Genetic diversity of an endemic terrestrial orchid assessed with ISSR polymorphisms

Background/Question/Methods Piperia yadonii Rand. Morgan and Ackerman (Orchidaceae), a mostly outbreeding, endemic perennial herb is restricted to a single California County, where the climate is Mediterranean. It occupies Northern coastal scrub, Pinus radiata forest, and Cupressus macrocarpa forest with some degree of geographic separation among populations. All populations are located within 80 km of each other, with a majority lying within 15 km of each other; however, habitats vary in elevation and plant community structure. To estimate the genetic relationships (population subdivision and population structure in space) among and within populations, we used nine primers to generate Inter-Simple Sequence Repeat (ISSR) band frequency data for up to 35 individuals from each of the seven sampled populations. Total number of loci scored for each primer, number of unique bands within each population, and percent polymorphic loci were determined from the two-state data. Dice similarity coefficients to establish pair wise similarities and Nei’s genetic distance between populations were calculated. Neighbor-joining trees followed by a majority rule consensus tree were generated to establish among and within population relationships. Results/Conclusions Preliminary analyses indicate most all bands (total of 636 across 9 primers and 7 populations) were polymorphic; percent ISSR polymorphism observed in this study ranged from 89 to 100% indicating a diversity of ISSR fragments in individuals within populations.  Percentage of unique loci across populations ranged from 21% to 27%. The mean Nei’s gene diversity (H_e) within a population was 0.058. Nei’s genetic distance measure indicated that several populations within geographic proximity of each other shared genetic similarity (e.g., 1.33 for the distinct group AP and JP; 1.36 for BC and SFB; 1.20 for BC and PQR), however we also observed high genetic similarity among some populations which are geographically distant (e.g., 1.23 for PL and MP).  PQR appears to be the closest common ancestor of BC and SFB.  Hierarchical AMOVA showed that 57% of the total variation was attributable to differences among individuals within populations while approximately 42% was distributed among the populations (F_ST = 0.42).The cladistic analyses indicate that AP, JP, BC, and SFB have diverged and are placed further from what might have been the original population with a comprehensive gene pool.  Habitat fragmentation, isolation of resulting populations, and limited gene flow among populations may be contributing to the observed pattern in ISSR polymorphisms within and across populations of Piperia yadonii.