Eukaryotic diversity? Morphology: United Nation Census, 2008 World Taxonomists Database Earth Total Eukaryotic Biodiversity: Tara Oceans 2009 - 2012 Map of two expeditions: TARA Polar Circle (2014) & TARA Ice Drift (2008) Tara-Oceans – 2009-2012 ILLUMINA sequencing of >½ billion rDNA V9 fragments from 36 stations. 3 depths, 4 organismal size-fractions (~300 samples) Tara Oceans Expedition, 2009-2012 381 samples from 49 locations depths surface 5-10 m deep chlorophyll maximum 17-184 m mesopelagic zone 268-852 m size fractions 0.8-5 µm 5-20 µm 20-180 µm 180-2000 µm 0.00 0.50 % e-06 40 A. B. C. D. E. F. 0.25 0.75 G. H. e-05 30 EUKARYOTIC LINEAGE # REFERENCE # OTUs - SWARMs # rDNA READs (million) % of reads per READS OTUs Phylogenetic patterning of 20 BARCODE plankton size-fractions e-04 Stations 0 0 0 % e-03 10 0 0 00 0000 20 30 40 500 0 10 200 30 400 600 8 1 100 600 1 2 3 4 5 0 20 40 60 80100 80-85% 85-90% 90-95% 95-<100% 100% 80-85% 85-90% 90-95% 95-<100% 100% total eukaryotic rDNA Other Discoba 7,475 Diplonemea 4,850 5.8 million Kinetoplastida EXCAVATA Euglenida diversity: from unknown to Heterolobosea Jakobida EXCAVATA - Discosea - Lobosa (86% to LKM74) O B Tubulinea (no Arcellinida) A E known-unknown eukaryotic O ZOA O Variosea Z Mycetozoa & Myxogastrea M AMOEBO A Metazoa 18,530 17,010 plankton lineages. Choanoflagellida 247 million Other Opisthokonta Mesomycetozoa Ascomycota OPISTHO- 4,444 Basidiomycota KONTA 1,832 -> Overall , eukaryotic plankton Chytridiomycota Entomophtoromycota KONTA OPISTHO- Microsporidiomycota diversity explodes in lineages with Other Mycota Other fungi 80% to Chlorophyta very poor taxonomy (species Chlorophyceae ARCHAE- Ulvophyceae PLASTIDA Trebouxiophyceae description) and even less Prasinophyceae Clade VII Mamiellophyceae Pyramimonadales ARCHAE- representatives in culture other Chlorophyta (Clade V) PLASTIDA Streptophyta 3,285 Rhodophyta 1,169 97 million collections. Only 0.9% of OTUs are Collodaria 5,636 Nasselaria & Eucyrtidium Spumellarida 100% similar to any Ref Seq !! RAD A 5.2 million RAD B RHIZARIA RAD C Other Radiolaria Acantharea 1,043 Foraminifera RHIZARIA Other Cercozoa 1,097 Phaeodarea d d -> By far the largest diversity Chlorarachnea Euglyphida 69.6 million Dinophyceae 24,813 occurs in the 2-5um organismal MALV-II 5,631 11.7 million MALV-I 2,035 MALV-III ALVEOLATA 17.9 million MALV-IV size fraction ( the pico-nano ), which MALV-V Other Alveolata 1,910 Ellobiopsidae fundamentally challenge the Perkinsea ALVEOLATA Apicomplexa 1,897 6.2 million Ciliophora 1,963 significance of the classical Bacillariophyta 1,232 3,276 Bolidophyceae and relatives 14.6 million Dictyochophyceae planktonic functional group HNF Pelagophyceae MOCH-1,2 MOCH-4 (Heterotrophic nanoflagellate). Chrysophyceae-Synurophyceae Phaeophyceae Pinguiophyceae Raphidophyceae MOCH-3 MOCH-5 MAST-1 Oomyceta Pirsonia STRAMENOPILA Labyrinthulea STRAMENOPILA MAST-4,6,7,8,9,10,11 MAST-3;12 Bicoecea MAST-16,22,24 Other Stramenopiles Other MAST Placididae & others Katablepharidophyta Cryptophyta Kata/Crypto (81% ID) Haptophyta 713 Telonemia Picozoa INCERTA SEDIS Centroheliozoa SEDIS INCERTA Apusozoa ... a few surprises there, e.g. low counts of streptophytes and haptophytes, only diatoms and dinos significant among primary producers. Obvious winners – metazoans and dino. Amazingly diverse & abundant Rhizarians but first and foremost, diplonemids . All this in photic zone, but they are deep, too de Vargas et al. Science , 2015 de Vargas et al. Science , 2015 Eukaryotic diversity in the World Ocean – still enigmatic operationalOTU taxonomic units (OTUs) Choanoflagellata Metazoa 17,010 B Ichthyosporea Fungi Opisthoko nta Amoebozoa UNIKON TA Diplonemida * 12,325 *(→45,197) Kinetoplastida Euglenida Euglenoz oa Heterolobosea Jakobida Parabasalida DISCO BA Embryophyta Chlorophyta Rhodophyta Glaucophyta Apicomplexa ARCHAEPLAS TIDA MALV Dinophycea 24,813 Alveola te SA R Ciliophora Rhizaria Stramenopila Haptophyta de Vargas et al. Science (2015); Flegontova et al. Curr Biol (2016) DIPLONEMIDS?! Diplonemids are Euglenozoan protists Glaucophytes Cryptophytes Cyanidiophytes Katablepharids Bangiophytes Picozoa Porphyridiophytes Palpitomonas Floridiophytes Centrohelids Trebouxiophytes Te lonemids Chlorophyceans Haptophytes Rappemonads Red Ulvophytes Dinoflagellates algae Prasinophytes Syndiniales Mesostigma Oxyrrhis Zygnemophyceans Perkinsus Green Charophyceans Apicomplexa Bryophytes Vitrella plants Tracheophytes Colpodellids Kinetoplastids Chromera Diplonemids Colponemids Alveolates Euglenids Ciliates Heteroloboseans Bicosoecids Jakobids Labyrinthulids Trimastix Thraustochytrids Oxymonads Blastocystis Parabasalids Opalinids Stramenopiles Oomycetes Retortamonads Actinophryids Diplomonads Diatoms Malawimonas Phaeophytes Collodictyon Dictyochophytes Pelagophytes Zygomycetes Ascomycetes Eustigmatophytes Basidiomycetes Pinguiophytes Fungi Chytrids Raphidophytes Microsporidia Chrysophytes Cryptomycota Chlorarachniophytes Nucleariids Phaeodarea Fonticula Cercomonads Ichthyosporea Euglyphids Rhizaria Filasterea Thaumatomonads Animals Choanoflagellates Phytomyxea Porifera Gromia Bilateria Haplosporidia Coelenterata Placozoa Mikrocytos Ancyromonads Acantharea Apusomonads Breviata Arcellinids Foraminifera Tubulinids you are here Archamoebae Leptomyxids Mycetozoa Vannellids Dactylopodids Acanthomyxids Current Biology Burki & Keeling, Curr Biol. 2014 EUGLENOZOA KINETOPLASTEA Trypanosoma brucei DIPLONEMEA Diplonema papillatum EUGLENIDA Euglena gracilis Faktorová et al., F1000 , 2016 Kinetoplastids Trypanosoma brucei Trypanosoma cruzi Leishmania extracellular parasite intracellular parasite intracellular parasite Tse-tse fly Reduviid bugs Sand fly Sleeping sickness Chagas disease Leishmaniases Summary of our knowledge of diplonemid morphology A Diplonema breviciliata (Simpson 1997); B Rhynchopus sp. (Simpson 1997); C-E Rhynchopus euleeides (Roy et al. 2007) Less than a dozen papers in total on diplonemids , as compared to tens of thousands of papers on kinetoplastids “Trans -splicing” of all mitochondrial transcripts Molecule1 Molecule2 Molecule3 Molecule3 Cox1.1 Cox1.2 Cox1.3 Cox1.4 ~200bp ~200bp ~200bp ~200bp Transcription E1 E2 E3 E4 mRNA cox1.1 mRNA cox1.2 mRNA cox1.3 mRNA cox1.4 Ligation and Translation ??? Cox1.1 Cox1.2 Cox1.3 Cox1.4 Cox1 protein Vl ček et al., Nucl. Acids Res. 2011 Valach et al., Nucl. Acids Res . 2014 Comparative analysis of the amount of kinetoplast DNA in kinetoplastids and diplonemids We used DAPI (AT-selective) and redDot1 (less sequence-selective) for staining. Using colour deconvolution, we separated signals of both fluorescent stains into kDNA and nucleus. We confirmed that Trypanosoma brucei contains ~5% of total DNA in its kDNA, contrasting it With Perkinsela , where ~93% (!!!) of total DNA is in the mitochondrion. Diplonemids have up to 270 Mbp of mtDNA. Lukeš et al., submitted Diplonemids are the 3 rd most diverse (after Dinophycea and Metazoa) and 6 th most abundant eukaryote in the world oceans! Lukeš et al. Curr Biol 2015 total abundance: 1,315,922 barcodes total abundance: 845,473 barcodes clade: novel diplonemid clade: planktonic diplonemid sample: 71% of total barcodes in the sample sample: 51% of total barcodes in the sample size fraction: 3 µm - infinity size fraction: 0.8-3 µm depth: 784 m depth: 791 m temperature: 0.5 C temperature: 4.2 C total abundance: 1,315,922 barcodes total abundance: 845,473 barcodes clade: novel diplonemid clade: planktonic diplonemid sample: 71% of total barcodes in the sample sample: 51% of total barcodes in the sample size fraction: 3 µm - infinity size fraction: 0.8-3 µm depth: 784 m depth: 791 m temperature: 0.5 C temperature: 4.2 C Vertical structuring of the diplonemid population SRF DCM PF4 185 875 765 Are the diplonemids from oceanic depths different from those 327 on surface? YES! 92 56 Very little sharing of barcodes and swarms between these zones 6272 MES = strong vertical stratification of swarms Flegontova et al., Curr Biol. 2016 Snímek 21 PF4 As far as I know, analysis of barcodes is not finished yet by Shruti, so I don't know where the left diagram is coming from. I've removed it. Pavel Flegontov; 24.5.2015 Are diplonemids everywhere? YES, they are DCM MES A SUR A A 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 Station Eveness Station 0.2 Station Station Eveness Station Station Eveness 0.2 0.2 0.0 0.0 0.0 2 4 6 9 2 8 11 13 15 17 19 22 24 26 2 14 20 26 32 38 44 50 56 62 68 13 24 34 45 56 67 78 89 99 110 121 Occupancy Occupancy Occupancy B B B 5 5 10 10 5 10 4 4 10 10 4 10 3 3 10 10 3 10 Abundance Abundance Abundance 2 2