The utilization of micro-and macromolecular data for the analyses of evolutionary problems : examples from Juniperus and Calocedrus

SUMMARY In the last decade, techniques based on DNA markers have been used to detect variation at DNA level and have proven to be reliable and very effective for distinguishing between closely related genotypes. The development of the random amplified polymorphic DNA (RAPD) markers (Williams et al., 1990; Welsh et al., 1991) allowed genome characterization in several plant groups. The RAPD technique has been useful in studying polymorphism, identifying genes of interest, and permitting the determination of phylogenetic relationships and intraspecific diversity at a molecular genetic level. In four minor studies, this thesis deals with the evolutionary relationships and genetic variation among different populations of plants in two genera of Juniperus and Calocedrus using the methodology of RAPD-polymerase chain reaction (PCR) together with morphological traits. By performing chemical analysis of leaf essential oils and comparing their oil compositions, these data are also utilized to define phylogeny and taxonomy of species in these genera. In this respect, the synthesis of data from different levels, from biochemical to genetic, attempts to increase our understanding in plant evolution and systematics. The basics of the methods employed will be described briefly and their applications also reviewed. The first study examined the varieties of the species Juniperus deppeana. Populations of J.deppeana var. deppeana, var. pachyphlaea, var. patoniana, var. robusta, var. zacatecensis, and J. gamboana were analyzed by DNA fingerprinting (RAPDs). By comparison with the closely related J. saltillensis, J. gamboana was found to conspecific with J. deppeana and recognized as a new variety: J. deppeana var. gamboana (Mart.) R. P. Adams. Juniperus deppeana var. pachyphlaea and J. d. var. zacatecensis were found to be a part of J. deppeana var. deppeana. Juniperus deppeana var. zacatecensis was reduced to a new forma: J. deppeana f. zacatecensis (Mart.) R. P. Adams. Considerable amounts of intra-population variation was present. Previous Pleistocene distributions and past gene flow seems to have contributed to the lack of populational differentiation. A key and revised distribution map of J. deppeana is also presented here. In the second study, another species of Juniperus, J. communis, was investigated to detect the genetic variation in Norwegian junipers, also using RAPD-DNA fingerprinting applications. It has been shown (Looman and Baerheim Svendsen, 1992) that the needle essential oils of J. communis from the mountains of Norway were dominated by sabinene, in contrast to the oils from the lowland characterized by having high percentage of á-pinene. RAPD data of these two populations, one from the mountains of central Norway and the other from the coastal areas, were analyzed to determine whether these differences are due to divergence in the plant genome or they are merely products of environmentally induced changes. Principal coordinate analysis did not show two distinctive groups as expected; the coastal samples (high in á-pinene) formed a uniform group but some individuals from the mountain group (high in sabinene) were more scattered and linked to the coastal group, resulting in a complex pattern. The varied oil concentration ratio of á-pinene: sabinene: in some individuals of Looman and Baerheim Svendsen report may be reflected in the intermediate juniper individuals seen here. Thus, there is not enough evidence to consider a new variety or forma of J.communis grown in Norway, so that influence of environmental factors may be the explanation and supporting Looman and Baerheim Svendsen s suggestion of the existence of several mountain juniper chemotypes. The third study discusses the leaf essential oil composition of the genus Calocedrus which consists of only three taxa. Leaf essential oils of C. decurrens, (west North America), C. macrolepis (southwest China, northern Vietnam, northern Thailand) and C. macrolepis var. formosana (Taiwan) were analyzed and compared to the oils of the closely related species, Platycladus orientalis (east, northeast China, Korea, far eastern Russia). The oils of C. macrolepis var. macrolepis and C. m. var. formosana were very similar, being high in á-pinene (57.2, 67.1%) and myrcene (11.2, 6.2), but they differed in many smaller components. The oils of C. decurrens, from two populations in Oregon and one disjunct population in south California, were high in ä-3-carene (15.2 - 20.2%), limonene (18.2 - 23.6), á-pinene (8.7 - 15.8), terpinolene (5.7 - 8.0), endo-fenchyl acetate (3.5 - 9.7), with some cedrol (0.8 - 1.2). No large differences in oil compositions were seen between the three C. decurrens populations. Platycladus orientalis (= Thuja orientalis, = Biota orientalis) was found to contain considerable amounts of ä-3-carene (29.8%), cedrol (22.2%), á-pinene (15.1) and terpinolene (4.9). Overall, the oil of C. decurrens is as different from C. macrolepis and C. m. var. formosana, as it is from Platycladus orientalis, suggesting considerable divergence between Calocedrus species but not between C. macrolepis and C. m. var. formosana The composition of the leaf oils from seven populations of Juniperus sabina L. and one population of J. sabina var. arenaria (E. H. Wilson) Farjon were examined for geographic variation in the final study. In addition, the leaf oils of J. chinensis L. and J. davurica Pall. are compared to J. sabina. In Juniperus sabina var. arenaria, the sand loving juniper, the oil was found to be very similar to that of J. davurica, Mongolia, and J. sabina, on sand dunes in Mongolia. This suggests that J. sabina var. arenaria might be conspecific with J. davurica and Farjon's move (2001) of J. sabina var. arenaria out of J. chinensis is therefore supported. Considerable differentiation was found in populations of J. sabina from the Iberian peninsula. Cedrol, citronellol, safrole, trans-sabinyl acetate, terpinen-4-ol and trans-thujone were found to be polymorphic in several populations