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Home , Perkinsela, Phaeodarea, Phytomyxea

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 Euglenida diversity: from unknown to Heterolobosea Jakobida EXCAVATA -

Discosea -

Lobosa (86% to LKM74) O

B (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 very poor (species Chlorophyceae ARCHAE- Ulvophyceae PLASTIDA Trebouxiophyceae description) and even less Prasinophyceae Clade VII Mamiellophyceae Pyramimonadales ARCHAE- representatives in culture other Chlorophyta (Clade V) PLASTIDA 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 RAD C Other

Acantharea 1,043 RHIZARIA Other 1,097 d d -> By far the largest diversity Chlorarachnea 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 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 INCERTA SEDIS

Centroheliozoa SEDIS INCERTA Apusozoa ... a few surprises there, e.g. low counts of streptophytes and , 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 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

Glaucophytes Cryptophytes Cyanidiophytes Bangiophytes Picozoa Porphyridiophytes Palpitomonas Floridiophytes Trebouxiophytes Te lonemids Chlorophyceans Haptophytes Rappemonads Red Ulvophytes Prasinophytes Syndiniales Mesostigma Oxyrrhis Zygnemophyceans Perkinsus Green Charophyceans Apicomplexa Bryophytes Vitrella Tracheophytes Colpodellids Kinetoplastids Chromera Diplonemids Colponemids Euglenids Heteroloboseans Bicosoecids Labyrinthulids Trimastix Thraustochytrids Oxymonads Blastocystis Parabasalids Opalinids Stramenopiles Retortamonads Actinophryids Diplomonads Diatoms Malawimonas Phaeophytes Collodictyon Dictyochophytes Pelagophytes Zygomycetes Ascomycetes Eustigmatophytes Basidiomycetes Pinguiophytes Fungi Chytrids Raphidophytes Microsporidia Chrysophytes Cryptomycota Nucleariids Phaeodarea Fonticula Cercomonads Ichthyosporea Euglyphids Rhizaria Thaumatomonads Animals Porifera Bilateria Haplosporidia Coelenterata Placozoa Mikrocytos Ancyromonads Apusomonads Arcellinids Foraminifera Tubulinids you are here Leptomyxids Mycetozoa Vannellids Dactylopodids Acanthomyxids Current Biology Burki & Keeling, Curr Biol. 2014

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 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 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 10 2 10 10 1 1 10 10 1 10 0 5 10 15 20 25 1 10 100 1000 0 20 40 60 80 100 120 1 10 100 1000 0 10 20 30 40 50 60 1 10 100 1000 Occupancy No. of ribotypes Occupancy No. of ribotypes Occupancy No. of ribotypes

Upper plots: swarm distribution among sampling sites. X-axis = number of stations the swarm was recovered from. Y-axis = evenness of distribution. The bigger the bubble, the more abundant the swarm. Bottom plots: mostly linear relationship between the occupancy and abundance. Flegontova et al., Curr Biol. 2016 100 Hemistasia_phaeocysticola_YPF1303 uncultured_eukaryote_KJ762701 93 Diplonemea_EU635614 Diplonemea_EU635615 81 Diplonemea_EU635671 Diplonemea_EU635670 93 Diplonemea_EU635669 Diplonemea_EU635667 Diplonemea_EU635668 100 Diplonemea_EU635595 100 80 Diplonemea_FJ032684 Greenland_17774_2_14 Diplonemea_EU635665 Diplonemea_EU635634 Diplonemea_EU635672 100 Diplonemea_EU635626 88 WorldOcean_960_3 84 WorldOcean_960_12 Verification using full-size 18S rRNAs WorldOcean_960_22 Diplonemea_EU635636 Diplonemea_EU635605 WorldOcean_1599_2 WorldOcean_1645_5 Diplonemea_EU635656 Diplonemea_EU635609 Diplonemea_EU635664 Diplonemea_DQ504322 Diplonemea_EU635613 Diplonemea_EU635643 WorldOcean_1638_8 Diplonemea_EU635642 94 Diplonemea_EU635610 WorldOcean_1645_11 WorldOcean_1638_3 WorldOcean_1638_2 Diplonemea_EU635611 Diplonemea_EU635645 ... as revealed also by ML phylogeny Diplonemea_EU635666 Diplonemea_EU635641 92 Diplonemea_EU635639 98 Diplonemea_EU635625 99 Diplonemea_EU635662 Diplonemea_EU635622 WorldOcean_960_32 94 WorldOcean_960_13 WorldOcean_2213_1 97 of complete 18S rRNA Diplonemea_EU635637 WorldOcean_960_5 97 WorldOcean_1638_4 100 Diplonemea_EU635648 Diplonemea_EU635618 Diplonemea_EU635603 Diplonemea_EU635640 84 Diplonemea_EU635644 Diplonemea_EU635657 81 Diplonemea_EU635647 Diplonemea_EU635646 Diplonemea_EU635604 Diplonemea_EU635612 94 WorldOcean_2213_5 WorldOcean_908_38 100 99 Diplonemea_EU635593 100 WorldOcean_960_24 WorldOcean_960_6 Diplonemea_EU635588 Diplonemea_EU635591 WorldOcean_1599_1 WorldOcean_960_17 WorldOcean_960_29 WorldOcean_960_33 WorldOcean_960_18 WorldOcean_960_4 WorldOcean_960_14 97 WorldOcean_960_27 WorldOcean_960_30 WorldOcean_960_34 Diplonemea_EU635658 Diplonemea_EU635659 Diplonemea_EU635619 Diplonemea_EU635620 Diplonemea_EU635660 Diplonemea_EU635596 Diplonemea_EU635617 Diplonemea_EU635592 Diplonemea_DQ504323 Diplonemea_EU635632 Diplonemea_EU635635 WorldOcean_1645_8 WorldOcean_1645_4 82 Diplonemea_EU635633 97 WorldOcean_1638_5 95 Diplonemea_EU635597 WorldOcean_1599_3 WorldOcean_1638_9 Diplonemea_EU635627 98 Diplonemea_EU635589 Diplonemea_EU635590 Greenland_17776_1_2 Greenland_17774_1_6 WorldOcean_960_9 91 WorldOcean_960_40 WorldOcean_960_1 WorldOcean_960_19 WorldOcean_960_28 Greenland_17776_14 Greenland_17774_7 Greenland_17775_1 Greenland_17774_1_7 Greenland_17776_1 Greenland_17774_2_9 Diplonemea_EU635621 100 Diplonemea_EU635628 Diplonemea_EU635629 WorldOcean_1884_2 Diplonemea_DQ504349 Diplonemea_DQ504321 100 90 Diplonemea_EU635608 Diplonemea_EU635607 Diplonemea_EU635631 100 WorldOcean_960_2 WorldOcean_960_42 82 Greenland_17776_1_3 Greenland_17774_13 100 Greenland_17776_1_12 95 Greenland_17776_1_6 Diplonemea_EU635651 90 WorldOcean_960_15 Greenland_17776_2 Diplonemea_EU635652 Diplonemea_EU635653 Greenland_17771_1 Greenland_17774_2_3 82 Diplonemea_EU635654 92 WorldOcean_1884_5 Greenland_17774_4_1 Greenland_17774_6 99 Greenland_17774_2_6 Greenland_17774_3_17 89 WorldOcean_908_4 WorldOcean_908_15 WorldOcean_908_13 Diplonemea_EU635650 Diplonemea_EU635649 Greenland_17774_3_20 Diplonemea_EU635661 Diplonemea_EU635624 Diplonemea_EU635630 Greenland_17776_4 Diplonemea_EU635655 WorldOcean_1884_4 Diplonemea_EU635599 WorldOcean_1645_6 Diplonemea_EU635600 Diplonemea_EU635601 11 DNA samples collected in the Labrador Diplonemea_EU635602 WorldOcean_908_2 100 WorldOcean_908_5 WorldOcean_908_10 100 Diplonemea_EU635678 92 Diplonemea_EU635677 94 uncultured_eukaryote_KJ760020 96 Diplonemea_EU635673 96 Diplonemea_EU635674 Diplonemea_EU635675 Diplonemea_EU635676 97 Diplonema_sp_AY425011 Sea = 104 clones/85 unique18S rRNAs! Diplonema_ambulator_AY425009 Diplonema_sp_AY425012 Diplonema_papillatum_KF633466 100 Rhynchopus_sp_AF380997 Rhynchopus_sp_AY425013 Rhynchopus_sp_AY425014 99 Rhynchopus_sp_AY490209 Diplonemea_DQ504350 Rhynchopus_sp_AY490210 Diplonema_sp_AY425010 Diplonemea_EU635681 Our unpubl. data Diplonemea_EU635682 100 Diplonemea_EU635679 Diplonemea_EU635680

0.09 ... they are now the 1 st most diverse and remain the 6 th most abundant marine

reads OTUs

diplonemids others other diatoms rarefaction other stramenopiles curve slope: others stramenopiles 7×10-6 diatoms unknown eukaryotes dinozoans rhizarians metazoans 1.4×10-7 × -7 unknown 5.3 10 dinozoans eukaryotes diplonemids

rhizarians metazoans

1.5×10-7

Fig. 7

Flegontova et al., Curr Biol. 2016 What are diplonemids doing in the ocean?

phototrophs ?

parasites symbionts Heterotrophs = predators detritovores bacteriovores ... Co-occurrence plot says very little if anything in case of diplonemids

Co-presences (green) Co-exclusions (red)

Lima-Mendez et al, Science 2015 Single-cell genomics of marine clade diplonemids

First diplonemids captured, photographed and sequenced

Gawryluk et al., Curr Biol . 2016 Conclusions

• A clade sister to classic diplonemids is the most abundant group of Excavata in the World Ocean plankton.

• As compared to other eukaryotes, it emerges as a hyper- diverse clade, the 1 rd by the number of OTUs and the 6 th most abundant.

• Marine planktonic diplonemids reach very high relative abundance in some stations of the deep non-photic zone, in small size-fractions up to 20 µm.

• This huge group represents an overlooked major player in marine ecosystems, however, almost nothing is known about their morphology and lifestyle Acknowledgements

Aleš Horák, Institute of Parasitology, České Budějovice

Pavel Flegontov, University of Ostrava

Julius Lukeš, Institute of Parasitology, České Budějovice

Shruti Malviya, CNRS, Paris

Chris Bowler, Ecole Normal Superieure, Paris

Colomban de Vargas, Station Biologique de Roscoff

Stephane Audic, Station Biologique de Roscoff