Comparative chemical profiling of leaf essential oils from Cinnamomum kanehirae and related species using steam distillation and solvent extraction: Implications for plant-based classification.

Cinnamomum kanehirae Hayata, belonging to Lauraceae family, is an indigenous and endangered species of considerable economic importance in Taiwan. It plays a crucial role as the host for the economically valuable saprotrophic fungus, Taiwanofungus camphorates. However, accurate species identification poses a challenge due to the similarity in morphological features and frequent natural hybridization with closely related species. Acquiring high-quality and pure leaf oils becomes imperative for precise species identification and producing superior goods. In this study, our objective was to establish methodologies for analyzing the chemical composition of leaf essential oils and subsequently apply this knowledge to differentiate among three Cinnamomum species. Gas chromatography-mass spectrometry (GC/MS) was employed to scrutinize the chemical makeup of leaf essential oils from three closely related species: C. kanehirae, C. micranthum, and C. camphora. We utilized Steam Distillation (SD) and steam distillation-solvent extraction (SDSE) methods, with the SDSE-Hexane approach chosen for optimization, enhancing extraction efficiency and ensuring essential oil purity. Through the SDSE-Hexane method, we identified seventy-four compounds distributed across three major classes: monoterpenes hydrocarbons (0.0-7.0 %), oxygenated monoterpenes (3.8-90.9 %), sesquiterpenes hydrocarbons (0.0-28.3 %), and oxygenated sesquiterpenes (1.6-88.1 %). Our findings indicated the presence of more than one chemotype in both C. kanehirae and C. camphora, whereas no specific chemotype could be discerned in C. micranthum. Furthermore, clustering based on chemotypes allowed for the differentiation of samples from the three species. Notably, we demonstrated that the chemical compositions of grafted C. kanehirae remained largely unaffected by the rootstock. Conversely, natural hybrids between C. kanehirae and C. camphora exhibited profiles more closely aligned with C. kanehirae. The optimized extraction method and the chemotype-based classification system established in this study present valuable tools for essential oil preparation, species identification, and further exploration into the genetic variation of Cinnamomum.

Complete chloroplast genome of Angelica hirsutiflora Liu et al. 1961 (Apiaceae).

Angelica hirsutiflora Liu et al.1961, is a perennial herb in the Apiaceae family that is endemic to Taiwan. In this study, the complete circular chloroplast genome of A. hirsutiflora was reconstructed and annotated using Illumina sequencing. The size of the chloroplast genome is 154,266 bp, consisting of two inverted repeats (IRs, 25,075 bp) separated by a large single-copy region (LSC, 86,569 bp) and a small single-copy region (SSC, 17,547 bp). The GC content of the chloroplast genome is 37.6%. There are 114 different genes in the chloroplast genome of A. hirsutiflora, including 80 protein-coding genes, 30 tRNA genes and four rRNA genes. A maximum-likelihood phylogenetic analysis showed that A. hirsutiflora forms a distinct clade, and separated from other species within the genus Angelica. This study provided insights into the evolutionary relationships among different species of Angelica.

Comparative Study of Heavy Metal Blood Serum Level Between Organic and Conventional Farmers in Eastern Taiwan.

Numerous studies have indicated that organic fertilizers (OFer) might contain heavy metals (HMs) that present health risks to organic farmers (OFar). This study compared the concentrations of six HMs (Zn, Ni, Cd, Cu, Pb, Cr) in the blood of two distinct groups of farmers: 30 OFar from a designated organic area in eastern Taiwan, and 74 conventional farmers (CFar) from neighboring non-organic designated regions. The findings revealed that the OFar exhibited higher levels of Zn (1202.70 ± 188.74 µg/L), Cr (0.20 ± 0.09 µg/L), and Ni (2.14 ± 1.48 µg/L) in their blood compared to the CFar (988.40 ± 163.16 µg/L, 0.18 ± 0.15 µg/L, and 0.77 ± 1.23 µg/L), respectively. The disparities in Zn, Cr, and Ni levels were measured at 214.3 µg/L, 0.02 µg/L, and 1.37 µg/L, respectively. Furthermore, among the OFar, those who utilized green manures (GM) displayed significantly elevated blood levels of Zn (1279.93 ± 156.30 µg/L), Cr (0.24 ± 0.11 µg/L), and Ni (1.94 ± 1.38 µg/L) compared to individuals who exclusively employed chemical fertilizers (CFer) (975.42 ± 165.35 µg/L, 0.19 ± 0.16 µg/L, and 0.74 ± 1.20 µg/L), respectively. The differences in Zn, Cr, and Ni levels were measured at 304.51 µg/L, 0.05 µg/L, and 1.20 µg/L, respectively. As a result, OFar should be careful in choosing OFer and avoid those that may have heavy metal contamination.

A taxonomic revision of the genus Angelica (Apiaceae) in Taiwan with a new species A. aliensis.

BACKGROUND: Angelica L. sensu lato is a taxonomically complex genus, and many studies have utilized morphological and molecular features to resolve its classification issues. In Taiwan, there are six taxa within Angelica, and their taxonomic treatments have been a subject of controversy. In this study, we conducted a comprehensive analysis incorporating morphological and molecular (cpDNA and nrDNA) characteristics to revise the taxonomic treatments of Angelica in Taiwan. RESULTS: As a result of our research, we have revised the classification between A. dahurica var. formosana and A. pubescens and merged two varieties of A. morrisonicola into a single taxon. A new taxon, A. aliensis, has been identified and found to share a close relationship with A. tarokoensis. Based on the morphological and molecular characteristics data, it has been determined that the former three taxa should be grouped into the Eurasian Angelica clade, while the remaining four taxa should belong to the littoral Angelica clade. Furthermore, Angelica species in Taiwan distributed at higher altitudes displayed higher genetic diversity, implying that the central mountain range of Taiwan serves as a significant reservoir of plant biodiversity. Genetic drift, such as bottlenecks, has been identified as a potential factor leading to the fixation or reduction of genetic diversity of populations in most Angelica species. We provide key to taxa, synopsis, phenology, and distribution for each taxon of Taiwan. CONCLUSIONS: Our comprehensive analysis of morphological and molecular features has shed light on the taxonomic complexities within Angelica in Taiwan, resolving taxonomic issues and providing valuable insights into the phylogenetic relationships of Angelica in Taiwan.

Genetic Diversity and Population Structure in Captive Populations of Formosan Sambar Deer (Rusa unicolor swinhoei).

Formosan sambar deer (Rusa unicolor swinhoei) are of great economic significance in Taiwan, resulting in a substantial increase in deer farming to meet the high demand for velvet antlers. Inbreeding depression and reduced genetic variability can lead to the deterioration of captive populations. In this study, 239 Formosan sambar deer were genotyped using 13 microsatellites to analyze their genetic diversity and population genetic structure. Our results indicate a high-resolution power of these microsatellites in individual discrimination and parentage analysis. However, captive populations exhibit a low level of genetic diversity, likely because of inbreeding and bottleneck effects. Both principal coordinate analysis (PCoA) and STRUCTURE analyses revealed two distinct and segregated genetic groups within the captive populations and indicated no clear population genetic structure among the captive populations. Introducing new genetic material from the wild through translocation offers a potential solution for mitigating the impact of inbreeding and enhancing genetic diversity. The comprehensive information obtained from these genetic analyses is crucial for the development of effective breeding strategies aimed at preserving and enhancing Formosan sambar deer populations.

New insights into polyploid evolution and dynamic nature of Ludwigia section Isnardia (Onagraceae).

BACKGROUND: While polyploids are common in plants, the evolutionary history and natural dynamics of most polyploid groups are still unclear. Owing to plentiful earlier systematic studies, Ludwigia sect. Isnardia (comprising 22 wetland taxa) is an ideal allopolyploid complex to investigate polyploid evolution and natural dynamics within and among taxa. With a considerable sampling, we concentrated on revisiting earlier phylogenies of Isnardia, reevaluating the earlier estimated age of the most recent common ancestor (TMRCA), exploring the correlation between infraspecific genetic diversity and ploidy levels, and inspecting interspecific gene flows among taxa. RESULTS: Phylogenetic trees and network concurred with earlier phylogenies and hypothesized genomes by incorporating 192 atpB-rbcL and ITS sequences representing 91% of Isnardia taxa. Moreover, we detected three multi-origin taxa. Our findings on L. repens and L. sphaerocarpa were consistent with earlier studies; L. arcuata was reported as a multi-origin taxon here, and an additional evolutionary scenario of L. sphaerocarpa was uncovered, both for the first time. Furthermore, estimated Isnardia TMRCA ages based on our data (5.9 or 8.9 million years ago) are in accordance with earlier estimates, although younger than fossil dates (Middle Miocene). Surprisingly, infraspecific genetic variations of Isnardia taxa did not increase with ploidy levels as anticipated from many other polyploid groups. In addition, the exuberant, low, and asymmetrical gene flows among Isnardia taxa indicated that the reproductive barriers may be weakened owing to allopolyploidization, which has rarely been reported. CONCLUSIONS: The present research gives new perceptions of the reticulate evolution and dynamic nature of Isnardia and points to gaps in current knowledge about allopolyploid evolution.

Effect of geographic isolation on genetic variation and population structure of Euphrasia nankotaizanensis, a threatened endemic alpine herb in Taiwan.

Euphrasia nankotaizanensis (Orobanchaceae) is a rare alpine herb that is endemic to Taiwan. Only four small populations remain in Xue, Nanhu, and Cilai Mountains of Taiwan. The distribution of alpine herbs is severely threatened by climate change, which influences genetic variation and population structure. In this study, we investigated the effects of the natural isolation of alpine habitats on the genetic diversity and geographic structure of populations of E. nankotaizanensis using chloroplast (cp) and nuclear DNA (nrDNA) markers. We found lower levels of genetic diversity in E. nankotaizanensis than in other alpine plants and little to no genetic variation within populations, which could be mainly attributed to the small population size and genetic drift. Only one nrDNA haplotype was present in each population. The lack of monophyly of the four populations in cpDNA probably resulted from lineage sorting or occasional long-distance seed dispersal. Phylogeographic analysis suggested that Nanhu Mountain was probably a refugium over the glacial maxima, agreeing with the potential refugia in central Taiwan. The STRUCTURE and AMOVA analyses revealed significant genetic differentiation in nrDNA among the mountains, which resulted from geographical isolation among these mountains. Estimates of the effective population size (Ne) and demography reflected lower Ne values and a recent population decline, probably implying a greater extinction risk for E. nankotaizanensis. We observed genetic depletion and considerable genetic differentiation among mountain populations, which should be considered in future conservation efforts for this species. In addition, this study provides important insights into the long-term potential of alpine herbs in Taiwan, which are useful for a better prediction of their responses to future climate change.

Effect of dietary supplementation of Bacillus subtilis TLRI 211-1 on laying performance, egg quality and blood characteristics of Leghorn layers.

OBJECTIVE: TLRI 211-1 is a novel Bacillus subtilis strain. This experiment was to investigate dietary supplementation of TLRI 211-1 on laying performance, egg quality and blood characteristics of layers. METHODS: One hundred and twenty 65-wk-old Leghorn layers were divided into four treatment groups for 8 weeks experiment. Each treatment had three replicates. The basal diet was formulated as control group with crude protein 17% and metabolizable energy 2,850 kcal/kg and supplemented with TLRI 211-1 0.1%, 0.3%, and commercial Bacillus amyloliquefaciens 0.1% as treatment 2, 3 and 4 groups, respectively. Both TLRI 211-1 and commercial Bacillus amyloliquefaciens were adjusted to contain 1×109 colony-forming unit (CFU)/mL (g), hence the 0.1% supplemental level was 1×109 CFU/kg. RESULTS: The results showed that TLRI 211-1 0.3% and commercial B. amyloliquefaciens groups had higher weight gain than the other groups; TLRI 211-1 0.1% group had better feed to eggs conversion ratio than the control and commercial B. amyloliquefaciens groups (p<0.05). Bacillus subtilis supplementation increased yolk weight (p<0.05). In egg quality during storage, TLRI 211-1 0.1% had higher breaking strength than the control group at the second week of storage (p<0.05). At the third week of storage, TLRI 211-1 0.3% had higher Haugh unit (p<0.05). Hens fed diets supplemented with TLRI 211-1 0.3% significantly decreased blood triglyceride levels and increased blood calcium levels (p< 0.05). TLRI 211-1 0.3% group had lower H2S (p<0.05) and hence had less unpleasant odor in excreta of hens. CONCLUSION: In conclusion, supplementation with 0.1% TLRI 211-1 can significantly improve feed to eggs conversion ratio. TLRI 211-1 supplementation also can maintain eggs at their optimum quality level during storage. The study showed that B. subtilis TLRI 211-1 can be used as feed additives for improving egg production performance and egg quality.

Complete chloroplast genome and phylogenetic analysis of Bupleurum kaoi Liu, Chao, and Chuang, 1977: an endemic species in Taiwan.

Bupleurum kaoi Liu, Chao, and Chuang is an endemic and endangered herb in Taiwan. In this study, the complete circular chloroplast genome of B. kaoi was reconstructed and annotated using Illumina sequencing. The genome size of B. kaoi is 155,938 bp, including a pair of inverted repeat regions (IRs: 26308 bp), separated by a large single-copy (LSC) region of 85,784 bp and a small single-copy (SSC) region of 17,538 bp. The GC content of the chloroplast genome is 37.6%. There are 113 different genes in the chloroplast genome of B. kaoi, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. A maximum-likelihood phylogenetic analysis showed that Bupleurum species is the monophyletic group, and B. kaoi belongs to subgenus Bupleurum and is closely related to B. scorzonerifolium.

Complete mitogenome of Antheraea formosana Sonan, 1937 (Lepidoptera: Saturniidae): an endemic silkmoth in Taiwan.

The complete mitogenome of an endemic silkmoth in Taiwan, Antheraea formosana, was determined using Illumina next-generation sequencing. The mitogenome is 15,318 bp in length and consists of 13 protein-coding genes (PCGs), two rRNAs, 22 tRNAs, and one non-coding control region. The overall base composition of the mitogenome showed a high A + T bias, and the A + T content (80.2%) was significantly higher than the G + C content (19.8%). All PCGs use the typical ATN as the initiation codon, with the exception of cox2, which begins with GTG, respectively. The complete mitogenome was used to reconstruct a phylogenetic tree, indicating that A. formosana is more closely related to Antheraea assamensis than other Antheraea species, with 93.19% nucleotide similarity.

The Ameliorative Effects of Fucoidan in Thioacetaide-Induced Liver Injury in Mice.

Liver disorders have been recognized as one major health concern. Fucoidan, a sulfated polysaccharide extracted from the brown seaweed Fucus serratus, has previously been reported as an anti-inflammatory and antioxidant. However, the discovery and validation of its hepatoprotective properties and elucidation of its mechanisms of action are still unknown. The objective of the current study was to investigate the effect and possible modes of action of a treatment of fucoidan against thioacetamide (TAA)-induced liver injury in male C57BL/6 mice by serum biochemical and histological analyses. The mouse model for liver damage was developed by the administration of TAA thrice a week for six weeks. The mice with TAA-induced liver injury were orally administered fucoidan once a day for 42 days. The treated mice showed significantly higher body weights; food intakes; hepatic antioxidative enzymes (catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD)); and a lower serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and C-reactive protein (CRP) levels. Additionally, a reduced hepatic IL-6 level and a decreased expression of inflammatory-related genes, such as cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) mRNA was observed. These results demonstrated that fucoidan had a hepatoprotective effect on liver injury through the suppression of the inflammatory responses and acting as an antioxidant. In addition, here, we validated the use of fucoidan against liver disorders with supporting molecular data.

Complete mitochondrial genome of Manispentadactylapentadactyla (Mammalia: Pholidota), an endemic subspecies of Chinese pangolin: mitogenome characterisation and phylogenetic implications.

The Chinese pangolin Manispentadactyla is critically endangered because of over-exploitation and illegal trafficking and includes three subspecies. However, the taxonomic status of the three subspecies of the Chinese pangolin has not been well resolved, which impedes regional conservation and illegal trade traces. In this study, the complete mitogenome sequence of M.p.pentadactyla, an endemic subspecies of the Chinese pangolin in Taiwan, was determined. The complete mitogenome of M.p.pentadactyla is 16,570 base pairs (bp) in length with 13 protein-coding genes (PCG), 23 transfer RNAs (tRNAs), two ribosomal RNAs and a 1164 bp control region. The overall base composition of the genome showed a slight A + T bias (59.9%), positive AT skew (0.1515) and negative GC skew (-0.3406), which is similar to that of other pangolins. All PCGs started with a typical ATN codon and all tRNAs were typical cloverleaf-shaped secondary structures, except for tRNA-Ser(GCU). Phylogenetic analysis indicated a monophyletic relationship for M.p.pentadactyla and M.p.aurita and was monophyletic for M.p.pentadactyla, but paraphyletic for M.p.aurita. The paraphyly of M.p.aurita resulted from an incomplete lineage sorting. This study enriched the mitogenome database of the Chinese pangolin and the molecular information obtained should be very useful for future research on mitogenome evolution and genetic diversification in M.pentadactyla.

Frequent gene flow blurred taxonomic boundaries of sections in Lilium L. (Liliaceae).

Gene flow between species may last a long time in plants. Reticulation inevitably causes difficulties in phylogenetic reconstruction. In this study, we looked into the genetic divergence and phylogeny of 20 Lilium species based on multilocus analyses of 8 genes of chloroplast DNA (cpDNA), the internally transcribed nuclear ribosomal DNA (nrITS) spacer and 20 loci extracted from the expressed sequence tag (EST) libraries of L. longiflorum Thunb. and L. formosanum Wallace. The phylogeny based on the combined data of the maternally inherited cpDNA and nrITS was largely consistent with the taxonomy of Lilium sections. This phylogeny was deemed the hypothetical species tree and uncovered three groups, i.e., Cluster A consisting of 4 taxa from the sections Pseudolirium and Liriotypus, Cluster B consisting of the 4 taxa from the sections Leucolirion, Archelirion and Daurolirion, and Cluster C comprising 10 taxa mostly from the sections Martagon and Sinomartagon. In contrast, systematic inconsistency occurred across the EST loci, with up to 19 genes (95%) displaying tree topologies deviating from the hypothetical species tree. The phylogenetic incongruence was likely attributable to the frequent genetic exchanges between species/sections, as indicated by the high levels of genetic recombination and the IMa analyses with the EST loci. Nevertheless, multilocus analysis could provide complementary information among the loci on the species split and the extent of gene flow between the species. In conclusion, this study not only detected frequent gene flow among Lilium sections that resulted in phylogenetic incongruence but also reconstructed a hypothetical species tree that gave insights into the nature of the complex relationships among Lilium species.

Multilocus Analyses Reveal Postglacial Demographic Shrinkage of Juniperus morrisonicola (Cupressaceae), a Dominant Alpine Species in Taiwan.

Postglacial climate changes alter geographical distributions and diversity of species. Such ongoing changes often force species to migrate along the latitude/altitude. Altitudinal gradients represent assemblage of environmental, especially climatic, variable factors that influence the plant distributions. Global warming that triggered upward migrations has therefore impacted the alpine plants on an island. In this study, we examined the genetic structure of Juniperus morrisonicola, a dominant alpine species in Taiwan, and inferred historical, demographic dynamics based on multilocus analyses. Lower levels of genetic diversity in north indicated that populations at higher latitudes were vulnerable to climate change, possibly related to historical alpine glaciers. Neither organellar DNA nor nuclear genes displayed geographical subdivisions, indicating that populations were likely interconnected before migrating upward to isolated mountain peaks, providing low possibilities of seed/pollen dispersal across mountain ranges. Bayesian skyline plots suggested steady population growth of J. morrisonicola followed by recent demographic contraction. In contrast, most lower-elevation plants experienced recent demographic expansion as a result of global warming. The endemic alpine conifer may have experienced dramatic climate changes over the alternation of glacial and interglacial periods, as indicated by a trend showing decreasing genetic diversity with the altitudinal gradient, plus a fact of upward migration.

Cross-Species, Amplifiable EST-SSR Markers for Amentotaxus Species Obtained by Next-Generation Sequencing.

Amentotaxus, a genus of Taxaceae, is an ancient lineage with six relic and endangered species. Four Amentotaxus species, namely A. argotaenia, A. formosana, A. yunnanensis, and A. poilanei, are considered a species complex because of their morphological similarities. Small populations of these species are allopatrically distributed in Asian forests. However, only a few codominant markers have been developed and applied to study population genetic structure of these endangered species. In this study, we developed and characterized polymorphic expressed sequence tag-simple sequence repeats (EST-SSRs) from the transcriptome of A. formosana. We identified 4955 putative EST-SSRs from 68,281 unigenes as potential molecular markers. Twenty-six EST-SSRs were selected for estimating polymorphism and transferability among Amentotaxus species, of which 23 EST-SSRs were polymorphic within Amentotaxus species. Among these, the number of alleles ranged from 1-4, the polymorphism information content ranged from 0.000-0.692, and the observed and expected heterozygosity were 0.000-1.000 and 0.080-0.740, respectively. Population genetic structure analyses confirmed that A. argotaenia and A. formosana were separate species and A. yunnanensis and A. poilanei were the same species. These novel EST-SSRs can facilitate further population genetic structure research of Amentotaxus species.

Inferring multiple refugia and phylogeographical patterns in Pinus massoniana based on nucleotide sequence variation and DNA fingerprinting.

BACKGROUND: Pinus massoniana, an ecologically and economically important conifer, is widespread across central and southern mainland China and Taiwan. In this study, we tested the central-marginal paradigm that predicts that the marginal populations tend to be less polymorphic than the central ones in their genetic composition, and examined a founders' effect in the island population. METHODOLOGY/PRINCIPAL FINDINGS: We examined the phylogeography and population structuring of the P. massoniana based on nucleotide sequences of cpDNA atpB-rbcL intergenic spacer, intron regions of the AdhC2 locus, and microsatellite fingerprints. SAMOVA analysis of nucleotide sequences indicated that most genetic variants resided among geographical regions. High levels of genetic diversity in the marginal populations in the south region, a pattern seemingly contradicting the central-marginal paradigm, and the fixation of private haplotypes in most populations indicate that multiple refugia may have existed over the glacial maxima. STRUCTURE analyses on microsatellites revealed that genetic structure of mainland populations was mediated with recent genetic exchanges mostly via pollen flow, and that the genetic composition in east region was intermixed between south and west regions, a pattern likely shaped by gene introgression and maintenance of ancestral polymorphisms. As expected, the small island population in Taiwan was genetically differentiated from mainland populations. CONCLUSIONS/SIGNIFICANCE: The marginal populations in south region possessed divergent gene pools, suggesting that the past glaciations might have low impacts on these populations at low latitudes. Estimates of ancestral population sizes interestingly reflect a recent expansion in mainland from a rather smaller population, a pattern that seemingly agrees with the pollen record.

Characterization of microsatellite loci from Litsea hypophaea (Lauraceae), a tree endemic to Taiwan.

PREMISE OF THE STUDY: Microsatellite primers were developed for the endemic tree Litsea hypophaea (Lauraceae) in Taiwan to investigate its genetic diversity and population genetic structure and to investigate species delimitation within Litsea. METHODS AND RESULTS: Fifteen new simple sequence repeat markers were developed from L. hypophaea with a magnetic bead enrichment method. Most loci were also amplified from three closely related species, L. coreana, L. lii, and L. acutivena. The number of alleles and observed and expected heterozygosities across loci varied with a range of 1-25, 0.000-1.000, and 0.000-0.956, respectively. CONCLUSIONS: The application of these microsatellite markers of L. hypophaea provides a tool for understanding genetic diversity and population differentiation. In addition, interspecific amplification suggests that these markers will also be useful for species identification of related taxa within Litsea in Taiwan.

Isolation and characterization of 15 microsatellite loci in four endangered Amentotaxus species (Taxaceae).

PREMISE OF THE STUDY: Fifteen microsatellite loci were developed in an endangered species, Amentotaxus formosana, and were tested in an additional three species, A. argotaenia, A. yunnanensis, and A. poilanei, to evaluate the population structure for conservation efforts and reconstruct the phylogeographic patterns of this ancient lineage. METHODS AND RESULTS: Polymorphic primer sets were developed from A. formosana; the number of alleles ranged from two to 10, with an observed heterozygosity ranging from 0 to 0.60. All of the loci were found to be interspecifically amplifiable. CONCLUSIONS: These polymorphic and transferable loci will be potentially useful for future studies that will focus on identifying distinct genetic units within species and establishing the phylogeographic patterns and the process of speciation among closely related species.

Evolutionary rates of commonly used nuclear and organelle markers of Arabidopsis relatives (Brassicaceae).

Recovering the genetic divergence between species is one of the major interests in the evolutionary biology. It requires accurate estimation of the neutral substitution rates. Arabidopsis thaliana, the first whole-genome sequenced plant, and its out-crossing relatives provide an ideal model for examining the split between sister species. In the study, rates of molecular evolution at markers frequently used for systematics and population genetics, including 14 nuclear genes spanning most chromosomes, three noncoding regions of chloroplast genome, and one intron of mitochondrial genome, between A. thaliana and four relatives were estimated. No deviation from neutrality was detected in the genes examined. Based on the known divergence between A. thaliana and its sisters about 8.0-17.6 MYA, evolutionary rates of the eighteen genes were estimated. Accordingly, the ratio of rates of synonymous substitutions among mitochondrial, chloroplast and nuclear genes was calculated with an average and 95% confidence interval of 1 (0.25-1.75): 15.77 (7.48-114.09): 74.79 (36.27-534.61). Molecular evolutionary rates of nuclear genes varied, with a range of 0.383-0.856×10(-8) for synonymous substitutions per site per year and 0.036-0.081×10(-9) for nonsynonymous substitutions per site per year. Compared with orthologs in Populus, a long life-span tree, genes in Arabidopsis evolved faster in an order of magnitude at the gene level, agreeing with a generation time hypothesis. The estimated substitution rates of these genes can be used as a reference for molecular dating.

Footprints of natural and artificial selection for photoperiod pathway genes in Oryza.

Asian rice, Oryza sativa, consists of two major subspecies, indica and japonica, which are physiologically differentiated and adapted to different latitudes. Genes for photoperiod sensitivity are likely targets of selection along latitude. We examined the footprints of natural and artificial selections for four major genes of the photoperiod pathway, namely PHYTOCHROME B (PhyB), HEADING DATE 1 (Hd1), HEADING DATE 3a (Hd3a), and EARLY HEADING DATE 1 (Ehd1), by investigation of the patterns of nucleotide polymorphisms in cultivated and wild rice. Geographical subdivision between tropical and subtropical O. rufipogon was found for all of the photoperiod genes in plants divided by the Tropic of Cancer (TOC). All of these genes, except for PhyB, were characterized by the existence of clades that split a long time ago and that corresponded to latitudinal subdivisions, and revealed a likely diversifying selection. Ssp. indica showed close affinity to tropical O. rufipogon for all genes, while ssp. japonica, which has a much wider range of distribution, displayed complex patterns of differentiation from O. rufipogon, which reflected various agricultural needs in relation to crop yield. In japonica, all genes, except Hd3a, were genetically differentiated at the TOC, while geographical subdivision occurred at 31°N in Hd3a, probably the result of varying photoperiods. Many other features of the photoperiod genes revealed domestication signatures, which included high linkage disequilibrium (LD) within genes, the occurrence of frequent and recurrent non-functional Hd1 mutants in cultivated rice, crossovers between subtropical and tropical alleles of Hd1, and significant LD between Hd1 and Hd3a in japonica and indica.