1. Consequences of
Combining mtDNAand
nucDNAData for Phylogenetic Trees
Leila Bahmani Kazerooni & John Malloy

2. What is a Phylogenetic Tree?
Leaves
• Evolutionary tree (Species)
Node
Human
Root Chimp
Gorilla
Orangutan
Branch (Edge)
• Visually defines species relationships
Deep divergences (e.g., The Tree of Life)
Recent divergences (e.g., species in a genus)

3. What is a Phylogenetic Tree? Leaves
• Evolutionary tree (Species)
Node
Human
Root Chimp
Gorilla
Orangutan
Branch (Edge)
• Information required to build a phylogeny ranges from
morphological data to purely genomic data, including mitochondrial
DNA (mtDNA) and nuclear DNA (nucDNA)

4. Molecular Phylogenies-mtDNA
• 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

5. Molecular Phylogenies-nucDNA
• 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)

6. Concatenation=(mtDNA+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

7. “Northern Oriole” Group: Gene Flow Between
Species
HYBRID ZONE
EASTERN
WESTERN
WESTERN Baltimore
Bullock’s
Bullock’s
MEXICAN
Black-backed
MEXICAN
Black-backed
•These three species illustrate one of the main challenges in determining relationshi
among recently diverged species.

8. Constructing the Phylogeny-MATERIALS
• 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-
92
 Six autosomal introns: β-ACT2, αENO8, FGB4, GAPDH11, RDP2, TGFβ5 3

9. Constructing the Phylogeny-TOOLS
• 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.

10. Tested Multiple Nuclear Introns (nucDNA)
against mtDNA – Deep divergence agreed:
CLADE A
nDNA (6 Z-introns combined) mtDNA
Jacobsen, Friedman &Omland 2010

11. Tested nucDNA against mtDNA – Deep
divergence agreed: CLADE B
nDNA (6 Z-introns combined) mtDNA
Jacobsen, Friedman &Omland 2010

12. Tested nucDNA against mtDNA – Deep
divergence agreed: CLADE C
nDNA (6 Z-introns combined) mtDNA
Jacobsen, Friedman &Omland 2010

13. Tested nucDNA against mtDNA – Deep divergence agreed
BUT one recent divergence did not
nDNA (6 Z-introns combined) mtDNA
Bullock’s
Black Backed Bullock’s
Black Backed
Baltimore
Baltimore
Jacobsen, Friedman &Omland 2010

14. Summary of the Conflicting Nodes in Clade C
combined nucDNA tree VS mtDNA gene tree
nucDNA Baltimore
mtDNA
Black-backed
Bullock’s
•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.

15. 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)

16. CONFLICT: mtDNA vs nDNA
mt Black-backed nuc Black-backed
Baltimore Bullock’s
Bullock’s
Baltimore

17. Concatenate 12nuc+1mt
mt Black-backed nuc Black-backed
Baltimore Bullock’s
Bullock’s
Baltimore
Black-backed
12nuc+mt
Baltimore • We combined 1 mitochondrial
gene and 12 nuclear genes
(6 sex-linked and 6 autosomal)
•The mitochondrial signal
overwhelmed the nuclear
signal.
Bullock’s

18. Concatenate 24nuc+1mt
mt Black-backed nuc Black-backed
Baltimore Bullock’s
Bullock’s
Baltimore
24nuc+mt Black-backed •To further test the influence of
Baltimore mtDNA on the branches, we
combined 24 nuclear genes and
1 mitochondrial gene, thereby
having doubled the nuclear
signal.
•The mitochondrial signal still
Bullock’s
overwhelmed the nuclear
signal.

19. Concatenate 48nuc+1mt
mt Black-backed nuc Black-backed
Baltimore Bullock’s
Bullock’s
Baltimore
48nuc+mt Black-backed
•Continuing the titration we
Baltimore combined 48 nuclear genes
and 1 mitochondrial gene,
thus quadrupling the nuclear
signal.
•The mitochondrial signal
continued to overwhelm the
nuclear signal.
Bullock’s

20. Concatenate 72nuc+1mt
mt Black-backed nuc Black-backed
Baltimore Bullock’s
Bullock’s
Baltimore
72nuc+mt Black-backed •We then combined 72
Bullock’s nuclear genes and one
mitochondrial gene having
Baltimore
multiplied the original
nuclear signal by six.
•Only at that point did the
nuclear signal overwhelm
the mitochondrial signal.

21. Titrate mtDNA with nucDNA
As the nucDNA was artificially doubled and quadrupled the
mtDNA signal continued to overwhelm the nucDNA signal
mt 12nuc+mt
72nuc+m
NODE 24nuc+mt 48nuc+mt nuc
t
BLACK- ___ ___
BACKED, Y Y Y Y
BALTIMORE
BLACK- __ ___ ___ ___
BACKED, Y Y
BULLOCK’S
Once the nucDNA was artificially multiplied by six the
nucDNA signal revealed itself.

22. Conclusion: mtDNA Potentially
Very Misleading
• 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.

23. Acknowledgments
Thanks to Dr. Kevin Omland, Dr. Matthias Gobbert, and Dr. Frode
Jacobsen 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.org
5Huelsenbeck, 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/