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 analysed 20 allozyme and 22 putative random amplified polymorphic DNA (RAPD) loci in two populations of Pinus sylvestris (L.) from northern Sweden. Genotypes for individual allozyme and RAPD loci were inferred from segregation patterns in haploid macrogametophytes. Therefore, it was possible to distinguish between homo-and heterozygotes carrying a RAPD fragment and to estimate directly the frequencies of RAPD fragments. The percentage of polymorphic loci and the expected and observed heterozygosity were lower for allozymes than for RAPDs. Average fixation indices for both types of markers were negative indicating a heterozygote excess over panmictic expectations. The apportionment of genetic variation within and among the investigated populations was similar for allozymes and RAPDs and showed that most of the variation resided within populations. RAPD genotypes inferred from haploid material were subsequently converted to diploid phenotypes and used to estimate indirectly the frequencies of RAPD fragments. Gene diversity measurements derived from indirectly estimated RAPD frequencies were distinctly lower than those based on directly estimated RAPD frequencies. This result was caused by the absence of the null homozygote at many loci which appeared as monomorphic in the diploid data set. Population differentiation coefficients based on the indirectly estimated RAPD frequencies were not concordant with those based on directly estimated RAPD frequencies. Our present results indicate that when complete genotype information can be obtained, RAPD analysis provides genetic information similar to that revealed by analysis of allozyme variation. On the other hand, our results are concordant with theoretical results suggesting that analysis of RAPD variation in diploid material can produce unreliable estimates of population-genetic parameters.

Although homoploid hybridization is increasingly recognized as an important phenomenon in plant evolution, its evolutionary genetic mechanisms are poorly documented and understood. Pinus densata, a pine native to the Tibetan Plateau, represents a good example of a homoploid hybrid speciation facilitated by adaptation to extreme environment and ecological isolation from the parents. Its ecologically and reproductively stabilized nature offers excellent opportunity for studying genetic processes associated with hybrid speciation. In this study, we investigated the levels and patterns of nucleotide variation in P. densata and its putative parents. Haplotype composition, gene genealogies, and the levels and patterns of nucleotide variation gave further support to the hybrid nature of P. densata. Allelic history, as revealed by our data, suggests the ancient nature of the hybrid preceding elevation of the Tibetan Plateau. We detected more deviations from neutrality in P. densata than in the parental species. Thus, at least some of the evolutionary forces that have shaped the genetic variation in P. densata are likely to be different from those acting upon parental species. We speculate that when populations of P. densata invaded new territories, they had elevated rates of response to selection in order to develop traits that help them to survive and adapt in the new environments.

Heterologous hybridization of chloroplast DNA (cpDNA) involving 30 endonucleaseprobe combinations was used to analyze cpDNA variation in multiple individuals and populations of Pinus tabulaeformis (Carr.), Pinus yunnanensis (Franchèt) and Pinus massoniana (Lamb.). Restriction fragment patterns detected by several combinations distinguished among the three species. The obtained cpDNA markers were subsequently used to examine cpDNA variation of Pinus densata (Masters), a putative tertiary hybrid between P. tabulaeformis and P. yunnanensis. The analysis demonstrated that P. densata populations harbor three different haplotypes. Two of these haplotypes are characteristic of P. tabulaeformis and P. yunnanensis. However, the third haplotype found in P. densata appears to be absent in other extant Asian Pinus species. It is suggested that the observed cpDNA composition of P. densata populations is a result of past hybridization involving P. tabulaeformis, P. yunnanensis, and a third unknown or extinct taxon. Chloroplast DNA polymorphism in P. densata was much greater than that for nuclear allozyme markers in this and the other Pinus species. Population differentiation was also substantial in P. densata and exceeded that for allozyme markers. In contrast, no cpDNA polymorphism was detected in populations of P. tabulaeformis, P. yunnanensis, and P. massoniana. The study suggests that interspecific gene exchange may lead to the creation of stable cpDNA polymorphism in conifer hybrids.

Restriction enzyme analysis was used to determine the inheritance of chloroplast DNA in conifers. The plant material studied included five individual trees of European larch (Larix decidua Mill.) and Japanese larch (Larix leptolepis Sieb. & Zucc.) and six hybrids from controlled crosses between these species. The chloroplast DNA fragment patterns generated by Bam-HI and Bcl-I were species-specific. Paternal inheritance of chloroplast DNA patterns was found in most Larix crosses. One hybrid showed maternal chloroplast DNA patterns. In addition, two other hybrids had mixed Bam-HI patterns suggesting recombination between maternal and paternal chloroplast DNA. The mechanisms favoring paternal inheritance in conifers are not known. Paternal inheritance of chloroplast DNA is suggested it to be a general phenomenon in conifers.