Leila Bahmani Kazerooni & John Malloy
Consequences of Combining mtDNA and
nucDNA Data for Phylogenetic Trees
• Evolutionary tree
What is a Phylogenetic Tree?
Human
Orangutan
Gorilla
Chimp
Root
Node
Branch (Edge)
Leaves (Species)
• Visually defines species relationships
Deep divergences (e.g., The Tree of Life)Recent divergences (e.g., species in a
genus)
• Evolutionary treeWhat is a Phylogenetic Tree?
Human
Orangutan
Gorilla
Chimp
Root
Node
Branch (Edge)
Leaves (Species)
• Information required to build a phylogeny ranges from morphological data to purely genomic data, including mitochondrial DNA (mtDNA) and nuclear DNA (nucDNA)
• Up until now, phylogenetic trees of closely related
animals have mostly been based on one region of
the genome - mitochondrial DNA (mtDNA)
High mutation rate (due to poor DNA repair
mechanism)
Passed down through the maternal lineage
Molecular Phylogenies-mtDNA
• Nuclear DNA (nucDNA) has recently become a
valuable tool alongside mitochondrial DNA
Slower mutation rate
Inherited from both parents
More versatile - contains both sex-linked and
autosomal genes (allows for increased validation)
Molecular Phylogenies-nucDNA
• Both mtDNA and nucDNA have unique advantages and
disadvantages
• A method used to give both regions of the genome an
opportunity to resolve the branches on the tree is
CONCATENATION
Concatenation: Combining a mitochondrial gene with
multiple nuclear genes to form one “BIG gene”
• This frequently used method may be flawed due to potential
gene flow between species.
• We have used the “Northern Oriole” group as a model group to
test concatenation
Concatenation=(mtDNA+nucDNA)
WESTERNBullock’s
MEXICANBlack-backed
WESTERNBullock’s
MEXICANBlack-backed
EASTERNBaltimore
HYBRID ZONE
•These three species illustrate one of the main challenges in determining relationships among recently diverged species.
“Northern Oriole” Group: Gene Flow Between Species
• Samples from 32 species of the genus Icterus were used
in the analyses
• The mtDNA used belong to the cytochrome b and ND2
regions and had been previously isolated and sequenced
by the Omland lab 1.
• The nucDNA had also been isolated and sequenced by
the Omland lab.
Six z-linked introns: ADAM-5, ALDO-5, BRM-15, CHD-18,
MUSK-3, SLC-9 2
Six autosomal introns: β-ACT2, αENO8, FGB4, GAPDH11,
RDP2, TGFβ5 3
Constructing the Phylogeny-MATERIALS
• The formatting and concatenation of the data files were
done using the modular file editor, Mesquite v2.75 4.
• Files from Mesquite were exported in the Nexus file
format and analyzed with MrBayes.
MrBayes is a tree building program that uses Bayesian
inference and Markov chain Monte Carlo methods to infer
phylogenetic trees 5.
• Output trace files from MrBayes were assessed using
Tracer, to ensure the convergence of each run 6.
• MrBayes outputs were converted to graphical trees using
FigTree, a tree viewing program7.
Constructing the Phylogeny-TOOLS
mtDNAnDNA (6 Z-introns combined)
Tested Multiple Nuclear Introns (nucDNA) against mtDNA – Deep
divergence agreed: CLADE A
Jacobsen, Friedman &Omland 2010
Jacobsen, Friedman &Omland 2010
Tested nucDNA against mtDNA – Deep divergence agreed: CLADE B
nDNA (6 Z-introns combined) mtDNA
mtDNA
Jacobsen, Friedman &Omland 2010
nDNA (6 Z-introns combined)
Tested nucDNA against mtDNA – Deep divergence agreed: CLADE C
Bullock’sBullock’sBlack Backed
Black Backed
Baltimore
Baltimore
mtDNA
Jacobsen, Friedman &Omland 2010
nDNA (6 Z-introns combined)
Tested nucDNA against mtDNA – Deep divergence agreed
BUT one recent divergence did not
combined nucDNA tree VS mtDNA gene treeBaltimore
Bullock’s
Black-backed
nucDNAmtDNA
Summary of the Conflicting Nodes in Clade C
•Previous analyses done by the Omland lab have shown strong support that the mtDNA tree is misleading, while there is strong support for the nucDNA tree1.•All analyses of nuclear DNA – z chromosome alone, autosomal chromosomes alone, and different analysis methods – all show mtDNA is the problematic outlier 1,2.
mtDNA and the problem with mindless CONCATENATION
• Why might mtDNA and nucDNA disagree for the Northern
Oriole Group?
Gene flow – mtDNA may have crossed between
species during hybridization
• What if we had mindlessly tried to resolve the conflict
between mtDNA and nucDNA by simply combining both
types of DNA into one large data matrix (Concatenation)?
Give both nucDNA and mtDNA an "equal" opportunity
to resolve the branches in the tree (very common
approach in molecular systematics)
Black-backed
Baltimore
Bullock’s
Black-backed
Bullock’s
Baltimore
mt nuc
CONFLICT: mtDNA vs nDNA
Black-backed
Baltimore
Bullock’s
Black-backed
Bullock’s
Baltimore
mt nuc
Concatenate 12nuc+1mt
Black-backed
Baltimore12nuc+mt
• We combined 1 mitochondrial gene and 12 nuclear genes (6 sex-linked and 6 autosomal)•The mitochondrial signal overwhelmed the nuclear signal.
Bullock’s
Black-backed
Baltimore
Bullock’s
Black-backed
Bullock’s
Baltimore
mt nuc
Concatenate 24nuc+1mt
Black-backed
Baltimore
Bullock’s
24nuc+mt •To further test the influence of mtDNA on the branches, we combined 24 nuclear genes and 1 mitochondrial gene, thereby having doubled the nuclear signal. •The mitochondrial signal still overwhelmed the nuclear signal.
Black-backed
Baltimore
Bullock’s
Black-backed
Bullock’s
Baltimore
mt nuc
Concatenate 48nuc+1mt
48nuc+mt
Bullock’s
Black-backed
Baltimore•Continuing the titration we combined 48 nuclear genes and 1 mitochondrial gene, thus quadrupling the nuclear signal. •The mitochondrial signal continued to overwhelm the nuclear signal.
Black-backed
Baltimore
Bullock’s
Black-backed
Bullock’s
Baltimore
mt nuc
Concatenate 72nuc+1mt
Bullock’s
Baltimore
72nuc+mt Black-backed •We then combined 72 nuclear genes and one mitochondrial gene having multiplied the original nuclear signal by six. •Only at that point did the nuclear signal overwhelm the mitochondrial signal.
NODE mt 12nuc+mt
24nuc+mt 48nuc+mt
72nuc+mt
nuc
BLACK-BACKED,
BALTIMOREY Y Y Y
___ ___
BLACK-BACKED,
BULLOCK’S
__ ___ ___ ___Y Y
Titrate mtDNA with nucDNA
As the nucDNA was artificially doubled and quadrupled the mtDNA signal continued to overwhelm the nucDNA signal
Once the nucDNA was artificially multiplied by six the nucDNA signal revealed itself.
• Although the number of nuclear nucleotides overwhelmed
mitochondrial nucleotides, the mitochondrial signal remained
dominant.
mtDNA is much more variable, due to its high mutation rate
• Simple concatenation, 12nuc+1mt, proceeded to result in the
misleading mtDNA signal.
• Only when the nucDNA signal was multiplied by six was the signal
strong enough to result in the well supported nuclear tree.
• These results consistently demonstrate the potential weaknesses
of using concatenation as a technique to build molecular
phylogenetic trees.
Conclusion: mtDNA Potentially Very Misleading
AcknowledgmentsThanks to Dr. Kevin Omland, Dr. Matthias Gobbert, and Dr.
FrodeJacobsen for the contributions and support for this project. This work was funded, in part, by the UBM program at UMBC, an interdisciplinary biology and math training grant funded by the National Science Foundation. Work Cited
1 Frode Jacobsen, Kevin E. Omland, Species tree inference in a recent radiation of orioles (Genus Icterus): Multiple markers and methods reveal cytonuclear discordance in the northern oriole group, Molecular Phylogenetics and Evolution, Volume 61, Issue 2, November 2011, Pages 460-469. 2 Jacobsen, F., Friedman, N. R. and Omland, K. E. 2010. Congruence between nuclear and mitochondrial DNA: combination of multiple nuclear introns resolves a well-supported phylogeny of New World orioles (Icterus). Molecular Phylogenetics and Evolution 56:419-427.3 Jacobsen, F. and Omland, K. E. 2012. Extensive introgressive hybridization within the northern oriole group (Genus Icterus) revealed by three-species isolation with migration analysis. Ecology and Evolution 2:2413-2429.4 Maddison, W. P. and D.R. Maddison. 2011. Mesquite: a modular system for evolutionary analysis. Version 2.75 http://mesquiteproject.org5Huelsenbeck, J. P. and F. Ronquist. 2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17:754-755.6 Rambaut A, Drummond AJ (2007) Tracer v1.4. 7 http://tree.bio.ed.ac.uk/software/figtree/
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