Recent advances in molecular phylogenies of actinopterygian fishes Guillermo Ortí University of Nebraska, USA.

Recent advances in molecular phylogenies of

actinopterygian fishes

Recent advances in molecular phylogenies of

actinopterygian fishes

Guillermo OrtíUniversity of Nebraska, USA

Molecular Systematics of Ray-finned Fishes

DeepFin will Advance The Phylogeny of “Fishes”

A Research Coordination Network



1. To promote fish phylogenetics (resolve the fish tree!)

2. To develop cyberinfrastructure, a portal for fish phylogenetics (www.deepfin.org) with networking tools and interconnected relational databases

3. To develop educational material to foster education on fish biodiversity, fish evolution, and current knowledge on the phylogenetic relationships of fishes

1. To promote fish phylogenetics—how far are we

from the “tree of all fishes”??

Integrate all sources of information: Morphology

GeneticsGenetics

Paleontology





Issues with molecular phylogenies based on a single

gene or few loci


gene or few loci• Low resolution or low support

(characters v taxa)• Conflicts among trees inferred

from different loci.– Analytical reasons (base

compositional bias / long branch attraction / heterotachy).

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

GC% at the 3rd codon position of RAG 1GC% at the 3rd codon position of RAG 1

Mean

0.5

1

Elas

mob

ranc

hii

Tetra

poda

Polyp

terif

orm

esBa

sal a

ctin

op.

Ost

eogl

osso

.El

opom

orph

aEl

opom

orph

a

Ost

ario

phys

iO

star

ioph

ysi

Clup

eom

orpg

ha

Protacanthop.Protacanthop.

Stom

iiform

esBa

sal n

eote

leos

tsBa

sal n

eote

leos

ts““ P

arac

anth

op.”

Para

cant

hop.

”

AcanthopterygiiAcanthopterygii

Ogcocephalus

Lophiiformes

Colisa

Arnoglossus

SparusTriglascorpaenids

Gonostomagalaxiids

Albula

Albuliformes

Megalops

Elops

Muraenesox

Zeus

Gasterosteus

0

Engraulis


gene or few loci


gene or few loci• Low resolution or low support

(characters v taxa)• Conflicts among trees inferred from

different loci.– Biological reasons (gene tree vs.

organismal tree)

LineageLineagesortingsorting

Gene Gene duplicationduplication

Horizontal Horizontal transfertransfer

Gene trees within organismal Gene trees within organismal treestrees

“Phylogenomics”: use many (genome-scale) loci to infer

phylogeny

“Phylogenomics”: use many (genome-scale) loci to infer

phylogeny

• Large number of characters will increase statistical power

• Analysis of many independent loci may reduce systematic error

• Genome-scale nuclear gene markers will be more likely to represent organismal evolution

How to collect “phylogenomic” data (from multiple loci)

How to collect “phylogenomic” data (from multiple loci)

• Using available genome databases (model organisms)

• Sequencing cDNA/EST libraries• Directly amplify and sequence

target fragments from genomic DNA using ‘universal’ nuclear markers

How can we find new ‘universal’How can we find new ‘universal’nuclear gene markers???nuclear gene markers???

How can we find new ‘universal’How can we find new ‘universal’nuclear gene markers???nuclear gene markers???

Three criteria to choose “good” nuclear gene markers*

Three criteria to choose “good” nuclear gene markers*

1) Orthologous genes should be easy to identify and amplify in all taxa of interest. To minimize the chance of “mistaken paralogy”, we seek only single-copy genes single-copy genes (so, what about gene duplications?)(so, what about gene duplications?)

* Chenhong Li (UNL) and Guoqing Lu (UN-Omaha)

a1 a3a2

ba

Gene duplication

Taxon 3Taxon 2Taxon 1

a1 a3a2

ba


Gene loss

a1 b3a3b2a2b1

ba

Gene duplication


Gene loss

Gene duplication

Gene loss

2 nd speciation

1st speciation

a cb

2) The amplicon (i.e. target sequences amplified by the PCR primers) should be of reasonable size (exons >800 bp).

zebrafish elongation factor 1-alpha (ef1a)

Three criteria to choose “good” nuclear gene markers


Gonadotropin-releasing hormone

3) The gene should be “reasonably” conserved, so universal primers can be designed and the sequences can be easily aligned.



If we agree with these 3 criteria (single copy, long exon, reasonable conservation) for good nuclear makers, “randomly testing” genes provides a poor chance to finding a good marker (additional criteria are possible)

Directly apply the 3 criteria to screen genomes of two model organisms, zebrafish (Danio rerio) and pufferfish (Takifugu rubripes).

If we agree with these 3 criteria (single copy, long exon, reasonable conservation) for good nuclear makers, “randomly testing” genes provides a poor chance to finding a good marker (additional criteria are possible)

Directly apply the 3 criteria to screen genomes of two model organisms, zebrafish (Danio rerio) and pufferfish (Takifugu rubripes).

Scheme of our marker-developing strategy 130 candidate loci were

identified ‘in silico’

109 are located on 24 of the 25 chromosomes (21 with no location information).Chi-square test did not reject the Poisson distribution of these markers (p=0.0746).

Distribution of 109 candidate markers in zebrafish chromosomes

Distribution of 109 candidate markers in zebrafish chromosomes

• Size range: from 802 bp to 5811 bp in zebrafish.

• Base composition: GC content ranges from 41.6% to 63.9% in zebrafish.

• Identity: of these markers between zebrafish and pufferfish ranges from 77.3% to 93.2%.

Summary of the 130 candidate loci

Summary of the 130 candidate loci

• A random sample of 15 candidate markers was examined in 52 ray-finned fish taxa (40/47 orders of Actinopterygii).

• PCR primers were designed to conserved regions (nested PCR strategy)

• 10 out of the 15 markers tested were successfully amplified by PCR from genomic DNA in most taxa

Experimental test of the candidate markersExperimental test of

the candidate markers

Marker* Exon ID† PCR Fragment Size (bp)

No. of PI sites‡

Average p-distance§

zic1 ENSDARE00000015655 945 344 0.156

myh6 ENSDARE00000025410 735 329 0.179

RYR3 ENSDARE00000465292 837 421 0.210

ptr ENSDARE00000145053 708 372 0.205

tbr1 ENSDARE00000055502 723 313 0.189

ENC1 ENSDARE00000367269 810 360 0.180

Gylt ENSDARE00000039808 882 510 0.211

SH3PX3 ENSDARE00000117872 708 317 0.167

plagl2 ENSDARE00000136964 690 345 0.173

sreb2 ENSDARE00000029022 987 387 0.149

New DataNew Data

10 genes8025 bp

52 taxa

ML tree

OSTEOGLOSSOMORPHAOSTEOGLOSSOMORPHA

PROTACANTHOPTERYGIIPROTACANTHOPTERYGII

ACIPENSERIFORMESACIPENSERIFORMES

SEMIONOTIFORMESSEMIONOTIFORMES

AMIIFORMESAMIIFORMES

ELOPOMORPHAELOPOMORPHA

CLUPEOMORPHACLUPEOMORPHA

OSTARIOPHYSIOSTARIOPHYSI

STOMIIFORMESSTOMIIFORMES

ATELEOPODIFORMESATELEOPODIFORMES

AULOPIFORMESAULOPIFORMES

MYCTOPHIFORMESMYCTOPHIFORMES

LAMPRIDIFORMESLAMPRIDIFORMES

POLYMIXIIFORMESPOLYMIXIIFORMES

PARACANTHOPTERYGIIPARACANTHOPTERYGII

ACANTHOPTERYGIIACANTHOPTERYGII

POLYPTERIFORMESPOLYPTERIFORMES

Nelson 94Nelson 94

AC

AN

THO

MO

RPH

A

NEO

TELEOSTEI

EUTELEO

STEI

TELEOSTEI

Basal lineagesBasal lineages

Basal teleostsBasal teleosts

Clupeo-OstarioClupeo-Ostario

ProtacanthoProtacantho

NeoteleostsNeoteleosts

AmiaAmia

garsgars

sturgeonssturgeons

PolypterusPolypterus

TeleosteiTeleostei

HolosteiHolostei

1.1. G. Nelson (1969) -- branchial arch morphology G. Nelson (1969) -- branchial arch morphology

NeopterygiiNeopterygii

AmiaAmia

garsgars

sturgeonssturgeons


TeleosteiTeleostei

HolosteiHolostei

2. 2. Jessen (1973) -- pectoral anatomyJessen (1973) -- pectoral anatomy

3. 3. Olsen (1984) -- skull and pectoral girdleOlsen (1984) -- skull and pectoral girdle

AmiaAmia

garsgars

sturgeonssturgeons


TeleosteiTeleosteiNeopterygiiNeopterygii

AmiaAmia

garsgars

sturgeonssturgeons


TeleosteiTeleostei

4.4. J. Nelson (1994) -- most reasonable J. Nelson (1994) -- most reasonable


ChondrosteiChondrostei

AmiaAmia

garsgars

sturgeonssturgeons


TeleosteiTeleostei

5.5. Bemis et al (1997) -- morphology Bemis et al (1997) -- morphology


AmiaAmia

garsgars

sturgeonssturgeons


TeleosteiTeleostei

6. 6. Lê et al (1993) -- 28S rRNA; Lê et al (1993) -- 28S rRNA;

Venkatesh et al (1999)-- 8 nuclear intronsVenkatesh et al (1999)-- 8 nuclear introns


AmiaAmia

garsgars

sturgeonssturgeons


TeleosteiTeleostei

HolosteiHolostei

7.7. Inoue et al -- mtDNA; Ortí et al Inoue et al -- mtDNA; Ortí et al

RAG-1, and rhodopsinRAG-1, and rhodopsin

Basal Actinopterygians

Chondrostei

Holostei

Teleostei

Raja radiataScyliorhinus canicula

Squalus acanthiaNeoceratodus forsteri

Lepidosiren paradoxa

Protopterus spLat im eria chalum nae

#Calam oichthys calabaricusPolypterus ornat ipinnis

#Polypterus congicusAm ia calva

#Lepisosteus platostom usLepisosteus oculatus

Acipenser sturioPsephurus gladius

Acipenser gueldenstaedt iiPseudoscaphirhynchus herm anni

Huso husoAcipenser t ransm ontanus#Polyodon spathula

Acipenser fulvescens#Scaphirhynchus albus

M egalops at lant icus#E lops saurusE lops hawaiiensis

Hiodon alosoides1#Hiodon alosoides2

Pantodon buchholzi1Pantodon buchholzi 2

Notopterus chitalaChitala ornata

#Arapaim a gigasOsteoglossum ferreirai

Osteoglossum bic irrhosumXenom ystus nigri

Gym narchus nilot icus1Gym narchus nilot icus2

Pet rocephalus sp.B rienom yrus batesii

Gnathonem us peters iiA lbula vulpes

#Halosauropsis m acrochirNotacanthus bonapartei

#S t rophodon sathete

#Echidna nebulosa#Gym nothorax ret icularis

#Nem ichthys scolopaceusS tem onidium hypom elas

Anguilla rost rataAnguilla japonica

Conger m yriaster#M uraenesox c inereus

#Ophichthus cephalazonaEchiophis punct ifer

Ophichthus rex#Ophichthus gom esii

POLYPTERIFORMES

ACIPENSERIFORMES

SEMIONOTIFORMESAMIIFORMES

ELOPIFORMES

OSTEOGLOSSIFORMES

Osteoglossoide iNotopteroide i

ALBULIFORMES

ANGUILLIFORMES

Anguillo ide i

Muraenoide iCongro ide i

9994

68

96

100

100

73100

100

71

10080

100

71100

100100

10082

93

100

74

98

69100

10061

89 7675

97

82

79

100

1.2.

1.

2.3.

1

1

2

3

1

2

3

2

2

3

58

60

0.02Euteleostei

HolosteiHolostei

mtDNA, 421 taxa, ME tree


















Nelson 94Nelson 94

AC

AN

TH

OM

OR

PH

A

NE

OT

EL

EO

ST

EI

EU

TE

LE

OS

TE

I

TE

LE

OS

TE

I

Basal teleostsBasal teleosts

Elopomorpha

10 genes8025 bp


















Nelson 94Nelson 94

AC

AN

TH

OM

OR

PH

A

NE

OT

EL

EO

ST

EI

EU

TE

LE

OS

TE

I

TE

LE

OS

TE

I

Clupeo-OstarioClupeo-Ostario

10 genes8025 bp


















Nelson 94Nelson 94

AC

AN

TH

OM

OR

PH

A

NE

OT

EL

EO

ST

EI

EU

TE

LE

OS

TE

I

TE

LE

OS

TE

IProtacanthoProtacantho

10 genes8025 bp

Table 1 | Summary Information of the 10 gene markers developed.

PI, parsimony informative sites; SDR, standard deviation of substitution rates among three codon positions; CI-MP, consistency index; , gamma distribution shape parameter; RCV, relative composition variability.Treeness, ratio of internal branch length to total branch length.

Gene* ENSEMBLEgene ID

No. ofbp

% ofvariablesites

% ofPI sites

Geneticdistance (%)

Sub.rate

SDR CI-MP RCV Tree-ness

FEM-1 00000010567 894 33.11 23.49 28(2.6-65.8) 0.64 1.00 0.61 1.64 0.13 0.23

PAK-6 00000041216 735 38.44 25.53 36(10.1-59.5) 1.35 2.06 0.54 0.68 0.11 0.22

RAB15 00000026484 825 37.04 27.26 36(10.1-58.1) 1.25 1.65 0.56 0.67 0.11 0.21

Frizzled-3 00000021664 705 34.85 22.80 41(6.1-93.6) 1.03 1.64 0.57 1.64 0.12 0.29

CENP-A 00000016745 666 43.12 33.19 28(3.1-79.1) 0.65 0.67 0.67 2.91 0.10 0.28

CutA 00000035396 810 41.13 32.06 38(8.4-78.0) 1.13 1.48 0.55 1.10 0.16 0.33

Gylt 00000010941 870 38.52 30.62 41(7.6-77.0) 1.18 1.35 0.60 1.70 0.12 0.27

HMG20A 00000031198 705 53.22 38.51 30(7.5-60.0) 1.11 1.70 0.55 1.53 0.14 0.22

UNK-1 00000020319 675 47.15 31.27 29(6.0-60.6) 0.81 1.04 0.61 0.92 0.10 0.33

UNK-2 00000038383 987 43.95 31.97 30(4.6-75.5) 0.85 1.33 0.61 0.88 0.11 0.23

RAG1ą - 1344 50.89 38.24 38(9.8-75.0) 1.28 1.51 0.57 1.68 0.05 0.23

SummarySummary

• Gene markers that satisfied the three criteria are widely distributed in zebrafish genome

• Ten out of 15 markers tested seem useful for phylogenetic inference. Their profiles are comparable to the popular RAG1 gene

• The strategy is successful! – The new markers developed will help to infer

the tree of ray-finned fishes– The bioinformatic tool developed can be used in

other taxonomic groups (S: similarity may vary)

www.deepfin.orgwww.deepfin.org

www.deepfin.orgwww.deepfin.orgMember data base (Directory) currently has 606 recordsMember data base (Directory) currently has 606 records

www.deepfin.orgwww.deepfin.orgLiterature data base currently has ~800 records

(for members only)Literature data base currently has ~800 records

(for members only)

www.deepfin.org -- Literature data base www.deepfin.org -- Literature data base

www.deepfin.orgwww.deepfin.orgLiterature data base: upload pdf files to share with other membersLiterature data base: upload pdf files to share with other members

““Collaboratories”: a virtual environment to share files and informationCollaboratories”: a virtual environment to share files and information

www.tolweb.orgwww.tolweb.org

Contribute tree pages!Contribute tree pages!

ContactContact

Join DeepFin ! Join DeepFin !

Questions, comments, suggestions?

Recent advances in molecular phylogenies of actinopterygian fishes Guillermo Ortí University of Nebraska, USA.

Documents

engraulis slide

usa slide

omaha slide

silico slide

good nuclear gene markers

single gene

organismal tree slide

finned fishes slide