Biased gene introgression and adaptation in the face of chloroplast capture in Aquilegia amurensis.

Chloroplast capture, a phenomenon that can occur through interspecific hybridization and introgression, is frequently invoked to explain cytonuclear discordance in plants. However, relatively few studies have documented the mechanisms of cytonuclear coevolution and its potential for driving species differentiation and possible functional differences in the context of chloroplast capture. To address this crucial question, we chose the Aquilegia genus, which is known for having minimal sterility among species, and inferred that A. amurensis captured the plastome of A. parviflora based on cytonuclear discordance and gene flow between the two species. We focused on the introgression region and its differentiation from corresponding regions in closely related species, especially its composition in a chloroplast capture scenario. We found that nuclear genes encoding cytonuclear enzyme complexes (CECs; i.e., organelle-targeted genes) of chloroplast donor species were selectively retained and displaced the original CEC genes in chloroplast-receiving species due to cytonuclear interactions during introgression. Notably, the intrinsic correlation of CEC introgression was a greater degree of evolutionary distance for these CECs between A. amurensis and A. parviflora. Terpene synthase activity genes (GO: 0010333) were overrepresented among the introgressed genes, and more than 30% of these genes were CEC genes. These findings support our observations that floral terpene release pattern is similar between A. amurensis and A. parviflora compared with A. japonica. Our study clarifies the mechanisms of cytonuclear coevolution, species differentiation and functional differences in the context of chloroplast capture and highlights the potential role of chloroplast capture in adaptation.

Characterizing complete mitochondrial genome of Aquilegia amurensis and its evolutionary implications.

BACKGROUND: Aquilegia is a model system for studying the evolution of adaptive radiation. However, very few studies have been conducted on the Aquilegia mitochondrial genome. Since mitochondria play a key role in plant adaptation to abiotic stress, analyzing the mitochondrial genome may provide a new perspective for understanding adaptive evolution. RESULTS: The Aquilegia amurensis mitochondrial genome was characterized by a circular chromosome and two linear chromosomes, with a total length of 538,736 bp; the genes included 33 protein-coding genes, 24 transfer RNA (tRNA) genes and 3 ribosomal RNA (rRNA) genes. We subsequently conducted a phylogenetic analysis based on single nucleotide polymorphisms (SNPs) in the mitochondrial genomes of 18 Aquilegia species, which were roughly divided into two clades: the European-Asian clade and the North American clade. Moreover, the genes mttB and rpl5 were shown to be positively selected in European-Asian species, and they may help European and Asian species adapt to environmental changes. CONCLUSIONS: In this study, we assembled and annotated the first mitochondrial genome of the adaptive evolution model plant Aquilegia. The subsequent analysis provided us with a basis for further molecular studies on Aquilegia mitochondrial genomes and valuable information on adaptive evolution in Aquilegia.

Effects of growth years on ginsenoside biosynthesis of wild ginseng and cultivated ginseng.

BACKGROUND: Ginsenoside, as the main active substance in ginseng, has the function of treating various diseases. However, the ginsenosides content of cultivated ginseng is obviously affected by the growth years, but the molecular mechanism is not clear. In addition, there are significant differences in morphology and physiology between wild ginseng and cultivated ginseng, and the effect of growth years on ginsenoside synthesis not yet understood in wild ginseng. RESULTS: Transcriptome sequencing on the roots, stems and leaves of cultivated ginseng and wild ginseng with different growth years was performed in this study, exploring the effect of growth years on gene expression in ginseng. The number of differentially expressed genes (DEGs) from comparison groups in cultivated ginseng was higher than that in wild ginseng. The result of weighted gene co-expression network analysis (WGCNA) showed that growth years significantly affected the gene expression of Mitogen-activated protein kinases (MAPK) signaling pathway and terpenoid backbone biosynthesis pathway in cultivated ginseng, but had no effects in wild ginseng. Furthermore, the growth years had significant effects on the genes related to ginsenoside synthesis in cultivated ginseng, and the effects were different in the roots, stems and leaves. However, it had little influence on the expression of genes related to ginsenoside synthesis in wild ginseng. Growth years might affect the expression of genes for ginsenoside synthesis by influencing the expression of these transcription factors (TFs), like my elob lastosis (MYB), NAM, ATAF1 and 2, and CUC2 (NAC), APETALA2/ethylene-responsive factor (AP2/ERF), basic helix-loop-helix (bHLH) and WRKY, etc., thereby affecting the content of ginsenosides. CONCLUSIONS: This study complemented the gaps in the genetic information of wild ginseng in different growth periods and helped to clarify the potential mechanisms of the effect of growth years on the physiological state in wild ginseng and cultivated ginseng, which also provided a new insight into the mechanism of ginsenoside regulation.

Transcriptome reveals insights into biosynthesis of ginseng polysaccharides.

BACKGROUND: Ginseng polysaccharides, have been used to treat various diseases as an important active ingredient. Nevertheless, the biosynthesis of ginseng polysaccharides is poorly understood. To elucidate the biosynthesis mechanism of ginseng polysaccharides, combined the transcriptome analysis and polysaccharides content determination were performed on the roots, stems, and leaves collected from four cultivars of ginseng. RESULTS: The results indicated that the total contents of nine monosaccharides were highest in the roots. Moreover, the total content of nine monosaccharides in the roots of the four cultivars were different but similar in stems and leaves. Glucose (Glc) was the most component of all monosaccharides. In total, 19 potential enzymes synthesizing of ginseng polysaccharides were identified, and 17 enzymes were significantly associated with polysaccharides content. Among these genes, the expression of phosphoglucomutase (PGM), glucose-6-phosphate isomerase (GPI), UTP-glucose-1-phosphate uridylyltransferase (UGP2), fructokinase (scrK), mannose-1-phosphate guanylyltransferase (GMPP), phosphomannomutase (PMM), UDP-glucose 4-epimerase (GALE), beta-fructofuranosidase (sacA), and sucrose synthase (SUS) were correlated with that of MYB, AP2/ERF, bZIP, and NAC transcription factors (TFs). These TFs may regulate the expression of genes involved in ginseng polysaccharides synthesis. CONCLUSION: Our findings could provide insight into a better understanding of the regulatory mechanism of polysaccharides biosynthesis, and would drive progress in genetic improvement and plantation development of ginseng.

The impacts of polyploidy, geographic and ecological isolations on the diversification of Panax (Araliaceae).

BACKGROUND: Panax L. is a medicinally important genus within family Araliaceae, where almost all species are of cultural significance for traditional Chinese medicine. Previous studies suggested two independent origins of the East Asia and North America disjunct distribution of this genus and multiple rounds of whole genome duplications (WGDs) might have occurred during the evolutionary process. RESULTS: We employed multiple chloroplast and nuclear markers to investigate the evolution and diversification of Panax. Our phylogenetic analyses confirmed previous observations of the independent origins of disjunct distribution and both ancient and recent WGDs have occurred within Panax. The estimations of divergence time implied that the ancient WGD might have occurred before the establishment of Panax. Thereafter, at least two independent recent WGD events have occurred within Panax, one of which has led to the formation of three geographically isolated tetraploid species P. ginseng, P. japonicus and P. quinquefolius. Population genetic analyses showed that the diploid species P. notoginseng harbored significantly lower nucleotide diversity than those of the two tetraploid species P. ginseng and P. quinquefolius and the three species showed distinct nucleotide variation patterns at exon regions. CONCLUSION: Our findings based on the phylogenetic and population genetic analyses, coupled with the species distribution patterns of Panax, suggested that the two rounds of WGD along with the geographic and ecological isolations might have together contributed to the evolution and diversification of this genus.

Phenotypic and genetic evidence for ecological speciation of Aquilegia japonica and A. oxysepala.

Natural selection is thought to be a driving force that can cause the evolution of reproductive isolation. The genus Aquilegia is a model system to address how natural selection promotes the process of speciation. Morphological differences between A. oxysepala, A. japonica and their hybrids were quantified for two vegetative (plant height and leaf area) and three floral morphological (sepal area, corolla length and diameter) traits. We also evaluated the genetic variability of the two species and their hybrids based on two chloroplast (1225 bp), four nuclear (5811 bp) genes and 15 microsatellites. Our results revealed that differentiation of A. japonica and A. oxysepala at the ecological and morphological levels also involved divergence at the genetic level. In addition, the analysis of nucleotide variation patterns showed that the two species possessed numerous fixation sites at nuclear genes gAA4, gA7 and gAA12. Furthermore, we found that all of the phenotypic hybrids also showed a genetically admixed ancestry. These findings suggest that natural selection has indeed facilitated the formation of distinct genetic variation patterns in the two Aquilegia species and habitat adaptation has been driving the ecologically based evolution of reproductive isolation.

Geographic-genomic and geographic-phenotypic differentiation of the Aquilegia viridiflora complex.

How species diverge into different lineages is a central issue in evolutionary biology. Despite the increasing evidence indicating that such divergences do not need geographic isolation, the correlation between lineage divergence and the adaptive ecological divergence of phenotype corresponding to distribution is still unknown. In addition, gene flow has been widely detected during and through such diverging processes. We used one widely distributed Aquilegia viridiflora complex as a model system to examine genomic differentiation and corresponding phenotypic variations along geographic gradients. Our phenotypic analyses of 20 populations from northwest to northeast China identified two phenotypic groups along the geographic cline. All examined traits are distinct from each other, although a few intermediate individuals occur in their contacting regions. We further sequenced the genomes of representative individuals of each population. However, four distinct genetic lineages were detected based on nuclear genomes. In particular, we recovered numerous genetic hybrids in the contact regions of four lineages. Gene flow is widespread and continuous between four lineages but much higher between contacting lineages than geographically isolated lineages. Gene flow and natural selection might result in inconsistency between heredity and phenotype. Moreover, many genes with fast lineage-specific mutations were identified to be involved in local adaptation. Our results suggest that both geographic isolation and local selection exerted by the environment and pollinators may together create geographic distributions of phenotypic variations as well as the underlying genomic divergences in numerous lineages.

Didymodonchangbaiensis (Pottiaceae, Musci), a new species from Changbai Mountain, China and its phylogenetic position based on molecular data.

Changbai Mountain, located in northeast China, is one of the areas with the most complete natural ecosystem preservation in China. A new species, Didymodonchangbaiensis C.Feng, J.Kou, H.-X. Xiao & T.-T.Wu from north slope of Changbai Mountain in Jilin Province of China is described and illustrated. It is characterised by ovate or ovate-lanceolate leaves that are appressed when dry, acute leaf apex, lamina red or reddish-orange with KOH, unistratose lamina throughout, plane and unistratose leaf margins, percurrent costa with one layer of guide cells and without ventral stereids, upper and middle laminal cells with elliptical papillae over the transverse walls between two immediately adjacent cells and basal laminal cells not differentiated from the median cells. Our morphological analyses and molecular results, based on DNA sequences of ITS, rps4 and trnM-trnV, confirm that D.changbaiensis is revealed to be sister to D.daqingii J. Kou, R.H. Zander & C. Feng. This new species is compared with similar species and its phylogenetic position and ecology are discussed.

Genomic and EST-derived microsatellite markers for Iris laevigata (Iridaceae) and other congeneric species.

PREMISE OF THE STUDY: The aims of this study are to develop and characterize genomic and expressed sequence tag (EST)-derived microsatellites from Iris laevigata and test their transferability in I. ensata, I. setosa, I. halophila, I. scariosa, I. potaninii, I. tenuifolia, I. bloudowii, and I. sanguinea. METHODS AND RESULTS: Ten genomic and six EST-derived microsatellites were characterized in I. laevigata. These microsatellite primers amplified one to five alleles in I. laevigata and some of these primers were also successfully amplified in congeneric species. CONCLUSIONS: These microsatellite primers provide us an initial set of molecular markers to explore the spatial population genetic structure of I. laevigata. In addition, these markers may also be useful in population and conservation genetic studies of closely related species.

Development of eighteen microsatellite markers in Anemone amurensis (Ranunculaceae) and cross-amplification in congeneric species.

Polyploidy plays an important role in the evolution of plant genomes. To enable the investigation of the polyploidy events within the genus Anemone, we developed eighteen microsatellite markers from the hexaploid species A. amurensis (Ranunculaceae), and tested their transferability in five closely related species. The number of total alleles (N(A)) for each resulting locus varied from one to eight. The polymorphism information content (PIC) and Nei's genetic diversity (N(GD)) for these microsatellites ranged from 0.00 to 0.71 and 0.00 to 0.91, respectively. For each population, the N(A) was one to seven, and the values of PIC and N(GD) varied from 0.00 to 0.84 and 0.00 to 0.95, respectively. In addition, most of these microsatellites can be amplified successfully in the congeneric species. These microsatellite primers provide us an opportunity to study the polyploid evolution in the genus Anemone.

Isolation and identification of fourteen microsatellite markers in Clivia miniata and Clivia nobilis (Amaryllidaceae).

Clivia is a genus of great horticultural importance and has been widely cultivated as ornamental plants in all over the world. In order to assess the phylogenetic relationships and genetic diversity of the wild Clivia species and cultivars, we isolated AC-enriched repeats using FIASCO from a single clone each of C. miniata Regel. and Clivia nobilis Lindl. Of the fourteen repeats, 10 were polymorphic and 4 were monomorphic. The polymorphic marker loci were characterized using 61 Clivia accessions. The number of alleles ranged from two to six, observed heterozygosity ranged from 0.04 to 1.00 and expected heterozygosity ranged from 0.04 to 0.83. These microsatellite marker loci provide tools for future studies of Clivia species and cultivars.

Structural characterization and anti-oxidation activity evaluation of pectin from Lonicera japonica Thunb.

Pectins are nutrient components of plants and are widely used in the food industry. In this study, one major pectin fraction (WLJP-A0.2b) with Mw of 40.6 kDa was purified from Lonicera japonica Thunb. The structural feature and antioxidant activity of it was investigated. Monosaccharide composition, Fourier transform infrared (FT-IR) spectra, enzymatic hydrolysis, and nuclear magnetic resonance (NMR) spectra analysis indicated that WLJP-A0.2b consisted of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and homogalacturonan (HG) domains, with mass ratio of 0.4:1.0:2.1. The RG-I domain contained highly branched α-L-1,5-arabinan, ß-D-1,4-galactan and type II arabinogalactan (AG-II) side chains. The HG domain was released in the form of un-esterified and partly methyl-esterified and/or acetyl-esterified oligogalacturonides with degree of polymerization 1-8 after degradation by endo-polygalacturonase. Radical scavenging assays indicated that WLJP-A0.2b exhibited antioxidant activity through the synergistic effects of different pectin domains. Oligogalacturonides, especially de-esterified oligogalacturonides, showed better antioxidant activities than RG-II and RG-I domains. Moreover, de-esterified oligogalacturonides remarkably reduced H2O2-induced reactive oxygen species production in HEK-293T cells. These results provide useful information for screening of natural antioxidants from Lonicera japonica Thunb. and application of pectin in functional food field.

Genomic and EST microsatellite markers for Aquilegia flabellata and cross-amplification in A. oxysepala (Ranunculaceae).

PREMISE OF THE STUDY: The goal of this study was to identify genomic and expressed sequence tag (EST)-derived microsatellite markers from the species Aquilegia flabellata and assess their transferability in A. oxysepala. METHODS AND RESULTS: Eleven genomic and nine EST-derived microsatellite loci were characterized in A. flabellata. These microsatellite primers amplified 66 alleles in all 20 loci. The observed heterozygosity (H(O)) for each population ranged from 0.00 to 0.94. CONCLUSIONS: All of the 20 loci were successfully amplified in A. oxysepala. These genomic and EST-derived microsatellite markers will be useful in further genetic structure, speciation, and adaptive evolution studies.

Genomic and EST microsatellites for Rhododendron aureum (Ericaceae) and cross-amplification in other congeneric species.

PREMISE OF THE STUDY: Our objective of this study was to develop genomic and expressed sequence tag (EST)-derived microsatellites for the endangered species Rhododendron aureum and access their transferability in R. dauricum and R. brachycarpum. • METHODS AND RESULTS: Twelve genomic microsatellites were isolated in R. aureum using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol and seven EST-derived microsatellites were characterized by screening the Rhododendron dbEST database of GenBank. The number of alleles per locus ranged from one to eight. The observed and expected heterozygosity varied from 0.00 to 1.00 and from 0.00 to 0.73, respectively. • CONCLUSIONS: A total of 19 microsatellite loci were developed for R. aureum. Sixteen and 10 of these loci were successfully amplified in R. brachycarpum and R. dauricum, respectively. These microsatellite markers will have potential applications in genetic diversity and conservation genetics studies.

Impact of different numbers of microsatellite markers on population genetic results using SLAF-seq data for Rhododendron species.

Microsatellites (simple sequence repeats, SSRs) are co-dominant nuclear markers that are widely used in population genetic studies. Population genetic parameters from different studies might be significantly influenced by differences in marker number. In our study, 265 sequences with polymorphic microsatellites were obtained from SLAF-seq data. Then, subpopulations containing different numbers (5, 6, 7,…, 15, 20, 25, 30, 35, 40) of markers were genotyped 10 times to investigate the impact of marker numbers on population genetic diversity results. Our results show that genotyping with less than 11 or 12 microsatellite markers lead to significant deviations in the population genetic diversity or genetic structure results. In order to provide markers for population genetic and conservation studies for Rhododendron, 26 SSR primers were designed and validated in three species.

Comparative chloroplast genomes and phylogenetic analysis of Aquilegia.

PREMISE: Aquilegia is an ideal taxon for studying the evolution of adaptive radiation. Current phylogenies of Aquilegia based on different molecular markers are inconsistent, and therefore a clear and accurate phylogeny remains uncertain. Analyzing the chloroplast genome, with its simple structure and low recombination rate, may help solve this problem. METHODS: Next-generation sequencing data were generated or downloaded for Aquilegia species, enabling their chloroplast genomes to be assembled. The assemblies were used to estimate the genome characteristics and infer the phylogeny of Aquilegia. RESULTS: In this study, chloroplast genome sequences were assembled for Aquilegia species distributed across Asia, North America, and Europe. Three of the genes analyzed (petG, rpl36, and atpB) were shown to be under positive selection and may be related to adaptation. The phylogenetic tree of Aquilegia showed that its member species formed two clades with high support, North American and European species, with the Asian species being paraphyletic; A. parviflora and A. amurensis clustered with the North American species, while the remaining Asian species were found in the European clade. In addition, A. oxysepala var. kansuensis should be considered as a separate species rather than a variety. DISCUSSION: The complete chloroplast genomes of these Aquilegia species provide new insights into the reconstruction of the phylogeny of related species and contribute to the further study of this genus.

Phylogeography of Iris loczyi (Iridaceae) in Qinghai-Tibet Plateau revealed by chloroplast DNA and microsatellite markers.

Quaternary climate oscillations and complex topography have tremendous effects on current distribution and genetic structure of species, and hence the Qinghai-Tibet Plateau (QTP), the largest plateau in the world, has become a hotspot for many phylogeographic studies. However, little is known about the phylogeographic pattern of herbaceous plants in QTP. Here, we investigate the genetic diversity, population structure and historical dynamics of Iris loczyi, using five chloroplast DNA (cpDNA) fragments and seven microsatellite markers. A total of 15 populations, and 149 individuals were sampled throughout the QTP. High genetic diversity was detected both in cpDNA (H d = 0.820) and SSR (H o = 0.689, H e = 0.699). Ten cpDNA haplotypes and 163 alleles were identified. AMOVA and clustering analyses revealed obvious differentiation between regions. The N st, G st and Mantel test showed significant phylogeographic structure of I. loczyi. The neutrality test and mismatch distribution analyses indicated that I. loczyi could not have undergone a historical population expansion, but population XS from the Qilian Mountain area could have experienced a local expansion. Bottleneck analyses indicated that I. loczyi had not experienced bottleneck recently. Based on cpDNA and SSR results, the Qilian Mountain area was inferred as a potential glacial refuge, and the southern Tibet valley was considered as a 'microrefugia' for I. loczyi. These findings provided new insights into the location of glacial refuges for the species distributed in QTP, and supplemented more plant species data for the response of QTP species to the Quaternary climate.

Optimization of SPME-GC-MS and characterization of floral scents from Aquilegia japonica and A. amurensis flowers.

BACKGROUND: The floral scents of plants play a key role in plant reproduction through the communication between plants and pollinators. Aquilegia as a model species for studying evolution, however, there have been few studies on the floral scents and relationships between floral scents and pollination for Aquilegia taxa. METHODS: In this study, three types of solid-phase micro-extraction (SPME) fiber coatings (DVB/PDMS, CAR/PDMS, DVB/CAR/PDMS) were evaluated for their performance in extracting volatile organic compounds (VOCs) from flowers of Aquilegia amurensis, which can contribute to the future studies of elucidating the role of floral scents in the pollination process. RESULTS: In total, 55 VOCs were identified, and among them, 50, 47 and 45 VOCs were extracted by the DVB/CAR/PDMS fiber, CAR/PDMS fiber and DVB/PDMS fibers, respectively. Only 30 VOCs were detected in A. japonica taxa. Furthermore, the relative contents of 8 VOCs were significant different (VIP > 1 and p < 0.05) between the A. amurensis and A. japonica. CONCLUSIONS: The results can be applied in new studies of the relationships between the chemical composition of floral scents and the processes of attraction of pollinator. It may provide new ideas for rapid evolution and frequent interspecific hybridization of Aquilegia.

A Genomewide Scan for Genetic Structure and Demographic History of Two Closely Related Species, Rhododendron dauricum and R. mucronulatum (Rhododendron, Ericaceae).

Understanding the processes of divergence and speciation is an important task for evolutionary research, and climate oscillations play a pivotal role. We estimated the genetic structure and demographic history of two closely related species of Rhododendron, R. dauricum, and R. mucronulatum, distributed in northeastern China using 664,406 single nucleotide polymorphic loci of specific-locus amplified fragment sequencing (SLAF-seq) and 4 chloroplast DNA (cpDNA) fragments, sampling 376 individuals from 39 populations of these two species across their geographic distributions. The geographical distribution of cpDNA haplotypes revealed that R. dauricum and R. mucronulatum have different spatial genetic structures and haplotype diversity. Analysis of molecular variance (AMOVA) results showed that these two species have significant genetic differentiation and that the phylogeny demonstrates that these two species clustered a monophyletic group based on SLAF data, respectively, but not in cpDNA data. The evidence of significant gene flow was also detected from R. mucronulatum to R. dauricum. A deep divergence between the two species was observed and occurred during the early Oligocene. The niche models showed that the two species have different demographic histories. Thus, our results imply that geography and climate changes played important roles in the evolutionary process of R. dauricum and R. mucronulatum, and although there was an interspecific gene flow, the divergence was maintained by natural selection.

Population genetic structures of two ecologically distinct species Betula platyphylla and B. ermanii inferred based on nuclear and chloroplast DNA markers.

Climatic oscillations during the last glacial maximum (LGM) significantly affected the distribution patterns and genetic structure of extant plants. Northeast China (NEC) is a major biodiversity center in East Asia, and the influence of historical climate change on NEC populations is critical for understanding species responses to future climate change. However, only a few phylogeographic studies of cool temperate deciduous tree species have been conducted in the area, and results are inconsistent for species with different niches or distribution areas. We employed multiple chloroplast and nuclear markers to investigate the genetic structure of two ecologically contrasting species, Betula platyphylla and B. ermanii, in NEC. Rare haplotypes were identified in the chloroplast genome of these species, and both exhibited high levels of nucleotide diversity based on a fragment of the nuclear gene G3PDH and microsatellites. Moreover, significant phylogeographic structure was detected for B. platyphylla, suggesting that these populations had recolonized from independent glacial refuges, whereas no genetic structure was found for B. ermanii. OPEN RESEARCH BADGES: The nSSR datasets used in the current study and the table of pairwise FST (below diagonal) and its standardized F'ST (above diagonal) among 25 populations based on seven SSRs are available from the Dryad (DOI: https://doi.org/10.5061/dryad.230d176). Sequences generated from this study were deposited in GenBank under Accession nos. KY199568-KY200162 and MK819541-MK819970.