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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.

Trailing edge populations of upward migrating species induced by postglacial climatic warming can be evolutionarily significant in the face of global warming. We tested for population size changes between lower- and higher-elevation populations located in the same mountainous regions of the upward-shifting temperate woody species of the Rhododendron pseudochrysanthum complex in Taiwan. We also tested whether natural selection evoked adaptive divergence in trailing edge populations of this species complex. We genotyped 26 expressed sequence tag-simple sequence repeat (EST-SSR) loci of 185 individuals from nine populations of the R. pseudochrysanthum complex including Rhododendron rubropunctatum, Rhododendron hyperythrum, Rhododendron morii, and R. pseudochrysanthum. R. rubropunctatum populations in the R. pseudochrysanthum complex possessed the lowest estimates of genetic diversity and effective population size. Higher-elevation R. pseudochrysanthum populations had lower effective population sizes than lower-elevation R. morii populations in Hohuanshan and Tahsueshan, as revealed by estimates using both MIGRATE-N and approximate Bayesian computation (ABC). R. rubropunctatum populations diverged significantly from populations of other members of the R. pseudochrysanthum complex. An outlier potentially under positive selection specific to R. rubropunctatum populations was identified and strongly associated with ecologically relevant environmental variables. Postglacial climatic warming has a significant influence on population isolation in the R. pseudochrysanthum complex. The results indicate adaptive evolution in the trailing edge populations, i.e., R. rubropunctatum populations of the upward shifting R. pseudochrysanthum complex.

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.

Ecological speciation has long been noted as a central topic in the field of evolutionary biology, and investigation into the relative importance of ecological and geographical factors is becoming increasingly emphasized. We surveyed genetic variation of 277 samples from 25 populations of nine Rhododendron species within Tsutsusi subgenus in Taiwan using simple sequence repeats of expressed sequence tags. Bayesian clustering revealed four genetic lineages: (1) the Rhododendron simsii, Rhododendron kanehirai, and Rhododendron nakaharae lineage (lineage 1); (2) the Rhododendron longiperulatum, Rhododendron breviperulatum, and Rhododendron noriakianum lineage (lineage 2); (3) the Rhododendron rubropilosum lineage (lineage 3); and (4) the Rhododendron oldhamii lineage (lineage 4). Asymmetric introgressions were found from lineage 3 into lineages 1 and 2 (introgressed lineages). Genetic admixture of non-R. oldhamii species was also revealed by a neighbor-joining tree. Variation partitioning showed that environment explained much larger portions of genetic variation than geography between non-introgressed lineages (i.e., between R. oldhamii and other lineages). However, the Mantel and partial Mantel tests and the multiple matrix regression with randomization found that isolation-by-distance played a more important role than isolation-by-environment (IBE) in contributing to genetic variation in most between lineage comparisons. Nevertheless, strong IBE was found when compared between non-introgressed lineages of R. oldhamii and R. rubropilosum, suggesting post-speciation ecological divergence. Several environmental variables, including annual mean temperature, aspect, isothermality, seasonal precipitation, slope, and soil pH, could be important ecological drivers involved in reproductive isolation between R. oldhamii and non-R. oldhamii species within the Tsutsusi subgenus.

To further understand natural variation and local adaptation in the evolution of plant defense, we analyzed polymorphism data of nucleotide-binding site (NBS) sequences of Rhododendron at both the species and population levels. Multiple duplication events were found in NBS sequence evolution in Rhododendron genomes, which resulted in six clades: A–F. Our results of several NBS clade pair comparisons showed significant evolutionary rate changes based on differences in substitution rates between NBS-encoding protein clades (type I functional divergence). Pairwise comparisons of NBS clades further revealed that many amino acids displayed radical biochemical property changes causing a shift in amino acid preferences between NBS-encoding protein clades (type II functional divergence). Such divergent evolution of NBSs is likely a consequence of positive selection related to differentiation of recognition signals in response to different pathogens. Primers specific to clades B and C, which differed in the number of radical amino acid changes causing type II functional divergence and levels of nucleotide diversities, were further used to amplify population clades B and C NBS sequences of Rhododendron formosanum populations. Higher levels of net nucleotide divergences (measured by D a) between R. formosanum populations were found based on NBS sequences of population clade B compared to population clade C, suggesting local adaptation of population clade B NBS sequences. Local adaptation can be further inferred for R. formosanum population clade B NBS sequences because of significant Φ ST based on variation in nonsynonymous substitutions. Furthermore, local adaptation was also suggested by no significant correlation of population pairwise F ST between population clades B and C in R. formosanum.

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.

Linking ecology with evolutionary biology is important to understand how environments drive population and species divergence. Phenotypically diverse Salix species, such as lowland riparian willow trees and middle- to high-elevation multistemmed shrubs and alpine dwarf shrubs, provide opportunities for studying genetic divergence driven by ecological factors. We used amplified fragment length polymorphism (AFLP) to quantify the genetic variation of 185 individuals from nine populations of four Salix species in Taiwan. Our phylogenetic analyses distinguished two riparian species and the separation of riparian species from multistemmed and dwarf shrub species. Variance partitioning for the total data found that environment explained a substantially larger proportion of genetic variation than geography. However, no genetic variation was explained by geography alone when only compared within and between species. Spatially structured regional environmental effects explained more variation than pure environments in most comparisons within and between species, suggesting that unmeasured environmental variables and/or past demographic histories played important roles in shaping population and species divergence. Based on forward selection analysis, annual mean temperature, aspect, and fraction of absorbed photosynthetically active radiation were the most influential ecological factors in shaping genetic variation within and between species. Nevertheless, different combinations of environmental variables correlated significantly with genetic variation within and between species. We identified eight AFLP loci that potentially evolved under selection intraspecifically using different outlier detection methods. These loci correlated with more than one environmental variable, suggesting local adaptation along environmental gradients at the population level.

Test of the relationship of genetic and particularly epigenetic variation with geographic isolation and environment is important to reveal potential environmental drivers for selection. Rhododendron oldhamii is widespread but inhabits fragmented subtropical forest landscapes, and populations across its range may exhibit different levels of genetic and epigenetic structuring correlated to their environmental conditions. Here, we investigated the genetic and epigenetic variations and their ecological correlates in R. oldhamii. Genetic and epigenetic variations were surveyed using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP), respectively. Using variation partitioning by redundant analysis (RDA), we examined the pure and spatially structured environmental effects and pure spatial factors on genetic and epigenetic variations among individuals collected from 18 localities across R. oldhamii distribution range in Taiwan. We found that environments compared to geographic isolation among sites explained more genetic and epigenetic variations. Patchy distribution of the contemporary R. oldhamii populations was revealed by correlograms with patch size of approximately around 20–30 km based on the total genetic and epigenetic data. Spatial variables derived from the method of principal coordinates of neighbor matrices (PCNM), including PCNM3, PCNM5, PCNM7, and PCNM8 representing biotic processes, such as individual dispersal, were found to be important influencing potentially adaptive genetic and epigenetic variations. Annual mean temperature, annual precipitation, precipitation of the warmest quarter, aspect, slope, and soil moisture were the most important environmental variables influencing potentially adaptive genetic and epigenetic variations and could be particularly important for the evolution of local adaptation in R. oldhamii.

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.

We investigated the changes in antioxidative enzyme activities of two sweet potato cultivars under waterlogging and high-light conditions in the growth chamber. The activities of antioxidative enzymes were measured from leaf crude extract of sweet potato during the first five days of the treatments. Activities of superoxide dismutase and catalase were consistently increased in Taoyuan 1 sweet potato over time under waterlogging and high-light conditions. However, decreases in both superoxide dismutase and catalase activities were observed for cultivar Yongtsai under waterlogging and high-light conditions. Waterlogging, together with high-light intensity, impairs superoxide dismutase and catalase activities in the cultivar Yongtsai indicating its greater susceptibility to waterlogging and high-light stress. In contrast, the increase in activities of superoxide dismutase and catalase in Taoyuan 1 indicated its greater ability to detoxify reactive oxygen species during the treatment and ensured its reduced susceptibility to waterlogging and high-light stress. The activities of peroxidase may be inactivated by high-light treatment and, therefore, may not be associated with tolerance of sweet potato plants to waterlogging and high-light stress. Differences in susceptibility to waterlogging and high-light conditions in the leafy vegetable Yongtsai and storage root Taoyuan 1 are discussed.

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.

The difference among florets within a panicle in grain filling and four hormone contents was compared by using two rice varieties differing in panicle type, i.e. compact-panicle variety Xiushui 994 and loose-panicle variety Xiushui 11. Meanwhile the effect of changing source (leaf cutting) and sink (floret removal) on grain filling and four hormone (indole-3-acetic acid, gibberellin, zeatin riboside and abscisic acid)level dynamics in developing grains was also examined. The results showed that Xiushui 994 had greater variation in grain filling rate (GFR), grain weight, filled grain percent (FGP) and four hormone contents during grain filling than Xiushui 11. Moreover, the former variety also showed the greater difference in the time of having the highest hormone contents between superior and inferior florets. The change in source and sink size had great effect on grain filling process and dynamics of four hormone contents, and the extent of the effect varied with variety and floret type. For Xiushui 994, floret-removal treatment had little effect on grain weight and FGP of the superior florets, but caused a marked decrease in GFR and four hormone contents, and a delay in the time when the highest GFR and hormone contents occurred. In the source-reducing treatment, GFR, grain weight and FGP for both superior and interior florets were all decreased. For Xiushui 11, both treatments had little effect on grain development in terms of grain filling properties and hormone content change in the two types of florets. The results indicated that the grain filling of compact-panicle variety is more source-limited than that of loose-panicle one, and relative level of hormone in both superior and inferior florets determined development of inferior florets.

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.