(Cronquist #11040) and Klamath Co., Oregon (Baldwin #380) were 

 utilized for the RAPD analysis. 



Electrophoretic Analysis 



Electrophoretic protocols employed were primarily those 

 described in Brunsfeld et al. (1991) and Soltis et al. (1983) . 

 Leaf tissue was ground in Tris-HCL grinding buffer, absorbed onto 

 filter paper wicks, loaded into starch gels, and electrophoresed 

 for approximately six hours. In preliminary tests seventeen 

 enzymes were stained: aconitase (ACN) , alcohol dehydrogenase 

 (ADH) , acid phosphatase (APH) , aspartate aminotransferase (AAT) , 

 esterase (EST) , glutamate dehydrogenase (GDH) , hexokinase (HK) , 

 isocitrate dehydrogenase (IDH) , leucine aminopeptidase (LAP) , 

 malate dehydrogenase (MDH) , malic enzyme (ME) , menadione 

 reductase (MNR) , phosphoglucoisomerase (PGI) , phosphoglucomutase 

 (PGM), 6-phosphogluconate dehydrogenase (6-PGD), shikimate 

 dehydrogenase (SKDH) , and triosephosphate isomerase (TPI) . Of 

 the enzymes tested, eleven (AAT, ADH, APH, IDH, LAP, MDH, PGI, 

 PGM, 6-PGD, SKDH, and TPI) produced enzyme activity and were 

 analyzed in all subsequent gel runs. 



The genetic basis of enzyme banding patterns was inferred 

 from observed segregation patterns in light of typical subunit 

 structure and subcellular compartmentalization (Gottlieb 1981, 

 Weeden and Wendel 1989). For enzymes with more than one locus 

 (TPI) , the isozymes were numbered sequentially, with the most 

 anodal isozyme designated 1. Allozymes were labeled 

 alphabetically starting with the fastest allozyme. Enzyme data 

 were analyzed using BIOSYS-1 (Swofford and Selander 1981) . Three 

 measures of genetic diversity were calculated: mean number of 

 alleles per locus (A) , percentage of loci polymorphic (P) , and 

 mean expected heterozygosity (HJ . Genetic divergence among 

 populations and species was analyzed using Nei's (1978) unbiased 

 genetic identity measures calculated by BIOSYS-1, and a UPGMA 

 cluster analysis of identity values was performed. 



DNA Analysis 



To gain better resolution of putative hybridization and 

 genetic relationships among Cirsium species, we conducted a 

 genetic analysis called "Randomly Amplified Polymorphic DNA" 

 (RAPD) , following the general methods of Williams et al. (1990) 

 and Welsh and McClelland (1990) . This technique involves 

 amplifying numerous random portions of the plant genome using the 

 polymerase chain reaction (PCR) . In PCR, a heat-stable enzyme 

 repeatedly replicates regions of the plant DNA, specifically 

 where the enzyme is "primed" by small pieces of synthetic DNA 

 (primers) . The amplified DNA products are separated in a gel. 



