Unicellular opisthokonts diversity and

distribution along the European coast

analyzed using high-throughput sequencingJ del Campo, D Mallo, R Massana, C de Vargas and I Ruiz-Trillo

ICOP XIVVancouver

Rigifilida

Collo

dictyo

nid

ae

Tsukubamonas

Jakobida

Euglenozoa

Heterolobosea

Preaxostyla

Fornicata

Parabasalia

Palpitomonas

PicozoaCryptophyceaeGlaucophyta

Em

briophyta

Charophyta

PalmophyllalesPrasinophytaeNephroselmis

Mamiellophyceae

PedinophyceaeUlvophyceae

Chlorophyceae

Trebouxiophyceae

Chlorodendrophyceae

Rhodophyceae

Centrohelida

Telonem

a

Hapto

phyta

Chrysophyceae

Synurales

Eustigmatales

Pinguiochrysidales

Phaeophycee

Schizocladia

Xanthophyceae

Phaeothamniophyceae

Raphidophyceae

Actinophryidae

Pelagophyceae

Dictyochophyceae

Diatomea

Bolidomonas

MAST 2MAST 1

Peronosporomycetes

Hyphochytriales

OpalinataBlastocystis

MAST 12

BicosoecidaMAST 3

Placidida

MAST 4 / 7 / 1

1

MAST 8 / 10

Labyrinth

ulomyc

etes

Cili

ophora

Per

kins

idae

Syn

dini

ales

Oxy

rrhis

Dinofla

gellata

Colp

odelli

da

Chro

mer

ida

Api

com

plex

a

Ch

lora

rachn

iop

hyta

Imbric

ate

aG

lissom

onadid

a

Cerc

om

onadid

a

Pansom

onadid

a

Thecofilo

se

a

Gra

nofilo

sea

Me

trom

onad

ea

Tre

mula

Phyto

myxea

Asceto

spore

a

Filo

reta

Gro

mia

Vam

pyre

llida

Fora

min

ifera

Aca

nth

aria

Polycistin

ea

Manta

monas

Apuso

monadid

a

Aphelid

a

Fun

gi

Roze

lla

Nucle

aria

Fontic

ula

Cora

lloch

ytr iu

mIc

hth

yo

spo

rea

Fila

ste

rea

Cho

ano

mon

ada

Meta

zoa

Bre

via

tea

Myxogastria

Sch

izopla

smodiid

a

Gra

cilip

odid

a

Fra

ctovi

telli

ida

Pro

tost

elii

da

Cav

oste

liida

Pro

tosp

oran

giid

a

Dicty

oste

lia

Multic

ilia

Archam

oebae

Tubulinea

Discose

a

Malawimonas

Ancyro

monadid

a

Opisthokonta

Amoebozoa

Excavata

Archaeplastida

Rhizaria

Alveolata

Stramenopila

Incertae sedis

Our phylogenetic framework

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 Biology2 del Campo et al. in prep.3 Not el al. 2009 PLoS ONE4 del Campo and Massana 2011 Protist

MARINE OPISTHOKONTA

METAZOA

FUNGI

MACHO 1

ACANTHOCORBIS

MONOSIGA

LAGENOECA

FRESCHO 1

URCEOLATA

SALPINGOECA

NAPIFORMIS

FRESCHO 2

FRESCHO 3

CLADE L

MAOP 2

MAOP 1

ECCRINALES

ICHTHYOPHONUS

AMOEBIDIUM

FRESHIP 1

ABEOFORMA

MAIP 1

ANUROFECA

PSEUDOPERKINSUS

DERMOCYSTIDIUM

SIMPLEX

THERMOPHILA

FONTICULA

FRESHOP

DIAPHANOECA

STEPHANOECA

ACANTHOECA

CORALLOCHYTRIUM

CAPSASPORA

MINISTERIA

FILASTEREA

FILASTEREA

CH

OA

NO

FLA

GELLA

TE

AIC

HTH

YO

SP

OR

EA

ICHTHYOPHONIDA

DERMOCYSTIDA

CRASPEDIDA

ACANTHOECIDA

0.09

PUTATIVECHOANOFLAGELLATES

*

*

*

*

*

*

PSOROSPERMIUM

PYXIDIUM

NUCLEARIIDA

FONTICULIDA

FRESHWATEROPISTHOKONTA

DIS

CIC

RIS

TO

ID

EA

*

*

*

*

*

*

*

*

*

*

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

del Campo and Ruiz-Trillo 2013 MBE

seqs

BioMarKsTotal number of reads

1076828

DNA: 496992

cDNA: 579836

Blanes

Naples Varna

Oslo

Roscoff

Xixón

Total Opisthokonts reads

10167 / 0.96

DNA: 3284 / 0.66

cDNA: 6883 / 1.19

BioMarKs DataBase

OTUs clustered at 97%

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

Blane

s

Xixon

Nap

les

Oslo

Ros

coff

Var

na

Varna

Ano

x

0

1

2

3

4

5

12

16

20

Perc

enta

ge o

f re

ads

HTS V4

18S DNA and cDNA

454 pyrosequencing

European Coast

MARINE OPISTHOKONTA

METAZOA

FUNGI

MACHO 1

ACANTHOCORBIS

MONOSIGA

LAGENOECA

FRESCHO 1

URCEOLATA

SALPINGOECA

NAPIFORMIS

FRESCHO 2

FRESCHO 3

CLADE L

MAOP 2

MAOP 1

ECCRINALES

ICHTHYOPHONUS

AMOEBIDIUM

FRESHIP 1

ABEOFORMA

MAIP 1

ANUROFECA

PSEUDOPERKINSUS

DERMOCYSTIDIUM

SIMPLEX

THERMOPHILA

FONTICULA

FRESHOP

DIAPHANOECA

STEPHANOECA

ACANTHOECA

CORALLOCHYTRIUM

CAPSASPORA

MINISTERIA

FILASTEREA

FILASTEREA

CH

OA

NO

FLA

GELLA

TE

AIC

HTH

YO

SP

OR

EA

ICHTHYOPHONIDA

DERMOCYSTIDA

CRASPEDIDA

ACANTHOECIDA

0.09

PUTATIVECHOANOFLAGELLATES

*

*

*

*

*

*

PSOROSPERMIUM

PYXIDIUM

NUCLEARIIDA

FONTICULIDA

FRESHWATEROPISTHOKONTA

DIS

CIC

RIS

TO

ID

EA

*

*

*

*

*

*

*

*

*

*

OUTGROUP

Diversity0 500 1000 1500 2000 2500 3000 3500

Sanger

453 seqs.

DNA V4

3284 reads

cDNA V4

6883 reads

Acanthoecida

Craspedida

Putative choanos

MAOP

Filasterea

Ichthyosporea

Discicristoidea

reads

Distribution

0

20

40

60

80

100

0

20

40

60

80

100

Pico DNA Pico cDNA Nano DNA Nano cDNA Micro DNA Micro cDNA Sed DNA Sed cDNA

Surface DNA Surface cDNA DCM DNA DCM cDNA Sed Ox DNA Sed OX cDNA Anox DNA Anox cDNA

Acanthoecida Craspedida Putative choanoflagellates

MAOP Filasterea Ichthyosporea Discicristoidea

0.6 1.1 0.6 1.3 0.2 0.1 1.3 2.7

0.4 0.8 0.7 1.1 1.0 0.9 3.3 1.4

Pe

rcen

tage o

f re

ad

sP

erc

en

tage o

f re

ad

s

% of total reads

% of total reads

Abundanceand Diversity

0

200

400

600

800

1000

1200

1400

1600499

1523

Only 16.6% of the reads are more than 97% similar to any 18S DNA

sequence from GenBank.

2.0 % -> 55 %0.4 % -> 23 % 5.0 %- > 75%

Nu

mb

er

of re

ad

s

OTUs

Num

ber

of re

ads

None of the 10 most abundant OTUs are more than 95% similar to

any culture 18S DNA sequence from GenBank.

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.cafonamental@gmail.com

@fonamental

Xavier Grau, Helena Parra, David López and Meritxell Antó

Our collaborators: Ramon Massana and Diego Mallo.

Acknowledgements

Thanks for you attention!