The sequence divergence of chloroplast rbcL, matK, trnV intron, and rpl20-rps18 spacer regions was analyzed among 32 Pinus species and representatives of six other genera in Pinaceae. The total aligned sequence length is 3570 bp. Of the four sequences examined, matK evolved much faster than rbcL in Pinus and in other Pinaceae genera. The two noncoding regions did not show more divergence than the two coding regions, especially within each Pinus subgenus. Phylogenetic analyses based on these four sequences gave consistent results and strongly supported the monophyly hypothesis for the genus Pinus and its two recognized subgenera. Pinus krempfii, the two-flat-needle pine endemic to Vietnam, was placed in subgen. Strobus and showed closer affinity to subsect. Gerardianae. The ancient character of sect. Parrya is further confirmed. However, monophyly of the sect. Parrya is not supported by our data. Among the Eurasian pines of subgen. Pinus, Mediterranean pines formed one clade and the Asian members of subsect. Sylvestres formed another. The Himalayan P. roxburghii showed considerable divergence from all the other hard pines from both regions. Pinus merkusii was distinctly separated from all the Asian members of subsect. Sylvestres. The implications of our results for Pinus classification are discussed.

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5' and 3' ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.

We investigated the molecular phylogeny of conifers using restriction endonuclease fragment length polymorphism of six polymerase chain reaction-amplified chloroplast genes - frxC, rbcL, psbA, psbD, trnK, and 16S. We detected 227 total site changes among species, representing 23, 26, 38, 48, 67, and 25 site changes in frxC, psbA, psbD, rbcL, trnK and 16S, respectively. The mean nucleotide substitution was 10.75% (SD 0.573) among species in five families. Forty maximally parsimonious trees were obtained using the Wagner parsimony method, and a 50% majority-rule consensus tree was obtained from them. Data analysis produced similar basic patterns when both the Wagner parsimony and the neighbor-joining methods were applied, and the main lineages were clearly separated. Taxaceae and Cephalotaxaceae species were used as the out-groups when applying Wagner parsimony methods. With the Wagner method, the consistency index was 0.510, the retention index was 0.879, and tree length was 435 steps. Our results indicated that Cupressaceae and Taxodiaceae are closely related families and that Sciadopitys verticillata is the basal lineage of Cupressaceae and Taxodiaceae. The neighbor-joining tree is similar to the 50% majority-rule consensus of the 40 Wagner parsimony trees except for the position of Keteleeria daversifolia, the Picea and Cedrus group, and the divergence within Cupressaceae.

Pollen flow and population genetic structure among 30 potentially flowering individuals of Neobalanocarpus heimii, a tropical emergent tree, were investigated in a lowland tropical rainforest of Malaysia using microsatellite polymorphism. The 248 offspring in the vicinity of five reproductive trees of the 30 potentially flowering trees were used in paternity analysis for pollen-flow study. Four primer pairs, developed in different species of dipterocarps, were adopted to detect microsatellite polymorphism. Based upon microsatellite polymorphism, pollen flow and seed migration were detected. Pollen-flow events of more than 400 m were observed directly, based on paternity analysis in the study plot. The estimated average mating distance of the five reproductive trees was 524 m. This result suggests that reproduction of this species is mediated by a long-distance pollinator. The haplotypes of some offspring were not compatible with the nearest reproductive tree. Thus, the results suggest that some seeds are dispersed by a seed dispersal vector. Investigation of genetic structure showed significant and negative correlation of genetic relatedness and spatial distances between the 30 potentially flowering trees, but this correlation was weak. We suggest that long-distance gene flow and seed migration are responsible for the poorly developed genetic structure of this species.

Mitochondrial (mt) DNA variation in Japanese beech, Fagus crenata (Fagaceae), was studied in 17 populations distributed throughout the species' range. Total genomic DNA of samples from single trees representing each of 12 populations were digested with 18 restriction enzymes and hybridized with three probes containing coxI, coxIII, and atpA gene sequences. Thirty-four of the 54 enzyme/probe combinations showed polymorphisms and all the individuals were subsequently analyzed with six combinations of three probes and two enzymes. Restriction fragment length polymorphisms were evident around all three genes, allowing the identification of eight distinct haplotypes. Haplotype diversity within the populations was found to be very low (HS = 0.031), but population differentiation to be much higher (GST = 0.963). The mtDNA variation was strikingly different from allozyme variation (HS = 0.209; GST = 0.039). Gene flow for maternally inherited mtDNA should be restricted to seed dispersal while nuclear gene flow occurs by both seed and pollen dispersal. Therefore, the difference in the variation between mtDNA and allozymes may be largely a result of the much higher rate of gene flow associated with pollen dispersal than with seed dispersal. The mtDNA variation displayed strong geographic structure, which may reflect the species' distribution in the last glacial maximum and subsequent colonization, and probably also reflects intraspecific phylogeography of the species.