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Minorisa minuta and the transition to secondary plastid endosymbiosisA 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, 2014Protist 2014
Banff, AB
Biases
Pedrós-Alió 2006 Trends in Microbiology
del Campo el al. 2013 The ISME Journal
Minorisa minuta, an atypical Chlorarachniophyte
CARD-FISH/DAPI
del Campo el al. 2013 The ISME Journal
An abundant and widespread predator
Cell size distribution
3 years 6 oceanic regions 154 stations 720 rosettes 1360 nets 28420 samples
Eukaryotic plankton diversity in photic oceans
Red Sea, January 2010
The deck
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
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
2 x SCG
2 x SEM
2mb into 5ml cryo+ 4ml RNAlater
2mb into 5ml cryo+ 4ml RNAlater
4 ml into 5 ml vials+ 0.6 ml
Glyc Betaine
Mb onto Petri Slideair dry
-20°C
3 L into 8L Nalgene
50ml Falcon +2 ml Gluta 2 x TEM4°C
50ml into 60 ml Brown bottles+ 100μl Lugol
2 x Lugol4°C
50mlPC 47mm 5μm
60ml Nalgene + 5 ml 37% neutr. Form
1 x HTM
Remove Form and add 40 ml PBS
PC 25mm 0.8 μm,Rinse DD H2O
50ml into 60 mlNalgene + 2ml
37% neutr. Form4°C
1h dark 4°C
-20°C
1 x DNAMb onto Petri Slide,
air dryRT
PROTIST-core / small fractions / 5 – 20 µm
• 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.
Single Cell Genomics
Rinke el al. 2013 Nature
del Campo el al. 2014 TREE
7 stations
903 SAGs
158 OTU97
Retrieved SAGs from Tara
0.1
FJ790721_Paracercomonas_produc
DQ504353_LC104_3EP_8
EF622548_Chlorarachniophyceae_
AJ514867_Rhogostoma_minus
EU567255_Limnofila_anglica
EF622538_Lotharella_oceanica
AF411283_Mesofila_limnetica
AB535_F18_CHLR_H
JF698748_MALINA_St390_3m_Pico_
RAD_AB246693_Larcopyle_butschl
AY266294_Aulacantha_scolymanth
FJ790709_Cercomonas_parincurva
AB535_A10_CHLR_H
AA538_D04_CHLR_H
AJ418792_Trinema_enchelys
AB196_E21_CERC_H
AF411269_Cercomonas_edax
AJ418786_Euglypha_filifera
AY180004_CCI66_18S
AF076171_Gymnochlora_stellata
DQ211593_Gyromitus_sp_HFCC94
AF411261_Thaumatomonas_sp_ATCC
U03479_Bigwlowiella_longifila
AF411259_Thaumatomonas_seravin
AJ561116_Pseudopirsonia_mucosa
FJ790724_Paracercomonas_oxonie
EF405665_Minimassisteria_diva
AF411285_Metopion_fluens
AF174374_Massisteria_marina
HQ121434_Nudifila_producta_CCA
AB232_J07_CHLR_P
FJ824122_Protaspa_obliqua
FJ824126_Cercozoa_sp_CC_2009a
EF622539_Lotharella_reticulosa
AF411278_Metopion_like_sp_SA
AA538_A15_CERC_H
AF310900_Sorosphaera_veronicae
AB504336_Chlorarachniophyceae_
AJ418789_Trachelocorythion_pul
FJ790717_Cavernomonas_stercori
AF076172_Norrisiella_sphaerica
U03477_Chlorarachnion_reptans
AY941201_Platyreta_germanica_G
EU567254_Limnofila_longa
EF514503_Corallomyxa_sp_tenera
AJ418790_Tracheleuglypha_denta
AY305010_Hedriocystis_reticula
JX457429_EukT1B3_1_12
AY305009_Clathrulina_elegans
RAD_AY268045_Sticholonche_sp
AF411276_Bodomorpha_minima
AB452995_Partenskyella_glossop
AB453002_Ovulinata_sp_YPF501
FJ790706_Cercomonas_magna_IVY8
AF310904_Phagomyxa_odontellae
EU484394_Auranticordis_quadriv
DQ314819_NW617_36
EU701015_Plasmodiophora_brassi
AB504333_Chlorarachniophyceae_
AF290540_Protaspa_longipes
HM369552_MS605_30
AY266292_Aulosphaera_trigonopa
DQ314817_NOR26_1
AF411265_Allantion_sp_ATCC_507
FJ790725_Paracercomonas_crassi
FJ973362_Bigelowiella_sp
AF076169_Lotharella_globosa
FJ790710_Cercomonas_celer_C_51
GQ863815_AMT15_15_10m_377
RAD_GU246570_Acanthometra_sp
EU709153_Sandona_ubiquita
AY748806_Agitata_tremulans
AF411267_Cercomonas_alexieffi
FJ790731_Paracercomonas_saepen
U02075_CSU02075_Chlorarachnion
AJ418793_Cyphoderia_ampulla
EF622547_Gymnochlora_dimorpha
AY620255_Metromonas_simplex
AF054890_Lotharella_vacuolata
AJ457815_Gromia_oviformis
AA538_O09_CERC_H
AY268040_Allas_sp_JJP_2003
AB453013_Chlorarachniophyceae_
AB535_P20_CHLR_H
FJ824131_Cercozoa_sp_CC_2009b
AF310898_Polymyxa_graminis
AF290539_Cryothecomonas_aestiv
EF622546_Chlorarachniophyceae_
AB233_K06_CHLR_P
FJ518816_Minchinia_mercenariae
U20858_Haplosporidium_costale
AA534_M22_CHLR_P
HQ121439_Tremula_longifila
AJ418791_Assulina_muscorum
JX272635_Minorisa_minuta
U20319_Minchinia_teredinis_199
AF411281_Spongomonas_minima_AT
AA539_F03_CERC_H
EF173002_SSRPE06
FJ153695_GoC4_B04
DQ369018_UEPAC33p4
U19538_Haplosporidium_nelsoni
U47852_Urosporidium_crescens
HQ121441_Micrometopion_nutans
AJ418794_Pseudodifflugia_cf_gr
AF411262_Allas_diplophysa
AY266293_Coelodendrum_ramosiss
AJ305043_Proleptomonas_faecico
X70809_C_reptans
X81811_P_chromatophora
U42447_Neoheteromita_globosa
FJ919772_Clautriaria_biflagell
AB232_I17_CHLR_P
DQ199663_Aurigamonas_solis
FJ790696_Cercomonas_ambigua_WA
FJ790702_Cercomonas_braziliens
DQ303922_Ebria_tripartita_1_18
FJ790704_Cercomonas_clavidefer
EU567256_Limnofila_oxoniensis
AF411271_Cercomonas_sp_Tempisq
AF289081_Nanofila_marina
AF076170_Amorphochlora_amoebif
AY268044_Filoreta_marina
DQ369015_UEPACLp3
AF054832_Bigelowiella_natans
JX457428_EukT3B3_1_5
100
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
87
83
68
100
78
100
100
80
100
100
47
57
100
65
100
59
100
90
96
100
81
85
92
63
100
67
85
57
93
96
79
71
91
(0/3/0)
(4/7/240)
(0/14/29)
(2/0/0)
(2/0/26)
(3/0/26)
(0/0/0)
(5/0/2)
(0/0/0)
(0/0/0)
(0/2/
0 .1
FJ790721_Paracercomonas_produc
DQ504353_LC104_3EP_8
EF622548_Chlorarachniophyceae_
AJ514867_Rhogostoma_minus
EU567255_Limnofila_anglica
EF622538_Lotharella_oceanica
AF411283_Mesofila_limnetica
AB535_F18_CHLR_H
JF698748_MALINA_St390_3m_Pico_
RAD_AB246693_Larcopyle_butschl
AY266294_Aulacantha_scolymanth
FJ790709_Cercomonas_parincurva
AB535_A10_CHLR_H
AA538_D04_CHLR_H
AJ418792_Trinema_enchelys
AB196_E21_CERC_H
AF411269_Cercomonas_edax
AJ418786_Euglypha_filifera
AY180004_CCI66_18S
AF076171_Gymnochlora_stellata
DQ211593_Gyromitus_sp_HFCC94
AF411261_Thaumatomonas_sp_ATCC
U03479_Bigwlowiella_longifila
AF411259_Thaumatomonas_seravin
AJ561116_Pseudopirsonia_mucosa
FJ790724_Paracercomonas_oxonie
EF405665_Minimassisteria_diva
AF411285_Metopion_fluens
AF174374_Massisteria_marina
HQ121434_Nudifila_producta_CCA
AB232_J07_CHLR_P
FJ824122_Protaspa_obliqua
FJ824126_Cercozoa_sp_CC_2009a
EF622539_Lotharella_reticulosa
AF411278_Metopion_like_sp_SA
AA538_A15_CERC_H
AF310900_Sorosphaera_veronicae
AB504336_Chlorarachniophyceae_
AJ418789_Trachelocorythion_pul
FJ790717_Cavernomonas_stercori
AF076172_Norrisiella_sphaerica
U03477_Chlorarachnion_reptans
AY941201_Platyreta_germanica_G
EU567254_Limnofila_longa
EF514503_Corallomyxa_sp_tenera
AJ418790_Tracheleuglypha_denta
AY305010_Hedriocystis_reticula
JX457429_EukT1B3_1_12
AY305009_Clathrulina_elegans
RAD_AY268045_Sticholonche_sp
AF411276_Bodomorpha_minima
AB452995_Partenskyella_glossop
AB453002_Ovulinata_sp_YPF501
FJ790706_Cercomonas_magna_IVY8
AF310904_Phagomyxa_odontellae
EU484394_Auranticordis_quadriv
DQ314819_NW617_36
EU701015_Plasmodiophora_brassi
AB504333_Chlorarachniophyceae_
AF290540_Protaspa_longipes
HM369552_MS605_30
AY266292_Aulosphaera_trigonopa
DQ314817_NOR26_1
AF411265_Allantion_sp_ATCC_507
FJ790725_Paracercomonas_crassi
FJ973362_Bigelowiella_sp
AF076169_Lotharella_globosa
FJ790710_Cercomonas_celer_C_51
GQ863815_AMT15_15_10m_377
RAD_GU246570_Acanthometra_sp
EU709153_Sandona_ubiquita
AY748806_Agitata_tremulans
AF411267_Cercomonas_alexieffi
FJ790731_Paracercomonas_saepen
U02075_CSU02075_Chlorarachnion
AJ418793_Cyphoderia_ampulla
EF622547_Gymnochlora_dimorpha
AY620255_Metromonas_simplex
AF054890_Lotharella_vacuolata
AJ457815_Gromia_oviformis
AA538_O09_CERC_H
AY268040_Allas_sp_JJP_2003
AB453013_Chlorarachniophyceae_
AB535_P20_CHLR_H
FJ824131_Cercozoa_sp_CC_2009b
AF310898_Polymyxa_graminis
AF290539_Cryothecomonas_aestiv
EF622546_Chlorarachniophyceae_
AB233_K06_CHLR_P
FJ518816_Minchinia_mercenariae
U20858_Haplosporidium_costale
AA534_M22_CHLR_P
HQ121439_Tremula_longifila
AJ418791_Assulina_muscorum
JX272635_Minorisa_minuta
U20319_Minchinia_teredinis_199
AF411281_Spongomonas_minima_AT
AA539_F03_CERC_H
EF173002_SSRPE06
FJ153695_GoC4_B04
DQ369018_UEPAC33p4
U19538_Haplosporidium_nelsoni
U47852_Urosporidium_crescens
HQ121441_Micrometopion_nutans
AJ418794_Pseudodifflugia_cf_gr
AF411262_Allas_diplophysa
AY266293_Coelodendrum_ramosiss
AJ305043_Proleptomonas_faecico
X70809_C_reptans
X81811_P_chromatophora
U42447_Neoheteromita_globosa
FJ919772_Clautriaria_biflagell
AB232_I17_CHLR_P
DQ199663_Aurigamonas_solis
FJ790696_Cercomonas_ambigua_WA
FJ790702_Cercomonas_braziliens
DQ303922_Ebria_tripartita_1_18
FJ790704_Cercomonas_clavidefer
EU567256_Limnofila_oxoniensis
AF411271_Cercomonas_sp_Tempisq
AF289081_Nanofila_marina
AF076170_Amorphochlora_amoebif
AY268044_Filoreta_marina
DQ369015_UEPACLp3
AF054832_Bigelowiella_natans
JX457428_EukT3B3_1_5
100
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
87
83
68
100
78
100
100
80
100
100
47
57
100
65
100
59
100
90
96
100
81
85
92
63
100
67
85
57
93
96
79
71
91
(0/3/0)
(4/7/240)
(0/14/29)
(2/0/0)
(2/0/26)
(3/0/26)
(0/0/0)
(5/0/2)
(0/0/0)
(0/0/0)
(0/2/
Core Cercozoa
Chlorarachnea
Haplosporidia + Gromia
Phytomixea
Radiolaria
17 SAGs26 GenBank env seqs330 HTS seqs
Chlorarachnea SAGs
primary endosymbiosis
primary endosymbiosis
secondary endosymbiosis
secondary endosymbiosis
secondary endosymbiosis
serial secondaryendosymbiosis
(green alga)
tertiary endosymbiosis(diatom)
stramenopiles
ciliates
Dinophysis
Lepididinium
euglenids
chlorarachniophytes
Paulinella
dinoflagellatesApicomplexa
green algae
Durinskia
Karlodinium
red algae
glaucophytes
tertiary endosymbiosis(cryptomonad)
tertiary endosymbiosis(haptophyte)
haptophytes
cryptomonads
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.
Secondary endosymbiosis (possible scenarios)
Keeling el al. 2010 Proc Trans R Soc B
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.
@fonamental
Future perspectives
Fabrice NotDaniel Vaulot
Colomban de Vargas
Irene Forn
Iñaki Ruiz-Trillo
Aknowledgements
The Keeling lab
The 18S collaborative annotation initiative
www.fonamental.cat/18S/
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![The eukaryotic tree of life: endosymbiosis takes its TOL · cells, a process known as ‘secondary’ endosymbiosis [9,10]. In the case of secondary endosymbiosis, the plastid acts](https://img.pdfslide.net/doc/110x75/5fbd1f246e87dd36774b7a8a/the-eukaryotic-tree-of-life-endosymbiosis-takes-its-cells-a-process-known-as-asecondarya.jpg)
