This document describes a study on Minorisa minuta, an atypical chlorarachniophyte alga. Single-cell genomics was used to analyze M. minuta cells collected from various ocean sites. Phylogenetic analysis of 158 operational taxonomic units identified from 903 single-amplified genomes placed M. minuta within the chlorarachniophytes and revealed that it is widespread and abundant in ocean plankton communities.

1. Minorisa minuta and the transition to secondary plastid endosymbiosis
A single cell genomics approach
Javier del Campo1, Michael E. Sieracki2, Ramon Massana3 and Patrick Keeling1
1 University of British Columbia, Vancouver, BC, Canada. 2 Bigelow Laboratory for Ocean Sciences, East
Boothbay, Maine, USA. 3 Institut de Ciències del Mar, CSIC, Barcelona, Catalonia, Spain
Agust 7, 2014
Protist 2014
Banff, AB

2. Biases
Pedrós-Alió 2006 Trends in Microbiology

3. Minorisa minuta, an atypical Chlorarachniophyte
1.E+05
8.E+04
6.E+04
4.E+04
2.E+04
del Campo el al. 2013 The ISME Journal
AF411280 Spongomonas minima
AF411276 Bodomorpha minima
AJ305043 Proleptomonas faecicola
FJ79079694 Cercomonas rapida
FJ790706 Cercomonas magna
FJ790717 Cavernomonas stercoris
AF411259 Thaumatomonas seravini
AJ418784 Euglypha rotunda
AJ418792 Trinema enchelys
AJ418790 Tracheleuglypha dentata
AJ418793 Cyphoderia ampulla
DQ199663 Aurigamonas solis
FJ790730 Paracercomonas paralaciniaegerens
66
96
AF290541 Cryothecomonas aestivalis
AJ418794 Pseudodifflugia gracilis
AJ514866 Gymnophrys cometa
AF174374 Massisteria marina
AB452995 Partenskyella glossopodia
X70809 Chlorarachnion reptans
79
93
100
84
51
96
81
100
56
100
100
AY620253 Metopion fluens
100
0.1
AJ561116 Pseudopirsonia mucosa
AY884324 Eocercomonas sp.
J514867 Lecythium sp.
AY305009 Clathrulina elegans
FJ973362 Bigelowiella sp.
AF054890 Lotharella vacuolata
AF076171 Gymnochlora stellata
AY426922 BL010625.12
EF173002 SSRPE06
Minorisa minuta candidatus
U20858 Haplosporidium costale
U47852 Urosporidium crescens
100
EF514503 Corallomyxa tenera
AF245217 Spongospora subterranea
AF310898 Polymyxa graminis
AF310904 Phagomyxa odontellae
AY941201 Platyreta germanica
100
100
AB252760 NAMAKO20
FJ518816 Minchinia mercenariae
AJ457811 Gromia oviformis
57
65
87
79
56
78
94
92
AY827842 Amastigomonas debruynei
Sarcomonadea
Imbricatea
Thecofilosea
Proteomyxidea
Chlorarachnea
Ascetosporea/
Gromiidea
Phytomyxea
CRN02
1 μm
Bacteria cells ml-1
Minorisa minuta
Minorisa minuta cells ml-1
Time / days
Bacteria
0
1.E+07
8.E+06
6.E+06
4.E+06
2.E+06
0
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

4. An abundant and widespread predator
CARD-FISH/DAPI
del Campo el al. 2013 The ISME Journal
<1.5 m
2.5-3 m
1.5-2.5 m
Cell size distribution

5. Eukaryotic plankton diversity in photic oceans
3 years 6 oceanic regions 154 stations 720 rosettes 1360 nets 28420 samples

6. The deck
Red Sea, January 2010

7. A40 pump 20Hz >300L record
time & flow rate: 5μm GPSS codends
1L cylinder A
“genomics”
4 filter towers
PC 47mm 5μm
4°C, ‘til station end
-20°C
1 x RNA
4°C, ‘til station end
-20°C
500ml into 2LNalgene
“morpho 5-20μm”
2 x Formol
50ml into 60 ml
Nalgene + 2ml
37% neutr. Form
50 ml Falcon
+ 5 ml 37%
neutr. Form
1h dark
4°C
1L cylinder B
“genomics”
4 filter towers
PC 47mm 5μm
Vacuum dry mb Vacuum dry mb
2 x FISH
Remove Form and
add 40 ml PBS
2 x SCG
-20°C
2 x SEM
2mb into 5ml cryo
+ 4ml RNAlater
2mb into 5ml cryo
+ 4ml RNAlater
60ml Nalgene + 5 ml
37% neutr. Form
4 ml into 5 ml vials
+ 0.6 ml
Glyc Betaine
Mb onto Petri Slide
air dry
3 L into 8L Nalgene
50ml Falcon +
2 ml Gluta
4°C 2 x TEM
50ml into 60 ml
Brown bottles
+ 100μl Lugol
4°C 2 x Lugol
50ml
PC 47mm 5μm
1 x HTM
PC 25mm 0.8 μm,
Rinse DD H2O
4°C
1h dark
4°C
-20°C
1 x DNA
Mb onto Petri Slide,
air dry
RT
PROTIST-core / small fractions / 5 – 20 μm

8. Single Cell Genomics
• Preservation using Glycine Betaine.
• 2 types of samples were preserved - whole water and the 5-20 μm fraction.
• 2 depths were sampled - surface and DCM.
• At each long station (CORE) - a total of 8 samples were collected.
Rinke el al. 2013 Nature

9. 7 stations
903 SAGs
158 OTU97
del Campo el al. 2014 TREE
Retrieved SAGs from Tara

10. Chlorarachnea SAGs
AJ418792_Trinema_enchelys
AJ418789_Trachelocorythion_pul
AJ418786_Euglypha_filifera
80
AJ418791_Assulina_muscorum
AJ418790_Tracheleuglypha_denta
X81811_P_chromatophora
AB453002_Ovulinata_sp_YPF501
AJ418793_Cyphoderia_ampulla
AY180004_CCI66_18S
AF411262_Allas_diplophysa
AF411261_Thaumatomonas_sp_ATCC
AF411259_Thaumatomonas_seravin
91
AY268040_Allas_sp_JJP_2003
HQ121434_Nudifila_producta_CCA
EU484394_Auranticordis_quadriv
AJ561116_Pseudopirsonia_mucosa
FJ919772_Clautriaria_biflagell
FJ824131_Cercozoa_sp_CC_2009b
AJ514867_Rhogostoma_minus
AF290539_Cryothecomonas_aestiv
AF290540_Protaspa_longipes
FJ824122_Protaspa_obliqua
FJ824126_Cercozoa_sp_CC_2009a
DQ303922_Ebria_tripartita_1_18
AY266292_Aulosphaera_trigonopa
AY266293_Coelodendrum_ramosiss
AJ418794_Pseudodifflugia_cf_gr
AA539_F03_CERC_H
DQ211593_Gyromitus_sp_HFCC94
AA538_O09_CERC_H
FJ153695_GoC4_B04
AF411285_Metopion_fluens
EU709153_Sandona_ubiquita
U42447_Neoheteromita_globosa
AF411278_Metopion_like_sp_SA
AF411276_Bodomorpha_minima
AJ305043_Proleptomonas_faecico
DQ199663_Aurigamonas_solis
AY748806_Agitata_tremulans
AF411265_Allantion_sp_ATCC_507
FJ790696_Cercomonas_ambigua_WA
FJ790710_Cercomonas_celer_C_51
FJ790704_Cercomonas_clavidefer
FJ790706_Cercomonas_magna_IVY8
85
87
AF411267_Cercomonas_alexieffi
FJ790702_Cercomonas_braziliens
AF411269_Cercomonas_edax
FJ790717_Cavernomonas_stercori
FJ790731_Paracercomonas_saepen
FJ790724_Paracercomonas_oxonie
FJ790721_Paracercomonas_produc
FJ790725_Paracercomonas_crassi
AF411271_Cercomonas_sp_Tempisq
EU567256_Limnofila_oxoniensis
EU567254_Limnofila_longa
AB196_E21_CERC_H
AF174374_Massisteria_marina
AF411283_Mesofila_limnetica
AY305010_Hedriocystis_reticula
AY305009_Clathrulina_elegans
AF289081_Nanofila_marina
EF405665_Minimassisteria_diva
HQ121441_Micrometopion_nutans
X70809_C_reptans
U03477_Chlorarachnion_reptans
AB452995_Partenskyella_glossop
GQ863815_AMT15_15_10m_377
DQ369018_UEPAC33p4
EF622546_Chlorarachniophyceae_
EF622547_Gymnochlora_dimorpha
AB453013_Chlorarachniophyceae_
(0/0/2)
(1/0/2)
(0/2/5)
(0/0/0)
AF076170_Amorphochlora_amoebif
AB504333_Chlorarachniophyceae_
U02075_CSU02075_Chlorarachnion
FJ973362_Bigelowiella_sp
AB232_I17_CHLR_P
AF054832_Bigelowiella_natans
AF076172_Norrisiella_sphaerica
AF076169_Lotharella_globosa
AF054890_Lotharella_vacuolata
AB233_K06_CHLR_P
AA534_M22_CHLR_P
JX272635_Minorisa_minuta
AB535_P20_CHLR_H
JX457429_EukT1B3_1_12
JX457428_EukT3B3_1_5
DQ369015_UEPACLp3
HM369552_MS605_30
DQ314819_NW617_36
DQ314817_NOR26_1
AY620255_Metromonas_simplex
HQ121439_Tremula_longifila
100
AJ457815_Gromia_oviformis
(0/0/0)
(5/0/2)
(0/0/0)
(3/0/26)
(2/0/26)
(4/7/240)
FJ518816_Minchinia_mercenariae
U20858_Haplosporidium_costale
EF514503_Corallomyxa_sp_tenera
0.1
AB504336_Chlorarachniophyceae_
AB232_J07_CHLR_P
AF076171_Gymnochlora_stellata
EF622548_Chlorarachniophyceae_
U03479_Bigwlowiella_longifila
EF622539_Lotharella_reticulosa
DQ504353_LC104_3EP_8
EU567255_Limnofila_anglica
EF622538_Lotharella_oceanica
AB535_F18_CHLR_H
JF698748_MALINA_St390_3m_Pico_
AF310898_Polymyxa_graminis
EU701015_Plasmodiophora_brassi
AF310904_Phagomyxa_odontellae
RAD_AB246693_Larcopyle_butschl
AY266294_Aulacantha_scolymanth
FJ790709_Cercomonas_parincurva
AB535_A10_CHLR_H
AA538_D04_CHLR_H
AA538_A15_CERC_H
AF310900_Sorosphaera_veronicae
AY941201_Platyreta_germanica_G
RAD_AY268045_Sticholonche_sp
RAD_GU246570_Acanthometra_sp
U20319_Minchinia_teredinis_199
AF411281_Spongomonas_minima_AT
EF173002_SSRPE06
U19538_Haplosporidium_nelsoni
U47852_Urosporidium_crescens
AY268044_Filoreta_marina
70
97
60
98
85
100
89
98
96
100
100
39
73
60
78
78
65
100
83
59
96
100
100
100
96
91
83
68
100
78
100
100
100
100
47
57
100
65
100
59
100
90
96
100
81
92
63
100
67
85
57
93
96
79
71
(0/3/0)
(0/14/29)
(2/0/0)
X70809_C_reptans
U03477_Chlorarachnion_reptans
AB504336_Chlorarachniophyceae_
AB232_J07_CHLR_P
AB452995_Partenskyella_glossop
GQ863815_AMT15_15_10m_377
DQ369018_UEPAC33p4
AF076171_Gymnochlora_stellata
EF622546_Chlorarachniophyceae_
EF622547_Gymnochlora_dimorpha
AB453013_Chlorarachniophyceae_
(0/0/2)
(1/0/2)
(0/2/5)
(0/0/0)
AF076170_Amorphochlora_amoebif
AB504333_Chlorarachniophyceae_
EF622548_Chlorarachniophyceae_
U02075_CSU02075_Chlorarachnion
U03479_Bigwlowiella_longifila
96
FJ973362_Bigelowiella_sp
AB232_I17_CHLR_P
AF054832_Bigelowiella_natans
AF076172_Norrisiella_sphaerica
AF076169_Lotharella_globosa
EF622539_Lotharella_reticulosa
EF622538_Lotharella_oceanica
AF054890_Lotharella_vacuolata
100
AB233_K06_CHLR_P
AB535_A10_CHLR_H
AA534_M22_CHLR_P
AB535_F18_CHLR_H
JX272635_Minorisa_minuta
EF173002_SSRPE06
AA538_D04_CHLR_H
AB535_P20_CHLR_H
JX457429_EukT1B3_1_12
JX457428_EukT3B3_1_5
DQ504353_LC104_3EP_8
DQ369015_UEPACLp3
HM369552_MS605_30
DQ314819_NW617_36
DQ314817_NOR26_1
JF698748_MALINA_St390_3m_Pico_
AA538_A15_CERC_H
AY620255_Metromonas_simplex
60
100
47
59
100
67
93
(0/0/0)
(5/0/2)
(0/0/0)
(3/0/26)
(2/0/26)
(4/7/240)
(0/3/0)
(0/14/29)
(2/0/0)
Core Cercozoa
17 SAGs
26 GenBank env seqs
330 HTS seqs
Chlorarachnea
Haplosporidia + Gromia
Phytomixea
Radiolaria

11. Secondary endosymbiosis (possible scenarios)
Apicomplexa dinoflagellates
primary endosymbiosis
secondary endosymbiosis
primary endosymbiosis
tertiary endosymbiosis
(diatom)
tertiary endosymbiosis
(haptophyte)
secondary endosymbiosis
secondary endosymbiosis
serial secondary
endosymbiosis
(green alga)
stramenopiles
ciliates
Durinskia
Karlodinium
Dinophysis
Lepididinium
Paulinella
chlorarachniophytes
euglenids
green algae
red algae
glaucophytes
tertiary endosymbiosis
(cryptomonad)
haptophytes
cryptomonads
land plants
?
Heterotrophic Chlorarachnea. Basal
Chlorarachnea have no chloroplast.
Glutton Chlorarachnea. Basal
Chlorarachnea have no chloroplast, but
they like to eat phototrophic organisms.
Phototrophic Chlorarachnea. Basal
Chlorarachnea have chloroplast but we
have not seen it.
Quantic Chlorarachnea. Basal
Chlorarachnea may have and may have
no chloroplast.
Keeling el al. 2010 Proc Trans R Soc B

12. Retrieve and analyse partial single cell genomes from the selected basal
Chlorarachnea to determine the presence or absence of chloroplast and/or
nucleomorph.
Sequence the four Minorisa minuta SAGs in order to retrieve a high quality
genome. The interest on this organisms rests in both its ecological and
evolutionary role.
Keep on culturing, because having SAGs is useful, but having cultures is even
better.
Minorisa minuta is one of the most abundant and the smallest predator of the
oceans. So, we will keep looking for it at HTS databases in order to know more
about its distribution and its related lineages.
javier.delcampo@botany.ubc.ca
www.fonamental.cat
@fonamental
Future perspectives

13. Iñaki Ruiz-Trillo
Fabrice Not
Daniel Vaulot
Colomban de Vargas
Irene Forn
Aknowledgements
The Keeling lab

14. The 18S collaborative annotation initiative
We want
YOU!
www.fonamental.cat/18S/
Join at: