The Utility of the Cytochrome b gene for Cypriniform Relationships

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The Utility of the Cytochrome b gene for
Cypriniform Relationships

• Nick Gidmark, Ryan Peterson,
• Rene Wolfe

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Introduction

• Cytochrome b is a widely used marker in molecular
  systematics
• Sequences are available on genbank for many
  species
• Easy to amplify and sequence
• Protein coding gene, simplifies alignment and
  analysis

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Cytochrome b

• Exhibits variation within and between populations
• Also used successfully to resolve relationships
  at higher taxonomic levels, i.e. between species
• Can be problematic due to saturation and
  inadequate taxon sampling

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Inadequate taxon sampling
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Mitochondrial genome
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Goals

• Amplify and sequence cytochrome b from CToL REU
  specimens
• Analyse sequences to determine phylogenetic
  signal
• Combine cytochrome b with other gene sequences to
  resolve family level relationships within
  Cypriniformes

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Methods

• Specimens
• Amplification
• Sequencing
• Alignments
• Analyses

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Specimens

• Cypriniform families
• 20 Cyprinidae
• 9 Cobitidae
• 6 Balitoridae
• 2 Catostomidae
• 1 Gyrinochelidae

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Specimens II

• Outgroup orders
• Gonorynchiformes (milkfish)
• Siluriformes (catfish)
• Gymnotoformes (knife fish)
• Characiformes (tetras and paranas)

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Amplification

• Fast amp PCR
• Primer combos LA/HD, LA/HC, HA/LD, HA/LC
• Difficulties
• Could not amplify entire gene with HA/LA primers
• Raised annealing temp with limited success
• LA/HC covers little of the gene despite good
  amplification
• Agarose gel used to check amplification qualities
• UV light bed reveals band indicating successful
  Cyt b amp

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Map of Cytochrome b with primers
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Sequencing

• Methods
• 2mL DNA mix, 2mL ddH20, 2mL primer loaded into 96
  well plates
• Mixture sent to AGAC at U of MN for sequencing

http//www.museum.tulane.edu/ictiobin/figures/elec
tropherogram.html
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Alignment

• Sequences aligned using Sequencher version 4.2
• Reference sequences were added from genbank to
  aid alignments

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Reference Sequences
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Analyses

• Parsimony analyses were performed using PAUP
• Applied differential weighting schemes based on
  codon positions
• Varied outgroup choice

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Weighting

• Equal weights
• Decrease weight of third position
• Eliminate third position

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Results

• Complete sequence for 17 species
• Incomplete sequence for 31 species
• Data matrix contained 48 taxa and 1141 nucleotide
  positions

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• All taxa
• First second codon positions only
• Outgroup - Chanos
• Consensus of 11 trees

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• All taxa
• Weighted by codon position 2-2-1
• Outgroup - Chanos
• Consensus of 72 trees

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• All taxa
• Weighted by codon position 3-3-1
• Outgroup - Chanos
• Consensus of 54 trees

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• All taxa
• Equal weights
• Outgroup - Chanos
• Consensus of 18 trees

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• Reduced taxa
• First second codon positions only
• Outgroup - Diplomystes
• Consensus of 5844 trees

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• Reduced taxa
• Weighted by codon position 2-2-1
• Outgroup - Diplomystes
• Consensus of 9 trees

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• Reduced taxa
• Weighted by codon position 3-3-1
• Outgroup - Diplomystes
• Consensus of 24 trees

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• Reduced taxa
• Equal weights
• Outgroup - Diplomystes
• Consensus of 38 trees

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Conclusions

• Need to establish reliable amplification
  protocols--specimens need to be reamplified
• Possible contamination in loading the plate
• REU specimens did not sequence well
• Couple other sequenced genes with cyt b analysis
  to determine phylogenetic relationships
• Cyt b not the best gene for determining
  cypriniform relationships?

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Acknowledgements

• Andrew Simons
• Liz Johnson
• The Simons lab group
• National Science Foundation
• University of Minnesota