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Population diversification can be shaped by a combination of environmental factors as well as geographic isolation interacting with gene flow. We surveyed genetic variation of 243 samples from 12 populations of Calocedrus formosana using amplified fragment length polymorphism (AFLP) and scored a total of 437 AFLP fragments using 11 selective amplification primer pairs. The AFLP variation was used to assess the role of gene flow on the pattern of genetic diversity and to test environments in driving population adaptive evolution. This study found the relatively lower level of genetic diversity and the higher level of population differentiation in C. formosana compared with those estimated in previous studies of conifers including Cunninghamia konishii, Keteleeria davidiana var. formosana, and Taiwania cryptomerioides occurring in Taiwan. BAYESCAN detected 26 FST outlier loci that were found to be associated strongly with various environmental variables using multiple univariate logistic regression, latent factor mixed model, and Bayesian logistic regression. We found several environmentally dependent adaptive loci with high frequencies in low- or high-elevation populations, suggesting their involvement in local adaptation. Ecological factors, including relative humidity and sunshine hours, that are generally not altitude related could have been the most important selective drivers for population divergent evolution in C. formosana. The present study provides fundamental information in relation to adaptive evolution and can be useful for assisted migration program of C. formosana in the future conservation of this species.

Demographic events are important in shaping the population genetic structure and exon variation can play roles in adaptive divergence. Twelve nuclear genes were used to investigate the species-level phylogeography of Rhododendron oldhamii, test the difference in the average GC content of coding sites and of third codon positions with that of surrounding non-coding regions, and test exon variants associated with environmental variables. Spatial expansion was suggested by R2 index of the aligned intron sequences of all genes of the regional samples and sum of squared deviations statistic of the aligned intron sequences of all genes individually and of all genes of the regional and pooled samples. The level of genetic differentiation was significantly different between regional samples. Significantly lower and higher average GC contents across 94 sequences of the 12 genes at third codon positions of coding sequences than that of surrounding non-coding regions were found. We found seven exon variants associated strongly with environmental variables. Our results demonstrated spatial expansion of R. oldhamii in the late Pleistocene and the optimal third codon position could end in A or T rather than G or C as frequent alleles and could have been important for adaptive divergence in R. oldhamii.

Genetic variation evolves during postglacial range expansion of a species and is important for adapting to varied environmental conditions. It is crucial for the future survival of a species. We investigate the nuclear DNA sequence variation to provide evidence of postglacial range expansion of Musa basjoo var. formosana, a wild banana species, and test for adaptive evolution of amplified fragment length polymorphic (AFLP) loci underlying local adaptation in association with environmental variables. Postglacial range expansion was suggested by phylogeographical analyses based on sequence variation of the second intron of copper zinc superoxide dismutase 2 gene. Two glacial refugia were inferred by the average FST parameter (mean FST of a population against the remaining populations). Using variation partitioning by redundancy analysis, we found a significant amount of explained AFLP variation attributed to environmental and spatially-structured environmental effects. By combining genome scan methods and multiple univariate logistic regression, four AFLP loci were found to be strongly associated with environmental variables, including temperature, precipitation, soil moisture, wet days, and surface coverage activity representing vegetation greenness. These environmental variables may have played various roles as ecological drivers for adaptive evolution of M. basjoo var. formosana during range expansion after the last glacial maximum.

Populations can be genetically isolated by differences in their ecology or environment that hampered efficient migration, or they may be isolated solely by geographic distance. Moreover, mountain ranges across a species’ distribution area might have acted as barriers to gene flow. Genetic variation was quantified using amplified fragment length polymorphism (AFLP) and 13 selective amplification primer combinations used generated a total of 482 fragments. Here, we tested the barrier effects of mountains on gene flow and environmentally dependent local adaptation of Cunninghamia konishii occur in Taiwan. A pattern of genetic isolation by distance was not found and variation partitioning revealed that environment explained a relatively larger proportion of genetic variation than geography. The effect of mountains as barriers to genetic exchange, despite low population differentiation indicating a high rate of gene flow, was found within the distribution range of C. konishii. Twelve AFLP loci were identified as potential selective outliers using genome-scan methods (BAYESCAN and DFDIST) and strongly associated with environmental variables using regression approaches (LFMM, Samβada, and rstanarm) demonstrating adaptive divergence underlying local adaptation. Annual mean temperature, annual precipitation, and slope could be the most important environmental factors causally associated with adaptive genetic variation in C. konishii. The study revealed the existence of physical barriers to current gene flow and environmentally dependent adaptive divergence, and a significant proportion of the rate of gene flow may represent a reflection of demographic history.

ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) of Arabidopsis thaliana is an AP2/ERF domain transcription factor that regulates jasmonate (JA) biosynthesis and is induced by methyl JA treatment. The regulatory mechanism of ORA47 remains unclear. ORA47 is shown to bind to the cis-element (NC/GT)CGNCCA, which is referred to as the O-box, in the promoter of ABI2. We proposed that ORA47 acts as a connection between ABA INSENSITIVE1 (ABI1) and ABI2 and mediates an ABI1-ORA47-ABI2 positive feedback loop. PORA47:ORA47-GFP transgenic plants were used in a chromatin immunoprecipitation (ChIP) assay to show that ORA47 participates in the biosynthesis and/or signaling pathways of nine phytohormones. Specifically, many abscisic acid (ABA) and JA biosynthesis and signaling genes were direct targets of ORA47 under stress conditions. The JA content of the P35S:ORA47-GR lines was highly induced under wounding and moderately induced under water stress relative to that of the wild-type plants. The wounding treatment moderately increased ABA accumulation in the transgenic lines, whereas the water stress treatment repressed the ABA content. ORA47 is proposed to play a role in the biosynthesis of JA and ABA and in regulating the biosynthesis and/or signaling of a suite of phytohormone genes when plants are subjected to wounding and water stress.

The question of what determines divergence both between and within species has been the central topic in evolutionary biology. Neutral drift and environmentally dependent divergence are predicted to play roles in driving population and lineage divergence. However, neutral drift may preclude adaptation if the rate of gene flow between populations is high. Here, we sampled populations of three Taiwania (Taiwania cryptomerioides) lineages occurring in Taiwan, the mainland of China (Yunnan-Myanmar border), and northern Vietnam, and tested the relative strength of neutral drift and divergent selection in shaping divergence of those populations and lineages. We quantified genetic and epigenetic variation, respectively, using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). Analysis of 1413 AFLP and 462 MSAP loci using frequency-based genome scan methods and generalized linear models (GLMs) found no potential selective outliers when only Taiwanese populations were examined, suggesting that neutral drift was the predominant evolutionary process driving differentiation between those populations. However, environmentally associated divergence was found when lineages were compared. Thirty-two potential selective outliers were identified based on genome scans and their associations with environmental variables were tested with GLMs, generalized linear mixed effect models (GLMMs), and model selection with a model averaging approach. Ten loci (six AFLP and four MSAP) were found to be strongly associated with environmental variables, particularly monthly temperature variation and normalized difference vegetation index (NDVI) using model selection and a model averaging approach. Because only a small portion of genetic and epigenetic loci were found to be potential selective outliers, neutral evolutionary process might also have played crucial roles in driving lineage divergence, particularly between geographically and genetically isolated island and mainland Asia lineages. Nevertheless, the vast amount of neutral drift causing genetic and epigenetic variations might have the potential for adaptation to future climate changes. These could be important for the survival of Taiwania in different geographic areas.

Double digest restriction site-associated DNA sequencing (ddRADseq) is a tool for delivering genome-wide single nucleotide polymorphism (SNP) markers for non-model organisms useful in resolving fine-scale population structure and detecting signatures of selection. This study performs population genetic analysis, based on ddRADseq data, of a coniferous species, Keteleeria davidiana var. formosana, disjunctly distributed in northern and southern Taiwan, for investigation of population adaptive divergence in response to environmental heterogeneity. A total of 13,914 SNPs were detected and used to assess genetic diversity, FST outlier detection, population genetic structure, and individual assignments of five populations (62 individuals) of K. davidiana var. formosana. Principal component analysis (PCA), individual assignments, and the neighbor-joining tree were successful in differentiating individuals between northern and southern populations of K. davidiana var. formosana, but apparent gene flow between the southern DW30 population and northern populations was also revealed. Fifteen of 23 highly differentiated SNPs identified were found to be strongly associated with environmental variables, suggesting isolation-by-environment (IBE). However, multiple matrix regression with randomization analysis revealed strong IBE as well as significant isolation-by-distance. Environmental impacts on divergence were found between populations of the North and South regions and also between the two southern neighboring populations. BLASTN annotation of the sequences flanking outlier SNPs gave significant hits for three of 23 markers that might have biological relevance to mitochondrial homeostasis involved in the survival of locally adapted lineages. Species delimitation between K. davidiana var. formosana and its ancestor, K. davidiana, was also examined (72 individuals). This study has produced highly informative population genomic data for the understanding of population attributes, such as diversity, connectivity, and adaptive divergence associated with large- and small-scale environmental heterogeneity in K. davidiana var. formosana.

Double digest restriction site-associated DNA sequencing (ddRADseq) is a tool for delivering genome-wide single nucleotide polymorphism (SNP) markers for non-model organisms useful in resolving fine-scale population structure and detecting signatures of selection. This study performs population genetic analysis, based on ddRADseq data, of a coniferous species, Keteleeria davidiana var. formosana, disjunctly distributed in northern and southern Taiwan, for investigation of population adaptive divergence in response to environmental heterogeneity. A total of 13,914 SNPs were detected and used to assess genetic diversity, FST outlier detection, population genetic structure, and individual assignments of five populations (62 individuals) of K. davidiana var. formosana. Principal component analysis (PCA), individual assignments, and the neighbor-joining tree were successful in differentiating individuals between northern and southern populations of K. davidiana var. formosana, but apparent gene flow between the southern DW30 population and northern populations was also revealed. Fifteen of 23 highly differentiated SNPs identified were found to be strongly associated with environmental variables, suggesting isolation-by-environment (IBE). However, multiple matrix regression with randomization analysis revealed strong IBE as well as significant isolation-by-distance. Environmental impacts on divergence were found between populations of the North and South regions and also between the two southern neighboring populations. BLASTN annotation of the sequences flanking outlier SNPs gave significant hits for three of 23 markers that might have biological relevance to mitochondrial homeostasis involved in the survival of locally adapted lineages. Species delimitation between K. davidiana var. formosana and its ancestor, K. davidiana, was also examined (72 individuals). This study has produced highly informative population genomic data for the understanding of population attributes, such as diversity, connectivity, and adaptive divergence associated with large- and small-scale environmental heterogeneity in K. davidiana var. formosana.

Elucidation of the evolutionary processes that constrain or facilitate adaptive divergence is a central goal in evolutionary biology, especially in non-model organisms. We tested whether changes in dynamics of gene flow (historical vs contemporary) caused population isolation and examined local adaptation in response to environmental selective forces in fragmented Rhododendron oldhamii populations. Variation in 26 expressed sequence tag-simple sequence repeat loci from 18 populations in Taiwan was investigated by examining patterns of genetic diversity, inbreeding, geographic structure, recent bottlenecks, and historical and contemporary gene flow. Selection associated with environmental variables was also examined. Bayesian clustering analysis revealed four regional population groups of north, central, south and southeast with significant genetic differentiation. Historical bottlenecks beginning 9168–13,092 years ago and ending 1584–3504 years ago were revealed by estimates using approximate Bayesian computation for all four regional samples analyzed. Recent migration within and across geographic regions was limited. However, major dispersal sources were found within geographic regions. Altitudinal clines of allelic frequencies of environmentally associated positively selected outliers were found, indicating adaptive divergence. Our results point to a transition from historical population connectivity toward contemporary population isolation and divergence on a regional scale. Spatial and temporal dispersal differences may have resulted in regional population divergence and local adaptation associated with environmental variables, which may have played roles as selective forces at a regional scale.

Nucleotide-binding site-leucine-rich repeats (NBS-LRR) gene families are one of the major plant resistance genes. Genomic NBS evolution was studied in many plant species for diverse arrays of NBS gene families. In this study, we focused on one family of NBS sequences in an attempt to understand how closely related NBS sequences evolved in the light of selection in domesticated plant species. A phylogenetic analysis revealed five major clades (A–E) and five subclades (A1–A5) within clade A of cloned NBS sequences. Positive selection was only detected in newly evolved NBS lineages in subclades of clade A. Positively selected codon sites were found among NBS sequences of clade A. A sliding-window analysis revealed that regions with Ka/Ks ratios of >1 were in the inter-motifs when paired clades were compared, but regions with Ka/Ks ratios of >1 were found across NBS sequences when subclades of clade A were compared. Our results based on a family of closely related NBS sequences showed that positive selection was first exerted on specific lineages across all NBS sequences after selective constraints. Subsequently, sequences with mutations in commonly conserved motifs were scrutinized by purifying selection. In the long term, conserved high frequency alleles in commonly conserved motifs and changes in inter-motifs were maintained in the investigated family of NBS sequences. Moreover, codons identified to be under positive selection in the inter-motifs were mainly located in regions involved in functions of ATP binding or hydrolysis.

Cinnamomum kanehirae Hayata (Lauraceae), the most valuable subtropical and temperate broadleaf timber tree in Taiwan, is rapidly disappearing from the wild. Taking advantage of a scion garden established by the Taiwan Forestry Research Institute, we examined patterns of chloroplast DNA (cpDNA) variations in 19 populations including 94 individuals. By sequencing two cpDNA fragments using universal primers (the trnL-trnF and petG-trnP intergenic spacers), we found eight polymorphic sites, six haplotypes, and extremely low nucleotide diversity (π = 0.00016) from 792 bp aligned sequences. The ancestral haplotype is widely distributed. Among the populations studied, three separated populations, at Yungfeng, Fuli, and Tahu have high nucleotide diversity. No phylogeographical structures of haplotypes were revealed because the tests of NST−GST for populations did not differ from zero in any situations; a ‘star-like’ genealogy is characteristic when all haplotypes rapidly coalesce and is a general outcome of population expansion. The neutrality test also suggested demographic expansion. The genetic divergence and diversity analyses suggested that two potential refugia existed during the last glaciation with a major one located in southeastern Taiwan and a minor one located in Tahu in north-central Taiwan in the Hsuehshan Range, west of the Central Mountain Range.

This study examined the spatial pattern of chloroplast DNA (cpDNA) variation in Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. (Fagaceae) in 140 trees from Taiwan (25 populations), Japan (three), Ryukyus (two), Hong Kong (one) and Mainland China (one). By sequencing three cpDNA intergenic spacer fragments using universal primers (trnT-trnL, trnV-trnM, including the trnV intron, and petG-trnP), we found a total of 1,980 bp and 15 polymorphic sites. Among them, 12 sites were caused by point mutation, and three resulted from insertion. This gives rise to a total of 13 cpDNA haplotypes. The level of differentiation among the populations studied is relatively high (GST = 0.612). Two ancestral haplotypes (A and B) are distributed widely in East Asia. Interestingly, all the derived cpDNA variations are found only in Taiwan but not in other areas. The Central Mountain Ridge (CMR) of Taiwan creates an unsurpassed barrier to the east-west gene flow of C. glauca. Among the populations on the west of CMR, only three separated populations, Yangmingshan, Wushe and Chinshuiying, have high haplotype diversity, each consisting of sister haplotypes all mutated from the same ancestral haplotype. Thus, they have probably originated from de novo mutation after the last glaciation. This inference agrees with the observation that no spatial autocorrelation existed on the west side. Two unrelated dominant lineages on the east of the CMR (haplotypes D and F) showed significant spatial genetic structure. Estimate of NST - GST was -0.090 and differed significantly from zero. Thus at the local scale, the phylogeographical component of the genetic structure is significant on the east of the CMR. Accompanied by published palynological records of the last glaciation, this study suggests the possibility that these two types were colonized northward from the southeastern part of Taiwan. 'Star-like' genealogy is characterized, with all the haplotypes coalescing rapidly and as a general outcome of population expansion (Page & Holmes 1998). A neutrality test also suggested a demographic expansion recovered from a bottleneck. We therefore inferred that the southeastern part of Taiwan might be a potential refugium for C. glauca.

Rice blast disease resistance to the fungal pathogen Magnaporthe grisea is triggered by a physical interaction between the protein products of the host R (resistance) gene, Pi-ta, and the pathogen Avr (avirulence) gene, AVR-pita. The genotype variation and resistant/susceptible phenotype at the Pi-ta locus of wild rice (Oryza rufipogon), the ancestor of cultivated rice (O. sativa), was surveyed in 36 locations worldwide to study the molecular evolution and functional adaptation of the Pi-ta gene. The low nucleotide polymorphism of the Pi-ta gene of O. rufipogon was similar to that of O. sativa, but greatly differed from what has been reported for other O. rufipogon genes. The haplotypes can be subdivided into two divergent haplogroups named H1 and H2. H1 is derived from H2, with nearly no variation and at a low frequency. H2 is common and is the ancestral form. The leucine-rich repeat (LRR) domain has a high πnon/πsyn ratio, and the low polymorphism of the Pi-ta gene might have primarily been caused by recurrent selective sweep and constraint by other putative physiological functions. Meanwhile, we provide data to show that the amino acid Ala-918 of H1 in the LRR domain has a close relationship with the resistant phenotype. H1 might have recently arisen during rice domestication and may be associated with the scenario of a blast pathogen–host shift from Italian millet to rice.

† 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.

Quercus glauca (Thunb. ex Murray) Oerst (Fagaceae) has a wide distributional range in Taiwan. In this study, the evolutionary history and the most genetically divergent sites of Q. glauca were studied using a nuclear gene marker, glyceraldehyde‐3‐phosphate dehydrogenase. Also, the consistency of the results obtained from nuclear gene and cytoplasmic loci was investigated. Using a genealogical approach (TCS software), we determined haplotypes and their relationships to one another. We used the level of divergence for each population from the remaining populations (calculated as mean values of pairwise population differentiation, FST, for each population) to locate the most genetically divergent areas in Taiwan. According to the average FST of each population in comparison with the remaining ones, a peak was found in the northern part of central Taiwan, and another was found in the southeastern region. The peak profiles of the mean FST values for all three DNA data sets (nDNA, cpDNA, and mtDNA) showed simil...

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.

In this study, we examined spatial patterns of chloroplast DNA (cpDNA) variation in a total of 30 populations of Castanopsis carlesii Hayata (Fagaceae), a subtropical and temperate tree species, including 201 individuals sampled throughout Taiwan. By sequencing two cpDNA fragments using universal primers (the trnL intron and the trnV-trnM intergenic spacer), we found a total of 1663 bp and 21 polymorphic sites. These gave rise to a total of 28 cpDNA haplotypes. The level of differentiation among the populations studied was relatively high (GST = 0.723). Two ancestral haplotypes are widely distributed. The Central Mountain Ridge (CMR) of Taiwan represents an insurmountable barrier to the east-west gene flow of C. carlesii. Among the populations studied, three separated populations, at Lienhuachih, Fushan and Lichia, have high nucleotide diversity. Estimates of NST-GST for populations on both sides of the CMR indicate that no phylogeographical structure exists. According to the genealogical tree, number of rare haplotype and population genetic divergence, this study suggests that two potential refugia existed during the last glaciation: the first refugium was located in a region to the north of Hsuehshan Range (HR) and west of the CMR; the second refugium was located in south, especially southeastern Taiwan. In fact, the second refugium happens to be the same as that reported for Quercus glauca. A 'star-like' genealogy is characteristic when all haplotypes rapidly coalesce and is a general outcome of population expansion. The neutrality test and mismatch distribution also suggest demographic expansion recovering from a bottleneck.

Luanta-fir (Cunninghamia konishii), an endemic to Taiwan, is an outcrossing, long-lived conifer. Populations of C. konishii are generally fragmented due to a once high intensity of timber exploitation. C. konishii and Cunninghamia lanceolata are two sibling taxa constituting derivative-progenitor species relationship. The amount of genetic variations within and between 11 and 10 populations of C. konishii and C. lanceolata, respectively, were assessed using amplified fragment length polymorphism (AFLP) markers in this report. Three AFLP primer pairs generated a total of 357 and 226 markers for C. konishii and C. lanceolata samples, of which 56.1 and 65.3% are polymorphic, respectively. Analysis of molecular variance indicates a 4.78% variation between C. konishii and C. lanceolata. A relatively high value of genetic variation (24.60%) was apportioned between the populations of C. konishii. In contrast, a lower divergence value (12.21%) between populations was found for C. lanceolata. The population with the highest genetic diversity was found in Nantou County, which concurred with the results of many other tree species investigated in Taiwan. The estimates of the number of migrants between populations (Nm), obtained from population pair-wise ΦST, suggest that gene flow in C. konishii is efficient in some adjacent populations but is restricted in the rest. Individual UPGMA tree, generated based on AFLP markers, suggests six evolutionary lineages for C. konishii. All evolutionary lineages of C. konishii were derived from C. lanceolata. In conclusion, the migration patterns of Cunninghamia from mainland China may have been established following multiple sources, migrant-pools, long-distance dispersal events, and via different directions.

Aim This paper described current phylogeographical patterns of chloroplastic DNA variation of Trochodendron aralioides, a temperate tree species, and inferred its possible refugium in Taiwan. This information was compared with the known phylogeographical pattern of subtropical tree species. Location A total of 24 populations were sampled including 20 from Taiwan, two each from the Ryukyus and Japan. Methods A haplotype network was constructed by computer program TCS, various parameters of genetic diversity were calculated and neutrality was tested by computer program DnaSP. To examine the similarity of genetic structure among populations, a maximum parsimony tree was reconstructed by computer program PAUP*. The results of isozyme of T. aralioides from a previous publication were incorporated into this study to infer the phylogeographical history. Results Nine haplotypes according to six substitutions, two indels and one inversion of the two cpDNA intergenic spacer fragments (petG-trnP and petA-psbJ) of T. aralioides were recognized. Genetic structure of the population of Japan is totally different from those of Taiwan and the Ryukyus. In Taiwan, the genetic structure was differentiated among populations revealed by Gst = 0.700 and Nst = 0.542, and the population genetics was clearly spatially structured. Two population groups were recognized. The first group was distributed islandwide and extended to the Ryukyus. The second group contained five of the seven known haplotypes, and was restricted to the area between latitude 24°46′ and 24°06′ N. Conclusions In Taiwan, north-central area between latitude 24°46′ and 24°06′ N is potentially a refugium during the last glaciations. This finding is contradicted to subtropical species as Cyclobalanopsis glauca.

The genus Chamaecyparis comprises five species and one variety native to Taiwan, Japan, Canada, and USA, which demonstrates a classical eastern Asian, western North American, and eastern North American disjunct distributional pattern. The phylogenetic relationships of the species of Chamaecyparis were inferred by comparing 1130 bp of the combined data set of chloroplast trnV intron and petG-trnP intergenic spacer. The phylogenetic tree shows that Chamaecyparis nootkatensis (Cupressus nootkatensis or Xanthocyparis nootkatensis) is clearly diverged from other Chamaecyparis species. For Chamaecyparis species, C. thyoides is sister to C. formosensis and C. pisifera and these together form a monophyletic group. C. lawsoniana is sister to C. obtusa and C. taiwanensis; and these form another monophyletic group. Homogeneity in evolutionary rates was found among species in these two monophyletic groups. Results indicate the divergent evolution of C. taiwanensis and C. formosensis and molecular evidence in this investigation supports C. taiwanensis as a variety of C. obtusa. Utility of cpDNA intergenic spacer petG-trnP in Chamaecyparis is also discussed. Several biogeographical implications were inferred: (1) at least two divergence events have produced the eastern Asian, and both western and eastern North American disjunct distribution in Chamaecyparis; (2) intercontinental sister species pairs are found in Chamaecyparis; (3) cpDNA divergence between two intercontinental sister pairs of C. thyoides and C. pisifera, and C. lawsoniana and C. obtusa is 2.8% and 1.1%, which suggest an estimated divergence time of 14 and 5.5 million years ago during middle and late Miocene, respectively; (4) cpDNA divergence of two Asian Chamaecyparis groups between C. obtusa and C. taiwanensis, and between C. pisifera and C. formosensis is 0.25% and 0.57%, which suggest an estimated divergence time of 1.3 and 2.9 million years ago during Pleistocene and late Pliocene, respectively; these estimated divergence times suggest a relatively recent migration of Chamaecyparis to Taiwan from the Japanese Archipelago; (5) that climatic deterioration caused the disappearance of Chamaecyparis in continental Asia is probable.

The present study investigated the genetic diversity, population structure, F ST outliers, and extent and pattern of linkage disequilibrium in five populations of Keteleeria davidiana var. formosana, which is listed as a critically endangered species by the Council of Agriculture, Taiwan. Twelve amplified fragment length polymorphism primer pairs generated a total of 465 markers, of which 83.74% on average were polymorphic across populations, with a mean Nei's genetic diversity of 0.233 and a low level of genetic differentiation (approximately 6%) based on the total dataset. Linkage disequilibrium and HICKORY analyses suggested recent population bottlenecks and inbreeding in K. davidiana var. formosana. Both STRUCTURE and BAPS observed extensive admixture of individual genotypes among populations based on the total dataset in various clustering scenarios, which probably resulted from incomplete lineage sorting of ancestral variation rather than a high rate of recent gene flow. Our results based on outlier analysis revealed generally high levels of genetic differentiation and suggest that divergent selection arising from environmental variation has been driven by differences in temperature, precipitation, and humidity. Identification of ecologically associated outliers among environmentally disparate populations further support divergent selection and potential local adaptation.

Based on fossil pollen, the distribution range of Taiwan fir [Abies kawakamii (Hay.) Ito] (Pinaceae) is smaller than it was 50 000 years ago. To characterize the present refuge populations of A. kawakamii, which survive only in subalpine forests in Taiwan, we surveyed nuclear genes and chloroplast intergenic spacers to assess the genetic diversity of Taiwan fir. Populations maintain high genetic diversity and contain similar numbers of haplotypes for the GapC (cytosolic glyceraldehyde 3-phosphate dehydrogenase) fragment. Haplotypes for GapC are generally widespread, and population-specific haplotypes accounted for 2.5% of the total. Differentiation among populations is very low (G(ST) = 0.01). Only three haplotypes were detected for the cpDNA marker, and every population had one or two haplotypes. In a neutrality test, the variation in nucleotides did not deviate from that expected with neutral evolution for either marker. A retreat route to higher elevations was not evident from either the GapC or cpDNA markers. Hsuehshan was the site of the most divergent population in Taiwan. We concluded that uniform genetic diversity, low differentiation, low numbers of population-specific haplotypes, and neutral evolution characterize contemporary refuge populations of Taiwan fir.

Background Species in the varied geographic topology of Taiwan underwent obvious demographic changes during glacial periods. Cinnamomum kanehirae has been exploited for timber and to obtain medicinal fungi for the past 100 years. Understanding anthropogenic factors influencing the demography of this species after the last glacial maximum (LGM) is critically important for the conservation of this species. Results Populations of C. kanehirae were classified into four geographic regions: northwestern (NW), west-central (WC), southwestern (SW), and southeastern (SE). In total, 113 individuals from 19 localities were sampled, and variations in the chalcone synthase gene (Chs) intron and leafy (Lfy) intron-2 sequences of nuclear DNA were examined in order to assess phylogeographic patterns, the timescales of demographic and evolutionary events, and recent anthropogenic effects. In total, 210 Chs and 170 Lfy sequences, which respectively constituted 36 and 35 haplotypes, were used for the analyses. Estimates of the migration rate (M) through time revealed a pattern of frequent gene flow during previous and the present interglacials. The isolation-by-distance test showed that there generally was no significant correlation between genetic and geographic distances. The level of among-region genetic differentiation was significant when comparing eastern to western populations. However, no significant among-region genetic differentiation was found in comparisons among the four geographic regions. Moreover, essentially no genetic structuring was found for the three regions west of the CMR. A fit of spatial range expansion was found for pooled and regional samples according to the non-significant values of the sum of squared deviations. Using the Bayesian skyline plot (BSP) method, a recent bottleneck after the LGM expansion was detected in both regional and pooled samples. Conclusions Common haplotype distributions among geographic regions and the relatively shallow genetic structuring displayed are the result of historical gene flows. Southward dispersals in an earlier time frame from the NW region and in a later time frame from the SE region were inferred. The BSP analysis suggested a postglacial expansion event. Recent trends, however, refer to a bottleneck due to human interventions observed for both pooled and regional C. kanehirae samples.

†Background and Aims Positive selection in the a-crystallin domain (ACD) of the chloroplast small heat shock protein (CPsHSP) gene was found in a previous study and was suggested to be related to the ecological adaptation of Rhododendron species in the subgenus Hymenanthes. Consequently, it was of interest to examine whether gene duplication and subsequent divergence have occurred in other sHSP genes, for example class I cytosolic sHSP genes (CT1sHSPs )i nRhododendron in Taiwan, where many endemic species have evolved as a result of habitat differentiation. †Methods A phylogeny of CT1sHSP amino acid sequences was built from Rhododendron, Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Vitis vinifera and other species for elucidation of the phylogenetic relationships among CT1sHSPs. Phylogenies of Rhododendron CT1sHSP nucleotide and amino acid sequences were generated for positive selection and functional divergence analysis, respectively. Positively selected sites and amino acid differences between types of Rhododendron CT1sHSPs were mapped onto the wheat sHSP16.9 protein structure. Average genetic distance (Dxy) and dN/dS ratios between types of Rhododendron CT1sHSP genes were analysed using sliding window analysis. Gene conversion was also assessed between types of Rhododendron CT1sHSPs. †Key Results Two types of Rhododendron CT1sHSP were identified. A high level of genetic similarity and diversity within and flanking the ACD, respectively, between types of Rhododendron CT1sHSP were found. Main differences between the two types of Rhododendron CT1sHSPs were: (1) increased hydrophobicity by two positively selected amino acid sites and a seven-amino-acid insertion in the N-terminal arm; and (2) increased structural flexibility and solubility by a seven-amino-acid insertion in the N-terminal arm and one positively selected amino acid site in the C-terminal extension. †Conclusions Functional conservation of the ACD of Rhododendron CT1sHSP genes was inferred because of strong purifying selection. However, sequence variations flanking the ACD inRhododendronCT1sHSP gene duplicates may have resulted in functional divergence and played important roles in chaperon function enhancement.

In this work, we attempted to study genetic differentiation between populations of Quercus glauca in Taiwan using nuclear microsatellite markers to infer the potential refugium in the last glaciation stage. Four microsatellite loci for 20 individuals each in 10 populations of Taiwan were analyzed. We found that Q. glauca has relatively high within-population diversity (H(E) = 0.741) and low population differentiation (F(ST) = 0.042) but shows isolation by distance. The most divergent populations, according to the average F(ST) for individual populations in comparison with every other population, were found in populations Cy, Sa, and Hy in southern Taiwan and Pa in north-central Taiwan. Moreover, populations Cy, Sa, and Pa were recognized as being the source populations for gene recolonization after the last glaciation stage. In addition, the three sites of Wu, Ym, and Cy exhibited the highest gene diversities that coincided with populations with the highest chloroplast DNA variations. This may have resulted from an admixture of colonization routes. In conclusion, observations of the most divergent populations and source populations suggest that southern and probably north-central Taiwan may have potentially been refugia for Q. glauca in the last glaciation. This agrees with the possible refugium in southern Taiwan revealed by a previous study using chloroplast DNA markers.

This study infers a single origin and a once-widespread distribution of the Rhododendron pseudochrysanthum species complex in Taiwan based on chloroplast DNA (cpDNA) variation. In total, 124 individuals from five endemic Rhododendron species were used for amplifications of two chloroplast intergenic spacers: trnL-trnF and atpB-rbcL. The haplotype and nucleotide diversities were much lower for the R. pseudochrysanthum complex, comprised of the species R. pseudochrysanthum, R. morii, Rhododendron rubropunctatum, and Rhododendron hyperythrum, than for Rhododendron formosanum. Two measures of pairwise population differentiation, N(ST) and F(ST), consistently revealed mostly non-significant levels of genetic divergence between populations of the R. pseudochrysanthum complex. No genetic difference was found among the four species of the R. pseudochrysanthum complex by analysis of molecular variance (AMOVA), which is concordant with the parsimonious topology of cpDNA haplotypes for the complex. Nested clade analysis (NCA) of the cpDNA haplotypes indicated that restricted gene flow with isolation-by-distance characterized the recolonization pattern of the R. pseudochrysanthum complex. In contrast, the NCA analysis indicated a contiguous range expansion for cpDNA haplotypes of R. formosanum. This research suggests a once-widespread distribution of the R. pseudochrysanthum complex probably via north-to-south colonization of mid-elevations during low-temperature periods of the Pleistocene. Population fragmentation followed the warmer climate which began in the Holocene and resulted in the present-day range contraction into high elevations.

Phylogeographical and mismatch analysis of chloroplast DNA (cpDNA) variation were used to infer the temporal dynamics of distributional and demographic history of Taiwan fir (Cunninghamia konishii). We examined 64 and 52 trees from 17 populations of C. konishii and 14 provenances of C. lanceolata, respectively, by sequencing three intergenic spacers and one intron using cpDNA universal primers. Of the aligned 1888 base pairs (bp) sequence, 30 varied among 28 haplotypes, which consisted of three transitions, 14 transversions and 13 indels. One ancestral haplotype was found in 86 individuals across the surveyed range of both species, C. konishii and C. lanceolata, which was distributed in all populations and provenances. The 28 haplotypes also included 15 C. konishii specific and 12 C. lanceolata-specific haplotypes. Ancestral haplotype was found fixed in five populations of C. konishii and five provenances of C. lanceolata. Other haplotypes occurred mainly as singletons. The levels of population differentiation studied are relatively low in both Cunninghamia species. The nucleotide diversity (theta) of chloroplast DNA sequences within C. konishii was slightly higher than that of C. lanceolata. Excess in singletons as well as star-like phylogeny of haplotypes suggested no clearcut migration patterns of C. konishii after glacial maximum. One probable demographic history of C. konishii is the postglacial population growth of C. konishii after a glacial bottleneck event. This inference is supported by the combined results of fossil pollen record, low nucleotide diversity, significant Tajima's d-value, phylogeographical analysis and unimodal mismatch distribution. Similarities and discrepancies between our results and those of Lu et al. (2001) are discussed.

Flooding is a frequently occurring environmental stress that can severely affect plant growth. This study shows that treatment of corn (Zea mays L.) seedlings with abscisic acid (ABA) increases their tolerance to anoxia 10-fold over untreated seedlings and twofold over seedlings treated with water. Corn seedlings stressed anoxically for 1 day showed only 8% survival when planted in vermiculite. Pretreatment of root tips with 100 micromolar ABA or water for 24 hours before the 1 day anoxic stress increased the anoxic survivability of seedlings to 87% and 47%, respectively. Cycloheximide (5 milligrams per liter), added together with ABA, reduced the seedling survival rate, indicating that the induction of anoxic tolerance in corn by ABA was partly a result of the synthesis of new proteins. ABA treatment induced a threefold increase in alcohol dehydrogenase enzyme activity in corn roots. However, after 24 h of anoxia, alcohol dehydrogenase enzyme activity between the ABA-pretreated and non-pretreated corn roots was not significantly different. The results indicated that ABA played an important role in inducing anoxic tolerance in corn and that the induced tolerance was probably mediated by an increase in alcohol dehydrogenase enzyme activity before the anoxic stress.

Background CBF/DREB duplicate genes are widely distributed in higher plants and encode transcriptional factors, or CBFs, which bind a DNA regulatory element and impart responsiveness to low temperatures and dehydration. Results We explored patterns of genetic variations of CBF1, -2, and -3 from 34 accessions of Arabidopsis thaliana. Molecular population genetic analyses of these genes indicated that CBF2 has much reduced nucleotide diversity in the transcriptional unit and promoter, suggesting that CBF2 has been subjected to a recent adaptive sweep, which agrees with reports of a regulatory protein of CBF2. Investigating the ratios of Ka/Ks between all paired CBF paralogus genes, high conservation of the AP2 domain was observed, and the major divergence of proteins was the result of relaxation in two regions within the transcriptional activation domain which was under positive selection after CBF duplication. With respect to the level of CBF gene expression, several mutated nucleotides in the promoters of CBF3 and -1 of specific ecotypes might be responsible for its consistently low expression. Conclusion We concluded from our data that important evolutionary changes in CBF1, -2, and -3 may have primarily occurred at the level of gene regulation as well as in protein function.

An allelopathic interaction of a pasture-forest intercropping system was evaluated by experiments conducted in field and by laboratory assays. A study site was situated in the farm of Hoshe Forestry Experiment Station at Nantou County, Taiwan. After deforestation of Chinese fir (Cunninghamia lanceolata), a split plot design of 4 treatments, namely litter removed, litter removed and kikuyu grass (Pennisetum clandestinum) planted, litter left, and litter left and grass planted, was composed. Field meaurements showed that the fir litter left on the ground did not significantly inhibit the growth of weeds, kikuyu grass, and fir seedlings in the first four months following deforestation, while kikuyu grass significantly suppressed the growth of weeds longer than four months but did not reduce growth of fir seedlings. The aqueous extracts of fresh fir leaves, fir litter, and kikuyu leaves were bioassayed by using lettuce and rice seeds and stolon cuttings ofBrachiaria mutica. Bioassays showed that fresh fir leaves produced significant phytotoxicity while fir litter and kikuyu grass gave limited toxicity. Nine phytotoxic phenolics and many unidentified flavonoids were found in the leaf and litter of Chinese fir and kikuyu leaves. A good correlation between the degree of phytotoxicity and phytotoxins was obtained, indicating an allelopathy was involved. This finding suggests that allelopathy may contribute benefits in the intercropping system to reduce the need for herbicides and to lessen the labor cost for weed control.