Recent advances in molecular phylogenies of actinopterygian fishes

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Recent advances in molecular phylogenies of
actinopterygian fishes

• Guillermo Ortí
• University of Nebraska, USA

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Molecular Systematics of Ray-finned Fishes
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DeepFin will Advance The Phylogeny of Fishes A
Research Coordination Network

• To promote fish phylogeneticshow far are we from
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• Morphology
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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).

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GC at the 3rd codon position of RAG 1
Muraenesox
Gonostoma
Albuliformes
Ogcocephalus
galaxiids
Colisa
Elops
Engraulis
Arnoglossus
Trigla
scorpaenids
Albula
Sparus
Gasterosteus
Megalops
Mean
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Zeus
Lophiiformes
Basal neoteleosts
0.5
Paracanthop.
Elopomorpha
Ostariophysi
Acanthopterygii
Protacanthop.
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Basal actinop.
Polypteriformes
Stomiiformes
Osteoglosso.
Clupeomorpgha
Elasmobranchii
Tetrapoda
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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)

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Gene trees within organismal trees
Lineage sorting
Gene duplication
Horizontal transfer
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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

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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 nuclear gene
markers???
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Three criteria to choose good nuclear gene
markers

• 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 (so, what about gene
  duplications?)

Chenhong Li (UNL) and Guoqing Lu (UN-Omaha)
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Three criteria to choose good nuclear gene
markers

• 2) The amplicon (i.e. target sequences
  amplified by the PCR primers) should be of
  reasonable size (exons gt800 bp).

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Three criteria to choose good nuclear gene
markers

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

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• 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).

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Scheme of our marker-developing strategy 130
candidate loci were identified in silico
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Distribution of 109 candidate markers in
zebrafish chromosomes
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 (p0.0746).
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Summary of the 130 candidate loci

• 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.

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Experimental test of the candidate markers

• 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

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New Data 10 genes 8025 bp 52 taxa ML tree
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POLYPTERIFORMES
ACIPENSERIFORMES
SEMIONOTIFORMES
AMIIFORMES
OSTEOGLOSSOMORPHA
ELOPOMORPHA
CLUPEOMORPHA
OSTARIOPHYSI
PROTACANTHOPTERYGII
TELEOSTEI
STOMIIFORMES
ATELEOPODIFORMES
EUTELEOSTEI
AULOPIFORMES
MYCTOPHIFORMES
NEOTELEOSTEI
LAMPRIDIFORMES
POLYMIXIIFORMES
ACANTHOMORPHA
PARACANTHOPTERYGII
Nelson 94
ACANTHOPTERYGII
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Basal Actinops
Polypterus
sturgeons
gars
Holostei
Amia
Teleostei
1. G. Nelson (1969) -- branchial arch morphology
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Basal Actinops
Polypterus
gars
Holostei
Amia
Teleostei
sturgeons
2. Jessen (1973) -- pectoral anatomy
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Basal Actinops
Polypterus
sturgeons
Amia
Teleostei
gars
3. Olsen (1984) -- skull and pectoral girdle
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Basal Actinops
Chondrostei
Polypterus
sturgeons
gars
Amia
Teleostei
4. J. Nelson (1994) -- most reasonable
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Basal Actinops
Polypterus
sturgeons
gars
Amia
Teleostei
5. Bemis et al (1997) -- morphology
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Basal Actinops
Polypterus
sturgeons
gars
Amia
Teleostei
6. Lê et al (1993) -- 28S rRNA Venkatesh
et al (1999)-- 8 nuclear introns
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Basal Actinops
Polypterus
sturgeons
gars
Holostei
Amia
Teleostei
7. Inoue et al -- mtDNA Ortí et al RAG-1, and
rhodopsin
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Basal Actinopterygians
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Basal Actinops
Holostei
mtDNA, 421 taxa, ME tree
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POLYPTERIFORMES
ACIPENSERIFORMES
SEMIONOTIFORMES
AMIIFORMES
OSTEOGLOSSOMORPHA
Basal teleosts
ELOPOMORPHA
CLUPEOMORPHA
OSTARIOPHYSI
PROTACANTHOPTERYGII
TELEOSTEI
STOMIIFORMES
ATELEOPODIFORMES
EUTELEOSTEI
AULOPIFORMES
MYCTOPHIFORMES
NEOTELEOSTEI
LAMPRIDIFORMES
POLYMIXIIFORMES
ACANTHOMORPHA
PARACANTHOPTERYGII
Nelson 94
ACANTHOPTERYGII
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Elopomorpha
10 genes 8025 bp
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POLYPTERIFORMES
ACIPENSERIFORMES
SEMIONOTIFORMES
AMIIFORMES
OSTEOGLOSSOMORPHA
ELOPOMORPHA
CLUPEOMORPHA
Clupeo-Ostario
OSTARIOPHYSI
PROTACANTHOPTERYGII
TELEOSTEI
STOMIIFORMES
ATELEOPODIFORMES
EUTELEOSTEI
AULOPIFORMES
MYCTOPHIFORMES
NEOTELEOSTEI
LAMPRIDIFORMES
POLYMIXIIFORMES
ACANTHOMORPHA
PARACANTHOPTERYGII
Nelson 94
ACANTHOPTERYGII
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10 genes 8025 bp
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POLYPTERIFORMES
ACIPENSERIFORMES
SEMIONOTIFORMES
AMIIFORMES
OSTEOGLOSSOMORPHA
ELOPOMORPHA
CLUPEOMORPHA
OSTARIOPHYSI
PROTACANTHOPTERYGII
TELEOSTEI
STOMIIFORMES
ATELEOPODIFORMES
EUTELEOSTEI
AULOPIFORMES
MYCTOPHIFORMES
NEOTELEOSTEI
LAMPRIDIFORMES
POLYMIXIIFORMES
ACANTHOMORPHA
PARACANTHOPTERYGII
Nelson 94
ACANTHOPTERYGII
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10 genes 8025 bp
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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.
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Summary

• 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)

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