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 relationshipsDeep 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/