Molecular Taxonomy of the Lichen Genus Heterodermia

Heterodermia is lichen consisting of a fungus and algae. Morphology and chemistry cannot provide definitive evidence for the identity of Heterodermia because various species have very similar morphologies that cannot always be distinguished. Other species only differ by the presence or absence of secondary chemicals detected. by specific chemical tests, which makes distinguishing between two species difficult. The objective of this project is to use Restriction Fragment Length Polymorphisms (RFLPs) to determine how closely related each Heterodermia species is to one another. RFLPs will also be used to determine how closely related a species is to its equal in another geographical location. ITS-I and ITS-2, the regions surrounding the 5.Ss ribosomal DNA sequence, is the location of interest because they arc noncoding regions and will accumulate mutations even among closely related taxa. Lichen material was homogenized in protcinase k enzyme and digested with TRI reagent (Sigma). Regions ITS-I and ITS-2 were isolated and purified using PCR with primers ITS-5 and ITS-4, which arc located in the 3\u27 end of the ssU rDNAon the+ strand and in the 5\u27 end of the lsU rDNA on the- strand, respectively. After the PCR step, the eight species of Heterodennia and one control, Anaptychia palmatula, were divided into two groups based on the sizes of the PCR products, which were e 600 hp andw 800 hp. Both groups were digested with EcoR I, qo I, and Hinfi. All species displayed different restriction patterns except for H. leucomela and H. barberifera. These two species will be further studied by DNA sequencing to see how close of a relationship they have. Five species were compared with their equals from other geographical locations. Three species displayed similar restriction patterns between the two geographical locations. One species, H. casarettiana displayed different restriction patterns. Another species, H speciosa, produced a PCR fragment of a size that did not match the other strain examined. Restriction digest analysis confirmed this finding. Based on these experiments, discovery of a new species or subspecies is possible. Future experiments will include DNA sequencing