Thursday, August 5, 2010

PS 74-79: Using microsatellites to assess genetic variability in Ceanothus megacarpus in the Santa Monica Mountains of California

Caitlin D. Ishibashi, Anthony R. Shaver, David P. Perrault, Rodney L. Honeycutt, and Stephen D. Davis. Pepperdine University


Chaparral vegetation of southern California is regularly subjected to freezing, as temperatures can drop well below 0 C in cold valleys as well as at high elevations.  Previous experiments established that Ceanothus species in the Santa Monica Mountains exhibit a broad range in cold tolerance, with lethal temperature for 50% leaf death (LT50) varying between -9 C to -22 C. We hypothesized that a 4.2 km gradient in minimum temperature between a warm coastal site (minimum of 0 C) and a cold inland site (minimum of -12 C) influenced genetic variability among C. megacarpus populations, especially at a cold-tolerance-ecotone, where hybridization might occur. We tested this hypothesis by using a panel of microsatellite loci, which was isolated from a genome library to Ceanothus megacarpus, optimized, and characterized.  Nuclear DNA was isolated from fresh tissue collected from individual plants using standard molecular techniques and specially designed primers to select for microsatellite regions.  The polymerase chain reaction (PCR) was used to amplify specific DNA segments at 5 unique loci.  Amplification of PCR product was quantified using agarose gel electrophoresis.  These DNA fragments were genotyped on a DNA sequencer, and statistical tests were run to examine levels of genetic diversity.


Our results showed that patterns of genetic variation at five loci revealed significant allelic and genotypic differences between two populations of C. megacarpus located only 4.2 kilometers apart, thus suggesting a reduction in gene flow.  Nei’s Unbiased Genetic Distance was 0.229, indicating 30% similarity between the two populations of C. megacarpus. An Analysis of Molecular Variance (AMOVA) showed 7% molecular variation based on allele frequencies between the two populations.  These results are significant, because they represent two contiguous populations, only 4.2 kilometers apart, yet separated genetically, possibly in response to a strong coastal to inland freezing gradient. We are exploring the possibility that observed genetic differences in the inland population are matched by increased freezing tolerance (LT50) that result from hybridization of C. megacarpus (LT50 = -9 C) with C. cuneatus (LT50 = -22 C) along a cold-tolerance-ecotone.