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
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
DeepFin will Advance The Phylogeny of “Fishes”
A Research Coordination Network
DeepFin will Advance The Phylogeny of “Fishes”
A Research Coordination Network
Issues with molecular phylogenies based on a single
gene or few loci
Issues with molecular phylogenies based on a single
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
Issues with molecular phylogenies based on a single
gene or few loci
Issues with molecular phylogenies based on a single
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
Taxon 3Taxon 2Taxon 1
Gene loss
a1 b3a3b2a2b1
ba
Gene duplication
Taxon 3Taxon 2Taxon 1
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
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.
Three criteria to choose “good” nuclear gene markers
Three criteria to choose “good” nuclear gene markers
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
PolypterusPolypterus
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
PolypterusPolypterus
TeleosteiTeleosteiNeopterygiiNeopterygii
AmiaAmia
garsgars
sturgeonssturgeons
PolypterusPolypterus
TeleosteiTeleostei
4.4. J. Nelson (1994) -- most reasonable J. Nelson (1994) -- most reasonable
NeopterygiiNeopterygii
ChondrosteiChondrostei
AmiaAmia
garsgars
sturgeonssturgeons
PolypterusPolypterus
TeleosteiTeleostei
5.5. Bemis et al (1997) -- morphology Bemis et al (1997) -- morphology
NeopterygiiNeopterygii
AmiaAmia
garsgars
sturgeonssturgeons
PolypterusPolypterus
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
NeopterygiiNeopterygii
AmiaAmia
garsgars
sturgeonssturgeons
PolypterusPolypterus
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
OSTEOGLOSSOMORPHAOSTEOGLOSSOMORPHA
PROTACANTHOPTERYGIIPROTACANTHOPTERYGII
ACIPENSERIFORMESACIPENSERIFORMES
SEMIONOTIFORMESSEMIONOTIFORMES
AMIIFORMESAMIIFORMES
ELOPOMORPHAELOPOMORPHA
CLUPEOMORPHACLUPEOMORPHA
OSTARIOPHYSIOSTARIOPHYSI
STOMIIFORMESSTOMIIFORMES
ATELEOPODIFORMESATELEOPODIFORMES
AULOPIFORMESAULOPIFORMES
MYCTOPHIFORMESMYCTOPHIFORMES
LAMPRIDIFORMESLAMPRIDIFORMES
POLYMIXIIFORMESPOLYMIXIIFORMES
PARACANTHOPTERYGIIPARACANTHOPTERYGII
ACANTHOPTERYGIIACANTHOPTERYGII
POLYPTERIFORMESPOLYPTERIFORMES
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
OSTEOGLOSSOMORPHAOSTEOGLOSSOMORPHA
PROTACANTHOPTERYGIIPROTACANTHOPTERYGII
ACIPENSERIFORMESACIPENSERIFORMES
SEMIONOTIFORMESSEMIONOTIFORMES
AMIIFORMESAMIIFORMES
ELOPOMORPHAELOPOMORPHA
CLUPEOMORPHACLUPEOMORPHA
OSTARIOPHYSIOSTARIOPHYSI
STOMIIFORMESSTOMIIFORMES
ATELEOPODIFORMESATELEOPODIFORMES
AULOPIFORMESAULOPIFORMES
MYCTOPHIFORMESMYCTOPHIFORMES
LAMPRIDIFORMESLAMPRIDIFORMES
POLYMIXIIFORMESPOLYMIXIIFORMES
PARACANTHOPTERYGIIPARACANTHOPTERYGII
ACANTHOPTERYGIIACANTHOPTERYGII
POLYPTERIFORMESPOLYPTERIFORMES
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
OSTEOGLOSSOMORPHAOSTEOGLOSSOMORPHA
PROTACANTHOPTERYGIIPROTACANTHOPTERYGII
ACIPENSERIFORMESACIPENSERIFORMES
SEMIONOTIFORMESSEMIONOTIFORMES
AMIIFORMESAMIIFORMES
ELOPOMORPHAELOPOMORPHA
CLUPEOMORPHACLUPEOMORPHA
OSTARIOPHYSIOSTARIOPHYSI
STOMIIFORMESSTOMIIFORMES
ATELEOPODIFORMESATELEOPODIFORMES
AULOPIFORMESAULOPIFORMES
MYCTOPHIFORMESMYCTOPHIFORMES
LAMPRIDIFORMESLAMPRIDIFORMES
POLYMIXIIFORMESPOLYMIXIIFORMES
PARACANTHOPTERYGIIPARACANTHOPTERYGII
ACANTHOPTERYGIIACANTHOPTERYGII
POLYPTERIFORMESPOLYPTERIFORMES
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?