Genetic
Coalescence in
North
American
White Pines
Kathleen Farrell
Dr. Aaron Liston, Dr. Richard Cronn,
John Syring
P. strobus
Coalescence is the pattern of common ancestry.
Coalescence theory: At some
point all alleles in the population
will be traceable to a single
common ancestral allele that
existed in the population at time
t0.
Factors:
Gene flow
Hybridization
Selection
Population size
Objective: to find low copy nuclear genes that accurately
demonstrate the relationships (phylogeny) between
closely related species, North American White Pines.
Why low copy genes?
Orthologs vs. Paralogs
Coalescing Tree:
A
A
B
B
C
C
Non-Coalescing Tree:
A
A
C
B
B
C
Objective: to find low copy nuclear genes that accurately
demonstrate the relationships (phylogeny) between
closely related species, North American White Pines.
Why low copy genes?
Orthologs vs. Paralogs
Coalescing Tree:
A
A
B
B
C
C
Non-Coalescing Tree:
A
A
C
B
B
C
Species Sampled:
North American White Pines
P. albicaulis
P. ayacahuite
P. chiapensis
P. flexilis
P. lambertiana
P. monticola
P. strobiformis
P. strobus
Whitebark Pine
Mexican White Pine
Chiapas White Pine
Limber Pine
Sugar Pine
Western White Pine
Southwestern White Pine
Eastern White Pine
P. flexilis
P. ayacahuite
P. lambertiana
P. albicaulis
Outgroups
P. monophylla
P. longaeva
P. longaeva
P. monophylla
Singleleaf Pinyon
Bristlecone Pine
Methods and Approach:
DNA Extraction from the megagametophyte tissue.
megagametophyte (1n)
embryo (2n)
shoot end
embryo root tip (radicle)
Polymerase Chain Reaction (PCR)
How it works:
DNA Denatures
Primers Anneal
Taq Polymerase builds
a complementary copy.
Polymerase Chain Reaction (PCR)
How it works:
DNA Denatures
Primers Anneal
Taq Polymerase builds
a complementary copy.
Polymerase Chain Reaction (PCR)
Results: Gel Electrophoresis
62 63
64
65
61
66 67
2.0 mM MgCl
68
Temperature (C) 
56 57 58 59 60
62 63
64
65
1.5 mM MgCl
61
Control
Temperature (C) 
56 57 58 59 60
Control
IFG8612 Optimization: MONTICOLA 02S1
66 67
68
Polymerase Chain Reaction (PCR)
Results: Gel Electrophoresis
62 63
64
65
61
66 67
2.0 mM MgCl
68
Temperature (C) 
56 57 58 59 60
62 63
64
65
1.5 mM MgCl
61
Control
Temperature (C) 
56 57 58 59 60
Control
IFG8612 Optimization: MONTICOLA 02S1
66 67
68
Polymerase Chain Reaction (PCR)
Results: Gel Electrophoresis
62 63
64
65
61
66 67
2.0 mM MgCl
68
Temperature (C) 
56 57 58 59 60
62 63
64
65
1.5 mM MgCl
61
Control
Temperature (C) 
56 57 58 59 60
Control
IFG8612 Optimization: MONTICOLA 02S1
66 67
68
Cycle Sequencing
Capillary Gel
Cycle Sequencing
Analyzing Results:
Clean Sequence
Sequence Noise
Alignment and Editing
------------------------GTAATT----AGATTTGAATGGCATGGAATGAATGGAAA
-GCaGGAtaTTGTCATTTCTTTCAGTAATT----AGATTTGAATGGCATGGAATGAATGGAAA
----GGAtaTTATCATTTTTTTCaGTAATTAATTAGATTTGAATGGCATGGAATGAATGGAAA
-GCaGGAtATTGTCATTTCTTTCAGTAATT----AGATTTGAATGGCATGGAATGAATGGAAA
Developing a Phylogenetic Tree



Export alignments to PAUP*
Search for phylogenetic trees based on maximum parsimony using
the branch and bound algorithm.
Calculate bootstrap values (measure of confidence for each branch)
and branch length (measure of relative divergence).
Results:
IFG8898
Cinnamyl Alcohol Dehydrogenase
IFG8612
Results:
IFG8898
Cinnamyl Alcohol Dehydrogenase
IFG8612
Results:
IFG8898
Cinnamyl Alcohol Dehydrogenase
IFG8612
IFG8612 alignment
IFG8612 alignment
IFG8612 alignment
IFG8612 alignment
Phylogenetic Tree
MONOPHYLLA JVS
LONGAEVA JVS
CHIAPENSIS JVS
100
CHIAPENSIS 02
CHIAPENSIS 01
65
FLEXILIS JVS
FLEXILIS 01S5
100
FLEXILIS 03S1
84
AYACAHUITE 04S2
62
AYACAHUITE 03S2
60
84
67
STROBIFORMIS 01S1
MONTICOLA JVS
70
MONTICOLA 02S3
ALBICAULIS 01S1
64
100
ALBICAULIS 03S1
ALBICAULIS 04S1
86
LAMBERTIANA
57
STROBUS 02S1
88
STROBUS JVS
100
STROBUS 03S1
53
LAMBERTIANA 08S2
LAMBERTIANA 02S1
Phylogenetic Tree
MONOPHYLLA JVS
LONGAEVA JVS
CHIAPENSIS JVS
100
CHIAPENSIS 02
CHIAPENSIS 01
65
FLEXILIS JVS
FLEXILIS 01S5
100
FLEXILIS 03S1
84
AYACAHUITE 04S2
62
AYACAHUITE 03S2
60
84
67
STROBIFORMIS 01S1
MONTICOLA JVS
70
MONTICOLA 02S3
ALBICAULIS 01S1
64
100
ALBICAULIS 03S1
ALBICAULIS 04S1
86
LAMBERTIANA JVS
57
STROBUS 02S1
Bootstrap
Support (%)
88
STROBUS JVS
100
STROBUS 03S1
53
LAMBERTIANA 08S2
LAMBERTIANA 02S1
Outgroups
Phylogenetic Tree
MONOPHYLLA JVS
LONGAEVA JVS
CHIAPENSIS JVS
100
CHIAPENSIS 02
CHIAPENSIS 01
65
FLEXILIS JVS
FLEXILIS 01S5
100
FLEXILIS 03S1
84
AYACAHUITE 04S2
62
AYACAHUITE 03S2
60
84
67
STROBIFORMIS 01S1
MONTICOLA JVS
70
MONTICOLA 02S3
ALBICAULIS 01S1
64
100
ALBICAULIS 03S1
ALBICAULIS 04S1
86
LAMBERTIANA JVS
57
STROBUS 02S1
88
STROBUS JVS
100
STROBUS 03S1
53
LAMBERTIANA 08S2
LAMBERTIANA 02S1
Phylogenetic Tree
MONOPHYLLA JVS
LONGAEVA JVS
CHIAPENSIS JVS
100
CHIAPENSIS 02
CHIAPENSIS 01
65
FLEXILIS JVS
FLEXILIS 01S5
100
FLEXILIS 03S1
84
AYACAHUITE 04S2
62
AYACAHUITE 03S2
60
84
67
STROBIFORMIS 01S1
MONTICOLA JVS
70
MONTICOLA 02S3
ALBICAULIS 01S1
64
100
ALBICAULIS 03S1
ALBICAULIS 04S1
86
LAMBERTIANA JVS
57
STROBUS 02S1
88
STROBUS JVS
100
STROBUS 03S1
53
LAMBERTIANA 08S2
LAMBERTIANA 02S1
Phylogenetic Tree
MONOPHYLLA JVS
LONGAEVA JVS
CHIAPENSIS JVS
100
CHIAPENSIS 02
CHIAPENSIS 01
65
FLEXILIS JVS
FLEXILIS 01S5
100
FLEXILIS 03S1
84
AYACAHUITE 04S2
62
AYACAHUITE 03S2
60
84
67
STROBIFORMIS 01S1
MONTICOLA JVS
70
MONTICOLA 02S3
ALBICAULIS 01S1
64
100
ALBICAULIS 03S1
ALBICAULIS 04S1
86
LAMBERTIANA JVS
57
STROBUS 02S1
88
STROBUS JVS
100
STROBUS 03S1
53
LAMBERTIANA 08S2
LAMBERTIANA 02S1
Phylogenetic Tree
MONOPYLLA JVS
LONGAEVA JVS
CHIAPENSIS JVS
CHIAPENSIS 02
CHIAPENSIS 01
FLEXILIS JVS
FLEXILIS 01S5
FLEXILIS 03S1
AYACAHUITE 04S2
AYACAHUITE 03S2
STROBIFORMIS 01S1
MONTICOLA JVS
MONTICOLA 02S3
ALBICAULIS 01S1
ALBICAULIS 03S1
ALBICAULIS 04S1
LAMBERTIANA JVS
STROBUS O2S1
STROBUS JVS
STROBUS 03S1
LAMBERTIANA 08S2
LAMBERTIANA 02S1
1
Phylogenetic Tree
MONOPYLLA JVS
LONGAEVA JVS
CHIAPENSIS JVS
CHIAPENSIS 02
CHIAPENSIS 01
FLEXILIS JVS
FLEXILIS 01S5
FLEXILIS 03S1
AYACAHUITE 04S2
AYACAHUITE 03S2
STROBIFORMIS 01S1
MONTICOLA JVS
MONTICOLA 02S3
ALBICAULIS 01S1
ALBICAULIS 03S1
ALBICAULIS 04S1
LAMBERTIANA JVS
STROBUS O2S1
STROBUS JVS
STROBUS 03S1
LAMBERTIANA 08S2
LAMBERTIANA 02S1
1
Future Research:
Continue screening low copy, nuclear genes to compare phylogenetic
patterns across the genome.
Increase sampling to five individuals from each species that represent the
geographic distribution of the species.
Extend research to include Asian White Pines.
Special Thanks to:
Howard Hughes Medical Institute
Portland Garden Club
Undergraduate Research, Innovation, Scholarship, and Creativity
(URISC)
National Science Foundation
P. strobus