1. Genetic Coalescence in North American White Pines Kathleen Farrell Dr. Aaron Liston, Dr. Richard Cronn, John Syring P. strobus

2. 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 t 0. Factors:  Gene flow  Hybridization  Selection  Population size

3. Objective: to find low copy nuclear genes that accurately demonstrate the relationships (phylogeny) between closely related species, North American White Pines. AABBCCAABBCC Coalescing Tree: AACBBCAACBBC Non-Coalescing Tree: Why low copy genes? Orthologs vs. Paralogs

4. Objective: to find low copy nuclear genes that accurately demonstrate the relationships (phylogeny) between closely related species, North American White Pines. AABBCCAABBCC Coalescing Tree: AACBBCAACBBC Non-Coalescing Tree: Why low copy genes? Orthologs vs. Paralogs

5. Species Sampled: North American White Pines P. albicaulis Whitebark Pine P. ayacahuite Mexican White Pine P. chiapensis Chiapas White Pine P. flexilis Limber Pine P. lambertiana Sugar Pine P. monticola Western White Pine P. strobiformis Southwestern White Pine P. strobus Eastern White Pine Outgroups P. monophylla Singleleaf Pinyon P. longaeva Bristlecone Pine P. longaeva P. monophylla P. flexilis P. ayacahuite P. albicaulis P. lambertiana

6. DNA Extraction from the megagametophyte tissue. megagametophyte (1n) embryo (2n) shoot end embryo root tip (radicle) Methods and Approach:

7. How it works:  DNA Denatures  Primers Anneal  Taq Polymerase builds a complementary copy. Polymerase Chain Reaction (PCR)

8. How it works:  DNA Denatures  Primers Anneal  Taq Polymerase builds a complementary copy. Polymerase Chain Reaction (PCR)

9. Results: Gel Electrophoresis IFG8612 Optimization: MONTICOLA 02S1 Temperature (C)  56 57 58 59 60 61 Temperature (C)  56 57 58 59 60 61 62 63 64 65 66 67 68 Control 2.0 mM MgCl1.5 mM MgCl

10. Polymerase Chain Reaction (PCR) Results: Gel Electrophoresis IFG8612 Optimization: MONTICOLA 02S1 Temperature (C)  56 57 58 59 60 61 Temperature (C)  56 57 58 59 60 61 62 63 64 65 66 67 68 Control 2.0 mM MgCl1.5 mM MgCl

11. Polymerase Chain Reaction (PCR) Results: Gel Electrophoresis IFG8612 Optimization: MONTICOLA 02S1 Temperature (C)  56 57 58 59 60 61 Temperature (C)  56 57 58 59 60 61 62 63 64 65 66 67 68 Control 2.0 mM MgCl1.5 mM MgCl

12. Cycle Sequencing Capillary Gel

13. Cycle Sequencing Analyzing Results: Clean Sequence Sequence Noise ------------------------GTAATT----AGATTTGAATGGCATGGAATGAATGGAAA -GCaGGAtaTTGTCATTTCTTTCAGTAATT----AGATTTGAATGGCATGGAATGAATGGAAA ----GGAtaTTATCATTTTTTTCaGTAATTAATTAGATTTGAATGGCATGGAATGAATGGAAA -GCaGGAtATTGTCATTTCTTTCAGTAATT----AGATTTGAATGGCATGGAATGAATGGAAA Alignment and Editing

14. 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).

15. Results: Cinnamyl Alcohol Dehydrogenase IFG8898 IFG8612

16. Results: Cinnamyl Alcohol Dehydrogenase IFG8898 IFG8612

17. Results: Cinnamyl Alcohol Dehydrogenase IFG8898 IFG8612

18. IFG8612 alignment

22. MONOPHYLLA JVS LONGAEVA JVS CHIAPENSIS JVS CHIAPENSIS 02 CHIAPENSIS 01 100 FLEXILIS JVS FLEXILIS 01S5 FLEXILIS 03S1 AYACAHUITE 04S2 AYACAHUITE 03S2 STROBIFORMIS 01S1 84 62 84 MONTICOLA JVS MONTICOLA 02S3 70 60 100 65 ALBICAULIS 01S1 ALBICAULIS 03S1 ALBICAULIS 04S1 64 LAMBERTIANA STROBUS 02S1 STROBUS JVS STROBUS 03S1 100 88 57 86 67 LAMBERTIANA 08S2 LAMBERTIANA 02S1 53 100 Phylogenetic Tree

23. Outgroups Bootstrap Support (%) MONOPHYLLA JVS LONGAEVA JVS CHIAPENSIS JVS CHIAPENSIS 02 CHIAPENSIS 01 100 FLEXILIS JVS FLEXILIS 01S5 FLEXILIS 03S1 AYACAHUITE 04S2 AYACAHUITE 03S2 STROBIFORMIS 01S1 84 62 84 MONTICOLA JVS MONTICOLA 02S3 70 60 100 65 ALBICAULIS 01S1 ALBICAULIS 03S1 ALBICAULIS 04S1 64 LAMBERTIANA JVS STROBUS 02S1 STROBUS JVS STROBUS 03S1 100 88 57 86 67 LAMBERTIANA 08S2 LAMBERTIANA 02S1 53 100

24. Phylogenetic Tree MONOPHYLLA JVS LONGAEVA JVS CHIAPENSIS JVS CHIAPENSIS 02 CHIAPENSIS 01 100 FLEXILIS JVS FLEXILIS 01S5 FLEXILIS 03S1 AYACAHUITE 04S2 AYACAHUITE 03S2 STROBIFORMIS 01S1 84 62 84 MONTICOLA JVS MONTICOLA 02S3 70 60 100 65 ALBICAULIS 01S1 ALBICAULIS 03S1 ALBICAULIS 04S1 64 LAMBERTIANA JVS STROBUS 02S1 STROBUS JVS STROBUS 03S1 100 88 57 86 67 LAMBERTIANA 08S2 LAMBERTIANA 02S1 53 100

25. Phylogenetic Tree MONOPHYLLA JVS LONGAEVA JVS CHIAPENSIS JVS CHIAPENSIS 02 CHIAPENSIS 01 100 FLEXILIS JVS FLEXILIS 01S5 FLEXILIS 03S1 AYACAHUITE 04S2 AYACAHUITE 03S2 STROBIFORMIS 01S1 84 62 84 MONTICOLA JVS MONTICOLA 02S3 70 60 100 65 ALBICAULIS 01S1 ALBICAULIS 03S1 ALBICAULIS 04S1 64 LAMBERTIANA JVS STROBUS 02S1 STROBUS JVS STROBUS 03S1 100 88 57 86 67 LAMBERTIANA 08S2 LAMBERTIANA 02S1 53 100

26. Phylogenetic Tree MONOPHYLLA JVS LONGAEVA JVS CHIAPENSIS JVS CHIAPENSIS 02 CHIAPENSIS 01 100 FLEXILIS JVS FLEXILIS 01S5 FLEXILIS 03S1 AYACAHUITE 04S2 AYACAHUITE 03S2 STROBIFORMIS 01S1 84 62 84 MONTICOLA JVS MONTICOLA 02S3 70 60 100 65 ALBICAULIS 01S1 ALBICAULIS 03S1 ALBICAULIS 04S1 64 LAMBERTIANA JVS STROBUS 02S1 STROBUS JVS STROBUS 03S1 100 88 57 86 67 LAMBERTIANA 08S2 LAMBERTIANA 02S1 53 100

27. Phylogenetic Tree 1 MONOPYLLA JVS LONGAEVA JVS CHIAPENSIS JVS CHIAPENSIS 02 CHIAPENSIS 01 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 FLEXILIS JVS

28. Phylogenetic Tree 1 MONOPYLLA JVS LONGAEVA JVS CHIAPENSIS JVS CHIAPENSIS 02 CHIAPENSIS 01 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 FLEXILIS JVS

29. 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.

30. Special Thanks to: Howard Hughes Medical Institute Portland Garden Club Undergraduate Research, Innovation, Scholarship, and Creativity (URISC) National Science Foundation P. strobus