University of Miami and Howard Hughes Medical Institute, Research in Ecology Program, Summer 2008

1. Saving the Endangered South Florida Slash Pines (Pinus elliottii var. densa)---- A study of Genetic Variations of Two Slash Pine Populations Super Awesome Pines (SAP): Renee Roache, Martin Alexis, Allegra delValle, Melissa Perez, O’Brian Anderson University of Miami and Howard Hughes Medical Institute, Research in Ecology Program, Summer 2008

2. Outline for our talk • Background on Florida Slash Pine • Importance of our study • Methods • Results • Discussion • Acknowledgements

3. Pinus elliottii var. densa • Evergreen conifer. • Pollen cones are lower on the trees than the seed cones.

4. Pinus elliottii var. densa Fire resistant to low intensity fires • Native to central and south Florida, including the Florida Keys. • It’s the keystone species in the pine rockland community.

5. Back Then… 1940’s Today • There used to be 65,000 acres of pine rockland habitat in the 1940’s but now there is less than 1% left.

6. Genetic diversity • Genetic variation is important for populations to adapt to a changing environment • Such as hurricane

7. Significant values of South Florida slash pine Over 225 types of native plants occur here and more than 20% of the plant species are found here and nowhere else in the world.

8. Other plants in our study sites

9. A A T T T T A A C C G G C C G G G G C C G G C C G G C C C C G G C C G G A G C T C G C G G T C A C T A G C C G G G A T C T C A G A T T A C T A G C G C G How can we distinguish tree DNA? Marker • We used nuclear microsatellite markers to study genetic variation. • Microsatellites are short segments of DNA that have a repeated sequence. Tree 1 Tree 2

10. Question:Whether fragmentation has effect on genetic diversity of South Florida Slash pine? • Null Hypothesis: Fragmentation has no effect on genetic diversity of Pinus elliottii var. densa • Prediction: Fragmentation decrease genetic diversity.

11. Study Sites Outside UM campus Everglades National Park

12. Sample Collection 10 samples per site

13. DNA Extraction

14. DNA Extraction

15. Polymerase Chain Reaction (PCR) • PCR is a technique used to amplify a piece of DNA. • PCR enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours.

16. DNA Gel Electrophoresis - Negative • DNA electrophoresis is an technique used to separate DNA fragments by weight. • An electric field forces the fragments to migrate from negative to positive. + Positive

17. Gene Scan • Gene Scan analysis is carried out on a LI-COR 4300 DNA analyzer.

18. Genotype results at locus PtTX3013 and RPTest01 PtTX3013 S S S EV UM RPTest01 S S S EV UM S: Size Marker, EV: Sample from Everglades, UM: Sample from UM

19. Results Genetic variation of two slash pine populations at locus PtTX3013 RPTest01 • At both PtTX3013 and RPTest01 locus, there are two different alleles in EV, but only one allele in UM. • EV and UM share same alleles. • EV have heterozygous trees, all trees are homozygous in UM, base on the samples we collected.

20. Discussion • EV and UM share two alleles implies some connectivity between them. • This may be due to historical connection or possibly dispersal by wind, people or animal. • Fragmentation increased inbreeding, and decrease heterozygocity. • Larger population size is more heterozygotic. • Large area population have more genetic variation than fragmented population.

21. Discussion • Efforts of preservation for such population should be equally applied to all populations, small (UM population) and large (Everglades population). • Local government should all participate in conservation of the remaining population. • We cannot afford to sacrifice any populations, even though the population size may be small. • Small populations still contain alleles of the original population.

22. Acknowledgement We would like to thank Dr. Gaines for organizing this program, Dr. Krempels for organizing us, Dr. Wang for instructing this research, Mr. Jiang, Ms. Suarez and Mr. Cheso for helping us. We thank our parents and friends, Howard Hughes Medical Institute and University of Miami.