Qu’a apporté la génomique à la phylogénie des animaux ?

Hervé Philippe

Département de Biochimie, Centre Robert Cedergren, Université de Montréal, Succursale Centre-Ville, Montréal, Québec H3C3J7, Canada

Cambrian explosion: a paleontological perspective

Marrella

HalkieriaPikaia

Aysheaia

Cambrian explosion: a molecular perspective

ChoanoflagellataPoriferaPoriferaPoriferaCnidariaCnidariaEcdysozoaEcdysozoaEcdysozoaLophotrochozoaLophotrochozoaLophotrochozoaLophotrochozoaDeuterostomiaDeuterostomiaDeuterostomiaDeuterostomia

700 600 500 400 300 200 100 0

Cambrian explosion

Molecular phylogenies should resolve series of

speciation events separated by a few

millions of years

MYa

(1) Inadequate selection of sequences (non-orthologous, saturated, etc.)

(2) Inadequate tree reconstruction method

(3) Inadequate taxon sampling

(4) Rapid diversification of species

Lack of resolution in molecular phylogenetics

Points (1), (2) and (3) are always mixed:

• A (simplistically) theoretical overview

• Analyses of several case studies

• A molecular dating approach

Cambrian explosion: a molecular perspective

ChoanoflagellataPoriferaPoriferaPoriferaCnidariaCnidariaEcdysozoaEcdysozoaEcdysozoaLophotrochozoaLophotrochozoaLophotrochozoaLophotrochozoaDeuterostomiaDeuterostomiaDeuterostomiaDeuterostomia

700 600 500 400 300 200 100 0

Cambrian explosion

MYa

18S Ribosomal RNA (~1000 positions): ~100 substitutions over 500 MY

resolution for branches with T 15 MY

Bootstrap support 95% requires 3 substitutions on the corresponding branch (Felsenstein, 1985)

T

50 genes (Rokas et al. 2005, 12060 positions): ~2400 substitutions over 500 MY

resolution for branches with T 0.7 MY

146 genes (Delsuc et al. 2006, 33800 positions): ~7000 substitutions over 500 MY

resolution for branches with T 0.25 MY

true history

1

2

3

Inferredtrees

1: 80%

2: 25%

3: 10%

1: 100%

2: 100%

3: 100%

21

3

12000positions

Phylogenetic signal

Phylogenetic signal

1000 positions

21 3

Comparison of ML SSU and LSU trees (A and B, respectively)

Medina M. et.al. PNAS 2001;98:9707-9712

Rokas et al. (2005) Animal evolution and the molecular signature of radiations compressed in time. Science, 310:1993-1998

50 genes (12,060 amino acid positions), ML RtREV+I+ / MP bootstrap support

true history

1

2

3

Inferredtrees

1: 80%

2: 25%

3: 10%

1: 100%

2: 100%

3: 100%

21

3

12000positions

Phylogenetic signal

Phylogenetic signal

1000 positions

21 3

Non-phylogenetic signal

Sequence evolves according to a very complex and heterogeneous process that our tree reconstruction method approximates as best as they can using elaborated model of

sequence evolutionReal complexities: mutation process is not homogeneous over time and across the genome, population structure is not homogeneous over time, selective pressures are not homogeneous over time and across the genome

Nucleotide compositions are heterogeneous across species, evolutionary rate is heterogeneous across positions and over time (heterotachy), substitution process is heterogeneous across positions and over time, positions are inter-dependent, etc.

All the complexities that are not adequately handled by our oversimplified models of sequence evolution can imply

systematic biases, which are referred here as non-phylogenetic signal

true history

1

2

3

Phylogenetic signal

Inferredtrees

1: 70%

2: 5%

3: 10%

1: 100%

2: 5%

3: 100%

21

3

1000 positions

12000positions

Phylogenetic signal and non-phylogenetic signal

21 3apparent

signal

21 3

21 3Non-phylogenetic

signal

Systematic error: the error in phylogenetic estimates that is due to the failure of the reconstruction method to account fully for multiple substitutions (in a probabilistic framework, the properties of the data)

Systematic errors (inconsistency)

LONG BRANCH ATTRACTION (Felsenstein, 1978)

A

DB

C

p

q q

p<q2

A

DB

C

A

D

CB

A

D

BC

Systematic errors will not disappear with phylogenomics, and may indeed

become more apparent

C

AA

C

Multiple substitutions at the same position

ACGC

AG

Tree building artefact

C

AA

C

Rokas et al. (2005) Science, 310:1993-1938

50 genes (12,060 amino acid positions), ML RtREV+I+ / MP bootstrap support

ML MP

99 56

94 55

97 51

72 36

84 54

100 75

43 74

true history

1

2

3

Phylogenetic signal

21

3

Inferredtrees

1: 100%

2: 99%

3: 84%

1: 75%

2: 56%

3: 54%

12000 positions

12000positions

Phylogenetic signal and non-phylogenetic signal

apparentsignal

21 3

21 3

21

3

Non-phylogeneticsignal

ML

MP

ML MP

99 56

94 55

97 51

72 36

84 54

100 75

43 74

QuickTime™ et undécompresseur TIFF (LZW)

sont requis pour visionner cette image.

Phylogenomics yields incongruent results

PLoS Biology Nature Current Biology

AmoebozoaAscomycotaBasidomycotaCalcareaHexactinellida

DemospongiaeScyphozoaAnthozoaTrichoplax adhaerens

ChoanoflagellataEchinodermata

MammaliaHexapodaCrustacea

BivalviaAnnelida

GastropodaHemichordata

0.1

Mitochondrial ATP synthase F0 subunit 6

Single gene phylogeny of Schierwater et al. (2009)

AnthozoaHexapoda

CrustaceaScyphozoa

PriapulidaBivalvia

EchinodermataHexactinellida

CalcareaDemospongiae

HydrozoaMammalia

CtenophoraTrichoplax adhaerens

ChoanoflagellataAmoebozoa

ExcavataAscomycotaBasidomycota

ChromalveolataAnnelida

0.1

Cytosolic HSP70

NON-HOMOLOGOUS

PARALOGOUS

ER HSP70

Single gene phylogeny of Schierwater et al. (2009)

RAC1

CDC42

BasidomycotaAscomycota

ChoanoflagellataCalcarea

AnthozoaHexactinellida

ChromalveolataMammalia

EchinodermataHexapoda

Trichoplax adhaerensDemospongiae

Amoebozoa0.1

Small RAS-like GTPase AmoebozoaAscomycotaBasidomycota

ChoanoflagellataTrichoplax_adhaerens

ExcavataChromalveolata

EchinodermataDemospongiae

HexactinellidaAnthozoa

MammaliaAnnelidaHexapoda

CubozoaCalcarea

PriapulidaGastropoda

Hydrozoa

0.1

GTP-binding nuclear protein Ran

Single gene phylogeny of Schierwater et al. (2009)

DemospongiaeCubozoa

ScyphozoaAnthozoa

MammaliaHexapoda

Trichoplax adhaerensEchinodermata

Hydrozoa

0.1

Pair box domain protein PAX-B

AnthozoaChromalveolata

ExcavataAscomycota

Trichoplax adhaerensMammaliaBasidomycota

AmoebozoaHexapoda

0.1

DNA directed RNA polymerase

Pol II

Pol III

Porifera

0.1

4

9

CnidariaBilateria

ExcavataCiliophora

Amoebozoa

AscomycotaBasidiomycota

Choanoflagellata

CalcareaDemospongiae

HexactinellidaCtenophora

Placozoa

5398

2762

0.1

38

36

44

40

23

ExcavataCiliophora

AmoebozoaAscomycota

BasidiomycotaChoanoflagellataCalcareaDemospongiae

HexactinellidaCtenophora

Placozoa 4

9

CnidariaBilateria

Porifera

Contaminated datasetSchierwater et al. (2009) PLoS Biol 7(1): e1000020

Clean datasetPhilippe et al. (2011) PLoS Biol in press

Dunn et al. : 150 genes 24,708 positions

Contaminations:Symsagitiferra: 13 genes (including 6 Chlorophyta, 2 Ciliophora, 2 Bacteria)

4 Neochildia (Microsporidia)2 Saccoglossus (Mus)2 Acanthoscurria (angiosperm)2 Hydra (Artemia)1 Oscarella (Pseudomonas)1 Asterina (Bacteria)1 Dugesia (Gallus)1 Xiphinema (Lumbricus)1 Monosiga (Rhizopus)1 Macrostomum2 Trichinella2 Priapulus1 Branchiostoma

Dunn et al.: 150 genes 24,708 positions

Frameshifts: 63 concerned speciesDrosophila 2Paraplanocera 3Echinoderes 4Xenoturbella 4Chaetopterus 5Cyanea 5Cristatella 6Platynereis 6Spinochordodes 6Cryptococcus 8Spadella 8Mnemiopsis 9Bugula 10Gnathostomula 10Hydra 10Sphaeroforma 10Turbanella 10Chaetoderma 15Myzostoma 15Scutigera 16Carcinus 18

Lumbricus 20Ptychodera 20Euperipatoides 21Carcinoscorpius22Symsagittifera 22Chaetopleura 23Homo 25Boophilus 30Hypsibius 30Richtersius 30Daphnia 32Asterina 35Anoplodactylus 40Argopecten 43Xiphinema 43Acropora 45Dugesia 46Brachionus 50Ciona 50Branchiostoma 52Hydractinia 53

Haementeria 54Flaccisagitta 55Strongylocentrotus 55Acanthoscurria 58Aplysia 58Saccoglossus 60Capsaspora 68Gallus 73Phoronis 87Capitella 93Echinococcus 100Ferrenopenaeus 112Monosiga 118Schmidtea 129Oscarella 141Mytilus 151Euprymna 201Trichinella 281Crassostrea 296Macrostomum 382Biomphalaria 384

Frameshifts: 3868 “invented” amino acids

Many single point errors :A total 970 errors(in large part due to the use of

erroneous mitochondrial genetic code!)

DUNN: 150 genes 21,152 positions55.6% of missing data

UPDUNN: 150 genes 18,463 positions35.6% of missing data

Several genes with paralogy issues:2-5 intractable problems10-20 tractable problems

5 introns:AnoplodactylusChaetopterusCionaThemisteTrichinella

SaccharomycesCryptococcus

SphaeroformaAmoebidium

CapsasporaMonosiga

AmphimedonOscarella

mertensiidMnemiopsis

HydractiniaHydra

CyaneaNematostelAcropora

0.2

SymsagittiferaNeochildia

HomoGallus

CionaBranchiostoma

XenoturbellaSaccoglossPtychodera

StrongylocentrotusAsterina

GnathostomulaSpadellaFlaccisagitta

PhilodinaBrachionus

MacrostomumParaplanocera

EchinococcusSchmidteaDugesia

PedicellinaCristatella

BugulaPhoronis

TurbanellaTerebrataliaCerebratulusCarinoma

ChaetopterusMyzostoma

ThemistePlatynereis

LumbricusHaementeria

UrechisCapitella

ChaetopleuraChaetodermaEuprymnaMytilusCrassostreaArgopecten

BiomphalariaAplysia

PriapulusEchinoderes

XiphinemaTrichinella

SpinochordodesRichtersius

HypsibiusEuperipatoides

DrosophilaDaphniaFenneropenaeusCarcinus

ScutigeraCarcinoscorpius

AnoplodactylusAcanthoscurriaBoophilus

30

86

58

62

84

BS=100%

70<BS<100

Cnidaria

PoriferaCtenophora

Bilateria

CAT+ model

150 genes 18,463 positions35.6% of missing data

Clean Dunn et al. dataset

00.01

0.020.03

0.040.050.06

0.070.08

0.090.1

A C D E F G H I K L M N P Q R S T V W

A C D E F G H I K L M N P Q R S T V W Y

+ 190 relative rates (ij = ji)+ 20 stationary probabilities (i)

E

CD

FGHIKLMNPQRSTVWY

jijijQ =

∑≠

−=ij

ijii QQ

ji ≠,

[ ]ablQeQlabp =),,|(bal

Model of sequence evolution

WAG matrix

amino acid profiles …

ACD...VWY ACD...VWY ACD...VWY ACD...VWY

Categories (modes): 1 2 3 … K

Heterogeneous (CAT) model : K distinct profiles Homogeneous (WAG) model1 substitution

matrix

Man

Zebrafish

Frog

Fly

Yeast

Amoeba

ParameciumGreen alga

M A E I G R L I E F S A M V D F W Q N R CM A E I G R L V E Y S A M V D F W Q N R CM A D L G K L I D Y S A L V D F W Q N R CM S D I G K L V E F S P M V E F W Q Q K CM S E I G R L V E F T P M V E F W Q N R CL S E L G R L V D F T A M V D F W N N R CL A E L G K L V E Y A P M I D F W Q A R CL S D L G K L I D F S A M I N F W Q N K C

The CAT model of sequence evolution

Lartillot & Philippe (2004) Mol Biol Evol. 21:1095-1109

A C D E F G H I K L M N P Q R S T V W

+ uniform relative rates (ij=ji)+ 20 stationary probabilities (i)

E

CD

FGHIKLMNPQRSTVWY

The CAT model of sequence evolution

Lartillot & Philippe (2004) Mol Biol Evol. 21:1095-1109

To keep the number of parameters low, a category is only defined by a set of stationary probabilities (the

relative rates are uniform), and the number of categories is inferred from the alignment

…A C D E F G H I K L M N P Q R S T V W Y

A C D E F G H I K L M N P Q R S T V W Y

Stable categories inferred by the CAT model

D N

E Q

The size of an amino acid is proportional to

its stationary probability

E

E

E

E

E

E

E

E

E

E

E

EE

E

E

E

EE

D

D

DD

D

D

DDD

D

D

DD

D

D

D

D

Multiple substitutions between two amino acids

# substitutions

What is predicted by evolutionary models?

WAG GTR CAT

1

2

3

4

5

6

7

0

E

E

E

E

E

E

E

E

E

E

E

EE

E

E

E

EE

D

D

DD

D

D

DDD

D

D

DD

D

D

D

D

These multiple substitutions are well handled by the CAT model because this position will be explained by this profile:

Multiple substitutions between two amino acids

# substitutions

What is predicted by WAG replacement matrix?

ChoanoflagellateDemosponge

Calcareous poriferanHexactinelid poriferan

Hydrozoan cnidarianAnthozoan cnidarian

NematodeArthropod

PriapulidAnnelid

MolluskTrematode platyhelminthTriclad platyhelminth

TunicateZebrafishMouseHuman

0.02

98

55

74

76

78

100100

100

95

99

10063

49

5276

Further reduction of non-phylogenetic signal

Alignment of Rokas et al. (2005): 50 genes (12,060 amino acid positions)

Model CAT+, inferred using phylobayes; 100 bootstrap replicates

0

10

20

30

40

50

60

70

80

90

100

MP rtREV+CAT+

Chordates

Protostomes

Ecdysozoa

Lophotrochozoa

Bilaterians

Cnidarians

Poriferans

Reduction of non-phylogenetic signal

Boots

trap

su

pp

ort

0.1

Choanoflagellata

CalcareaHomoscleromorpha

DemospongiaePlacozoa

CtenophoraCnidaria

Bilateria

2

2

3

4

3

9

2298

90

64

62

62

78

3

5 FungiIchthyosporea

99

PoriferaHexactinellida

0.1

80

91

98

45

55

99

38

Choanoflagellata

Calcarea

HomoscleromorphaHexactinellida

Demospongiae

Placozoa

Ctenophora

CnidariaBilateria

2

3

9

22

3

5

3

2

4

FungiIchthyosporea

128 genes30,257 positionsPhilippe et al. (2009) Curr. Biol.

Model CAT+

Philippe et al. (2011) PLOS Biol.

Model WAG+

Improvement of phylogenetic resolution

true history

1

2

3Phylogenetic

signal2

13

Non-phylogeneticsignal

To improve resolution, one has to use the same methods as to avoid systematic errors:1) Complex model of sequence evolution2) Rich taxon sampling3) Removal of fast evolving positions

and taxa

Phylogenomics: phylogenetic signal

as well as non-phylogenetic signal

are abundant

0.1Ctenophora

Placozoa

Hexactinellida

Calcarea

Demospongiae

Choanoflagellata

Cnidaria

86

94

70

44

56

53

3

9 Bilateria

0.1

Porifera

Choanoflagellata

CalcareaHomoscleromorpha

HexactinellidaDemospongiaePlacozoa

CtenophoraCnidaria

Bilateria

2

2

3

4

3

9

2294

96

91

62

90

93

47 species128 genes30,257 positionsPhilippe et al. (2009) Curr. Biol.

Model CAT+

Philippe et al. (2011) PLOS Biol.

Model CAT+

18 speciesSame sampling asSchierwater et al.

M A D I G R L I E F S A M V D F WM G E I G R L V E Y S A M V D F WM A E L G K L I D Y S A L V D F WM T D I G K L V E F S P M V E F WM W D I G R L V E F T P M V E Y WM S D L A R L V D F T A M V D F WM Y D L G K L I D F S A M I N F WM A D I G R L I E F S A M V D Y WM E D I G R L V E Y S A M V D F WM R D L G K L I D Y S A L V D F W

• Hétérogénéité entre les sitesHétérogénéité entre les sites• loi gamma, modèle CAT loi gamma, modèle CAT

• Hétérogénéité au cours du tempsHétérogénéité au cours du temps• modèle covarion, points de changementsmodèle covarion, points de changements

• Hétérogénéité des états de caractères• matrices d’échange : Dayhoff, WAG … LG, GTR

Hétérogénéité des modèles

Hypothèse

QuickTime™ et undécompresseur TIFF (LZW)

sont requis pour visionner cette image.

HétéropécilieHétéropécilievariation temporelle du processus variation temporelle du processus

de substitution en acides aminés de substitution en acides aminés

pour un site donnépour un site donné (( = to vary)= to vary)

HétéropécilieHétéropécilievariation temporelle du processus variation temporelle du processus

de substitution en acides aminés de substitution en acides aminés

pour un site donnépour un site donné (( = to vary)= to vary)

Roure & Philippe (2011) BMC Evol Biol Roure & Philippe (2011) BMC Evol Biol 11:1711:17

13 protéines mitochondriales13 protéines mitochondriales

68 espèces68 espèces

sites retirésaligneme

ntPIPn nb % taille

- - - 1927= 0 168 8.7 1759

≥ e-12 165 8.6 1594≥ e-8 177 9.2 1417≥ e-6 177 9.2 1240≥ e-

4.5 201 10.4 1039

• Sites retirés suivant une Sites retirés suivant une valeur croissante de PIPvaleur croissante de PIPnn

Choanoflagellata

Protostomia

Deuterostomia

Porifera

Cnidaria

CAT+4

• Inférence par CAT+ΓInférence par CAT+Γ44 avec les avec les

jeux réduitsjeux réduits

DonnéesDonnées ProtocoleProtocole

Retrait progressif des sites hétéropécilles

taille de l’alignement

Cnidaria

Choanoflagellata

Protostomia

Deuterostomia

Porifera

Bilateria

4141Roure & Philippe (2011) BMC Evol Biol Roure & Philippe (2011) BMC Evol Biol 11:1711:17

Retrait progressif des sites hétéropécilles

Cnidaria

Choanoflagellata

Protostomia

Deuterostomia

Porifera

taille de l’alignement

Roure & Philippe (2011) BMC Evol Biol Roure & Philippe (2011) BMC Evol Biol 11:1711:17

Retrait progressif des sites hétéropécilles

Cnidaria

Choanoflagellata

Protostomia

Deuterostomia

Porifera

Porifera

Cnidaria

Choanoflagellata

Protostomia

Deuterostomia

Porifera

taille de l’alignement

Roure & Philippe (2011) BMC Evol Biol Roure & Philippe (2011) BMC Evol Biol 11:1711:17

Retrait progressif des sites hétéropécilles

taille de l’alignement

Choanoflagellata

Porifera

Protostomia

Deuterostomia

Cnidaria

Choanoflagellata

Protostomia

Deuterostomia

Porifera

Porifera

Cnidaria

Choanoflagellata

Protostomia

Deuterostomia

Porifera

Roure & Philippe (2011) BMC Evol Biol Roure & Philippe (2011) BMC Evol Biol 11:1711:17

Retrait progressif des sites hétéropécilles

Sites hétéropéciles

taille de l’alignement

Sites à évolution rapide

taille de l’alignement

Le regroupement incorrect des Cnidaires et des Porifères n’est pas dû à la présence de sites à évolution rapide, mais à la présence de sites

hétéropéciles qui est erronément interprétée comme une synapomorphie pour regrouper Cnidaires et Porifères

Roure & Philippe (2011) BMC Evol Biol Roure & Philippe (2011) BMC Evol Biol 11:1711:17

Retrait progressif des sites