3 Marine4 Freshwater5 Choanoflagellates 0.5 1.1 0.6 1.2 0.8 1.3 3.7 Ichthyosporeans 0.1 0.7 0.06 0.8 - - - 1 Pawlowski et al. 2012 PLoS Biology 2 del Campo et al. in prep. 3 Not el al. 2009 PLoS ONE 4 del Campo and Massana 2011 Protist
- 4. ACANTHOECIDA CRASPEDIDA MARINE OPISTHOKONTA METAZOA FUNGI ACANTHOECA STEPHANOECA DIAPHANOECA MACHO 1 ACANTHOCORBIS MONOSIGA LAGENOECA FRESCHO 1 URCEOLATA SALPINGOECA NAPIFORMIS FRESCHO 2 PYXIDIUM FRESCHO 3 CLADE L MAOP 2 MAOP 1 CORALLOCHYTRIUM ECCRINALES ICHTHYOPHONUS AMOEBIDIUM FRESHIP 1 ABEOFORMA MAIP 1 ANUROFECA PSEUDOPERKINSUS DERMOCYSTIDIUM CAPSASPORA MINISTERIA SIMPLEX THERMOPHILA FONTICULA FRESHOP FILASTEREA FILASTEREA CHOANOFLAGELLATEA ICHTHYOSPOREA ICHTHYOPHONIDA DERMOCYSTIDA 0.09 PUTATIVE CHOANOFLAGELLATES * * * * * * PSOROSPERMIUM NUCLEARIIDA FONTICULIDA FRESHWATER OPISTHOKONTA DISCICRISTOIDEA * * * * * * * * * * OUTGROUP 18S meta-analysis Complete sequences ML analysis - 199 Opisthokonts seqs. - 227 seqs tree. - 1436 informative positions. - Environmental and Culture seqs. Marine Freshwater Symbiont Only environmental seqs. 0 20 40 60 80 seqs del Campo and Ruiz-Trillo 2013 MBE
- 5. BioMarKs Total number of reads 1076828 DNA: 496992 cDNA: 579836 Blanes Oslo Naples Varna Roscoff Xixón BioMarKs DataBase OTUs clustered at 97% Total Opisthokonts reads 10167 / 0.96 DNA: 3284 / 0.66 cDNA: 6883 / 1.19 Pico (0.8 – 3 μm) Nano (3 – 20 μm) Micro (20 – 200 μm) Total (Sediment) Surface (Oxic) DCM (Oxic) Sediments (Oxic) Anoxic (Varna) KeyDNATools at PR2 Opisthokonts ref DB ML phylogenies 20 16 12 5 4 3 2 1 Blanes Xixon Naples Oslo Roscoff Varna Varna Anox 0 Percentage of reads HTS V4 18S DNA and cDNA 454 pyrosequencing European Coast
- 6. ACANTHOECIDA CRASPEDIDA MARINE OPISTHOKONTA METAZOA FUNGI ACANTHOECA STEPHANOECA DIAPHANOECA MACHO 1 ACANTHOCORBIS MONOSIGA LAGENOECA FRESCHO 1 URCEOLATA SALPINGOECA NAPIFORMIS FRESCHO 2 PYXIDIUM FRESCHO 3 CLADE L MAOP 2 MAOP 1 CORALLOCHYTRIUM ECCRINALES ICHTHYOPHONUS AMOEBIDIUM FRESHIP 1 ABEOFORMA MAIP 1 ANUROFECA PSEUDOPERKINSUS DERMOCYSTIDIUM CAPSASPORA MINISTERIA SIMPLEX THERMOPHILA FONTICULA FRESHOP FILASTEREA FILASTEREA CHOANOFLAGELLATEA ICHTHYOSPOREA ICHTHYOPHONIDA DERMOCYSTIDA 0.09 PUTATIVE CHOANOFLAGELLATES * * * * * * PSOROSPERMIUM NUCLEARIIDA FONTICULIDA FRESHWATER OPISTHOKONTA DISCICRISTOIDEA * * * * * * * * * * OUTGROUP Diversity 0 500 1000 1500 2000 2500 3000 3500 Sanger 453 seqs. DNA V4 3284 reads cDNA V4 6883 reads reads Acanthoecida Craspedida Putative choanos MAOP Filasterea Ichthyosporea Discicristoidea
- 7. Distribution % of total reads 100 80 60 40 20 0 100 80 60 40 20 0 Acanthoecida Craspedida Putative choanoflagellates MAOP Filasterea Ichthyosporea Discicristoidea 0.6 1.1 0.6 1.3 0.2 0.1 1.3 2.7 Pico DNA Pico cDNA Nano DNA Nano cDNA Micro DNA Micro cDNA Sed DNA Sed cDNA 0.4 0.8 0.7 1.1 1.0 0.9 3.3 1.4 Surface DNA Surface cDNA DCM DNA DCM cDNA Sed Ox DNA Sed OX cDNA Anox DNA Anox cDNA Percentage of reads Percentage of reads % of total reads
- 8. Abundance and Diversity 1600 1400 1200 1000 800 600 400 200 0 499 1523 Only 16.6% of the reads are more than 97% similar to any 18S DNA sequence from GenBank. 0.4 % -> 23 % 2.0 % -> 55 % 5.0 %- > 75% Number of reads OTUs Number of reads None of the 10 most abundant OTUs are more than 95% similar to any culture 18S DNA sequence from GenBank.
- 9. Some ideas to bring back home The presence of Unicellular Opisthokonts in BioMarKs samples is significant. They are not the most abundant organisms but are quite important in some particular fractions and templates such as the DCM nanoplankton cDNA or the Anoxic samples. Developing meta-analysis of environmental surveys for the different protist groups is necessary and useful. The reference trees and reference sequence databases that you generate in this kind of analysis are going to be the backbone for the future high-throughput sequencing (HTS) studies. HTS analysis allows us (in terms of diversity) to improve our resolution and to go deeper into the rare biosphere because of the huge amount of data generated. Also using HTS we can process faster our samples allowing us to analyse a higher variety of environments so we can have access to new sources of diversity. From an ecological point of view HTS helps us to identify the most abundant organisms in the community, those who probably are responsible for most of the processes. Then we can target them as candidates for isolation and culturing and eventually for genomic or transcriptomics studies. javier.delcampo@botany.ubc.ca fonamental@gmail.com @fonamental
- 10. Acknowledgements Xavier Grau, Helena Parra, David López and Meritxell Antó Our collaborators: Ramon Massana and Diego Mallo. Thanks for you attention!