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† Background and Aims Evolutionary and ecological roles of the chloroplast small heat shock protein (CPsHSP) have been emphasized based on variations in protein contents; however, DNA sequence variations related to the evolutionary and ecological roles of this gene have not been investigated. In the present study, a basal angiosperm, Machilus, together with the eudicot Rhododendron were used to illustrate the evolutionary dynamics of gene divergence in CPsHSPs. † Methods Degenerate primers were used to amplify CPsHSP-related sequences from 16 Rhododendron and eight Machilus species that occur in Taiwan. Manual DNA sequence alignment was carried out according to the deduced amino acid sequence alignment performed by CLUSTAL X. A neighbour-joining tree was generated in MEGA using conceptual translated amino acid sequences from consensus sequences of cloned CPsHSP genes from eight Machilus and 16 Rhododendron species as well as amino acid sequences of CPsHSPs from five monocots and seven other eudicots acquired from GenBank. CPsHSP amino acid sequences of Funaria hygrometrica were used as the outgroups. The aligned DNA and amino acid sequences were used to estimate several parameters of sequence divergence using the MEGA program. Separate Bayesian inference of DNA sequences of Rhododendron and Machilus species was analysed and the resulting gene trees were used for detection of putative positively selected amino acid sites by the Codeml program implemented in the PAML package. Mean hydrophobicity profile analysis was performed with representative amino acid sequences for both Rhododendron and Machilus species by the Bioedit program. The computer program SplitTester was used to examine whether CPsHSPs of Rhododendron lineages and duplicate copies of the Machilus CPsHSPs have evolved functional divergence based on the hydrophobicity distance matrix. † Key Results Only one copy of the CPsHSP was found in Rhododendron. However, a higher evolutionary rate of amino acid substitutions in the Hymenanthes lineage of Rhododendron was inferred. Two positively selected amino acid sites may have resulted in higher hydrophobicity in the region of the a-crystallin domain (ACD) of the CPsHSP. By contrast, the basal angiosperm, Machilus, possessed duplicate copies of the CPsHSP, which also differed in their evolutionary rates of amino acid substitutions. However, no apparent relationship of ecological relevance toward the positively selected amino acid sites was found in Machilus. † Conclusions Divergent evolution was found for both Rhododendron lineages and the paralogues of CPsHSP in Machilus that were directed to the shift in hydrophobicity in the ACD and/or methionine-rich region, which might have played important roles in molecular chaperone activity.

Background and Aims The genetic variation and divergence estimated by allozyme analysis were used to reveal the evolutionary history of Castanopsis carlesii in Taiwan. Two major questions were discussed concerning evolutionary issues: where are the diversity centres, and where are the most genetically divergent sites in Taiwan? Methods Twenty-two populations of C. carlesii were sampled throughout Taiwan. Starch gel electrophoresis was used to assay allozyme variation. Genetic parameters and mean FST values of each population were analysed using the BIOSYS-2 program. Mean FST values of each population against the remaining populations, considered as genetic divergence, were estimated using the FSTAT program. Key Results Average values of genetic parameters describing the within-population variation, the average number of alleles per locus (A = 25), the effective number of alleles per locus (Ae = 138), the allelic richness (Ar = 238), the percentage of polymorphic loci (P = 69%), and the expected heterozygosity (He = 0270) were estimated. High levels of genetic diversity were found for C. carlesii compared with other local plant species. Genetic differentiation between populations was generally low. Conclusions From the data of expected heterozygosity, one major diversity centre was situated in central Taiwan corroborating previous reports for other plant species. According to the mean FST value of each population, the most divergent populations were situated in two places. One includes populations located in north central Taiwan between 2480N and 2420N. The other is located in south-eastern Taiwan between 2240N and 2310N. These two regions are approximately convergent with the most divergent locations determined for several other plant species using chloroplast DNA markers published previously. An important finding obtained from this study is that unordered markers like allozymes can be used to infer past population histories as well as chloroplast DNA markers do.

Seeds of Taxus mairei are known for their deep dormancy which can only be broken by a procedure involving warm stratification followed by cold stratification. Treatments with alternating temperatures of 25}15 or 23}11 ∞C (12 h light) for 6 months followed by 5 ∞C for 3 months were successful in overcoming seed dormancy. After 6 months of warm stratification, cytological changes observed included: enlargement of the embryo; a decrease in the number of lipid bodies; appearance of ER; and increases in mitochondria, plastids, dictyosomes, vacuoles and microbodies in the shoot apical meristem. Cold stratification following the warm treatment induced cell division, and one or two distinct nucleoli in the shoot apical meristem cells were observed. Both warm and cold stratification reduced endogenous ABA concentrations from the original 8888 pg per freshly harvested seed to 392 and 536 pg, respectively. Treatment with exogenous gibberellins after seeds had been warm-stratified showed that GA % and GA ( were eective at promoting seed germination, but GA $ was not. These results suggest that the strong seed dormancy of T. mairei could be caused by a high ABA content and underdevelopment of the embryos in freshly shed seeds. We conclude that warm stratification with alternating temperatures increases the growth of embryos by cell expansion and enlargement and decreases ABA content, but seeds still remain ungerminated. Cold stratification may induce the response to GAs and initiate cell division resulting in release from physiological dormancy and subsequent germination of T. mairei seeds. # 1998 Annals of Botany Company

Biochemical properties, i.e. endogenous abscisic acid, proline, sugars, respiration, adenosine phosphates and adenylate energy charge, and growth and moisture content were measured during the development of seeds of Machilus thunbergii. As dry matter accumulated in the embryo during development, moisture content, ABA, proline, respiration and sugars all declined. At maturity, the dry mass of the seeds failed to attain a plateau before the period of natural seed shedding; the axis and cotyledons reached moisture contents of 58 and 45%, respectively. Drying of immature seeds at 73% relative humidity and 25 °C for 30 d resulted in a complete loss of viability at all developmental stages tested with the exception of mature seeds that were able to tolerate a 5% decrease in moisture content before germination declined. ABA was detected in all embryos tested, with a maximum value 16.·16 μg g -1 d. wt about midway through development. Although the presence of ABA induced no tolerance to desiccation of mature seeds, it did coincide with decreased content of water in the developing seeds and decreased respiration. Desiccation damage of M. thunbergii seeds occurred when moisture content was still high (45%) and this damage was not related to the absence of oligosaccharides in the mature seeds. We conclude that developing embryos and mature seeds of M. thunbergii have properties common to many recalcitrant seeds, with seeds being sensitive to desiccation at all stages, having a prominent ABA peak, little proline, lacking oligosaccharides, and specifically, little dormancy and a moderate rate of respiration of mature seeds (0·9 μmol O 2 min -1 g -1 f. wt). Adenosine triphosphate content and energy charge decreased from stage four to stage eight of seed development, then increased again to 103 nmol g -1 d. wt and 0·73, respectively, in mature seeds. The moderate energy charge observed in mature seeds indicates that continuous metabolism is also a characteristic of recalcitrant seeds.