Regulation of exogenous sugars on the biosynthesis of key secondary metabolites in Cyclocarya paliurus.

The biosynthesis and accumulation of secondary metabolites play a vital role in determining the quality of medicinal plants, with carbohydrate metabolism often influencing secondary metabolism. To understand the potential regulatory mechanism, exogenous sugars (sucrose, glucose/fructose) were applied to the leaves of Cyclocarya paliurus, a highly valued and multiple function tree species. The results showed that exogenous sugars enhanced the accumulation of soluble sugar and starch while increasing the enzyme activity related to carbohydrate metabolism. In addition, the plant height was increased by a mixture of exogenous mixed sugars, the addition of sucrose promoted the net photosynthetic rate, while all types of exogenous sugars facilitated the accumulation of flavonoids and terpenoids. Based on weighted gene co-expression network analysis (WGCNA), two key gene modules and four candidate transcription factors (TFs) related to carbohydrate metabolism and secondary metabolite biosynthesis were identified. A correlation analysis between transcriptome and metabolome data showed that exogenous sugar up-regulated the expression of key structural genes in the flavonoid and terpenoid biosynthetic pathway. The expression levels of the four candidate TFs, TIFY 10A, WRKY 7, EIL 3 and RF2a, were induced by exogenous sugar and were strongly correlated with the key structural genes, which enhanced the synthesis of specific secondary metabolites and some plant hormone signal pathways. Our results provide a comprehensive understanding of key factors in the quality formation of medicinal plants and a potential approach to improve the quality.

Nine new nor-3,4-seco-dammarane triterpenoids from the leaves of Cyclocarya paliurus and their hypoglycemic activity.

This manuscript describes the isolation of nine new nor-3,4-seco-dammarane triterpenoids, norqingqianliusus A-I (1-9) and one known nortriterpenoid (10) from Cyclocarya paliurus leaves. Norqingqianliusus A and B (1 and 2) possess a unique 3,4-seco-dammarane-type C26 tetranortriterpenoid skeleton. The compounds were structurally characterized through modern spectroscopic techniques. Moreover, the potential mechanism of hypoglycemic activity was further explored by studying the effects on glucosamine-induced insulin resistant HepG2 cells. In vitro hypoglycemic effects of all of the isolates were investigated using insulin resistant HepG2 cells. The glucose consumption was significantly promoted by compound 10, in a dose-dependent manner, thus alleviating damage in IR-HepG2 cells. Besides, it reduced the PEPCK and GSK3ß gene expression, involved in glucose metabolism. The anti-diabetic effects of the plant, utilized traditionally, can hence be attributed to the presence of nor-3,4-seco-dammarane triterpenoids in the leaves.

Transcriptome profiling reveals the protective mechanism of sulfated Cyclocarya paliurus polysaccharides against oxidative damage of IEC-6 cell.

Our previous studies have shown that sulfated Cyclocarya paliurus polysaccharides (SCP3) can alleviate intestinal oxidative stress (OS) damage by improving the antioxidant capacity of the intestine, but its mechanism still needs further exploration. This study aimed to reveal the possible underlying protective mechanism of SCP3 against OS damage of intestinal epithelial cells (IEC-6) based on transcriptome profiling. The results showed that SCP3 could increase the activity of superoxide dismutase and reduce the production of malondialdehyde and reactive oxygen species. In addition, the SCP3 could also alleviate the H2O2-induced high apoptosis rate and mitochondrial membrane potential decrease in IEC-6 cells. RNA-sequencing results showed that there were 2152 differentially expressed genes between the control group and the SCP3 group, and the mitogen-activated protein kinases (MAPK) and PI3K-Akt signaling pathways are the main signaling pathways that contributed to SCP3 protecting IEC-6 cells from OS damage. In summary, the SCP3 plays a role in improving intestinal cell damage by inhibiting OS, which may be closely related to the PI3K/Akt and MAPK signaling pathways. PRACTICAL APPLICATION: This study provides a theoretical basis for the practical application of Cyclocarya paliurus polysaccharides as an antioxidant ingredient in auxiliary medicines and functional foods.

Effects of Foliar Dressing with Chemical Nano-Selenum and Na2SeO3 on the Antioxidant System and Accumulation of Se and Bioactive Components in Cyclocarya paliurus (Sweet Tea Tree).

Selenium (Se)-rich Cyclocarya paliurus is popular for its bioactive components, and exogenous Se fortification is the most effective means of enrichment. However, the effects of exogenous Se fortification on the nutritional quality of C. paliurus are not well known. To investigate the nutrient contents and antioxidant properties of C. paliurus following Se treatment, we used a foliar spray to apply Se in two forms-chemical nano-Se (Che-SeNPs) and sodium selenite (Na2SeO3). Sampling began 10 days after spraying and was conducted every 5 days until day 30. The Se, secondary metabolite, malondialdehyde contents, antioxidant enzyme activity, Se speciation, and Se-metabolism-related gene expression patterns were analyzed in the collected samples. Exogenous Se enhancement effectively increased the Se content of leaves, reaching a maximum on days 10 and 15 of sampling, while the contents of flavonoids, triterpenes, and polyphenols increased significantly during the same period. In addition, the application of Se significantly enhanced total antioxidant activity, especially the activity of the antioxidant enzyme peroxidase. Furthermore, a positive correlation between the alleviation of lipid peroxidation and Se content was observed, while methylselenocysteine formation was an effective means of alleviating Se stress. Finally, Na2SeO3 exhibited better absorption and conversion efficiency than Che-SeNPs in C. paliurus.

α-Glucosidase inhibitory flavonol glycosides from Cyclocarya paliurus (Batalin) Iljinskaja and their kinetics characteristics.

Seven previously undescribed flavonol glycosides including four rare flavonol glycoside cyclodimers, dicyclopaliosides A-C (1-3) with truxinate type and dicyclopalioside D (4) with truxillate type, as well as three kaempferol glycoside derivatives cyclopaliosides A-C (5-7), were obtained from the leaves of Cyclocarya paliurus. Their structures were elucidated by extensive spectroscopic methods and chemical analyses. All compounds were evaluated for their inhibitory α-glucosidase activities. Among them, compounds 1-4 display strong inhibitory activities with IC50 values of 82.76 ± 1.41, 62.70 ± 4.00, 443.35 ± 16.48, and 6.31 ± 0.88 nM, respectively, while compounds 5-7 showed moderate activities with IC50 values of 4.91 ± 0.75, 3.64 ± 0.68, and 5.32 ± 0.53 µΜ, respectively. The structure-activity relationship analysis assumed that the cyclobutane cores likely contribute to the enhancement of α-glucosidase inhibitory activities of dimers. Also, the interaction mechanism between flavonol glycoside dimers and α-glucosidase were explored by the enzyme kinetic assay, indicating that compounds 1-3 exhibited mixed-type inhibition, while 4 showed uncompetitive inhibition. Additionally, the active compounds have also undergone molecular docking evaluation.

Discovery and identification of natural alkaloids with potential to impact insulin resistance syndrome in Cyclocarya paliurus. (Batal) leaves by UPLC-QTOF-MS combined with HepG2 cells.

Cyclocarya paliurus (Batal.) leaves, which contain a range of bioactive compounds, have been used as a traditional Chinese medicine homologous food since ancient times. However, there is a paucity of literature on comprehensive studies of alkaloids in the leaves of Cyclocarya paliurus (Batal.). For the first time, this study aimed to discover and identify alkaloids extracted from Cyclocarya paliurus (Batal.) leaves by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-QTOF-MS). A total of ten alkaloids have been identified from Cyclocarya paliurus (Batal.) leaves based on accurate mass spectra (mass accuracy, isotopic spacing and distribution) and comparison to fragmentation spectra reported in the literature. In vitro, alkaloids alleviated insulin resistance by increasing glucose consumption and glycogen content in insulin resistance HepG2 cells. The RNA-seq and western blotting results showed that alkaloids could upregulate the expression of phosphatidylinositol 3-kinase (PI3K), and increase the phosphorylation of insulin receptor protein kinase B (AKT). This study not only clarified the chemical constituents and revealed that diverse alkaloids also presented from Cyclocarya paliurus (Batal.) leaves, also, it will provide chemical information on potential compounds for developing new drugs.

Characterization and antioxidant properties of three exopolysaccharides produced by the Cyclocarya paliurus endophytic fungus.

In recent years, the considerable potential of endophytic bacteria and fungi as prolific producers of exopolysaccharides (EPSs) have attracted interest. In this study, 56 endophytes were isolated from Cyclocarya paliurus, and the secondary metabolites of EPSs were extracted from Monascus purpureus, Penicillium citrinum and Aspergillus versicolor, screened, and named MPE, PCE and AVE, respectively. In this work, the physicochemical properties and antioxidant activities of three EPSs, their cell proliferation activity on IEC-6 and RAW264.7 were investigated. The three EPSs were mainly composed of neutral sugar and differ in microstructure. However, MPE had a loose structure, and PCE exhibited a dense and sheet-like structure. In addition, the three EPSs performed ordinary antioxidant activity in vitro but showed excellent cell proliferation activity on IEC-6 and RAW264.7. The cell proliferation activity of PCE was 1.4-fold that of the controls at a concentration of 800 µg/mL on IEC-6, and MPE exhibited 1.3-fold increase on RAW264.7. This study provided scientific evidence and insights into the application of endophytes as a novel plant resource possessing huge application potential.

Pseudostellaria heterophylla polysaccharide mitigates Alzheimer's-like pathology via regulating the microbiota-gut-brain axis in 5 × FAD mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by neuroinflammation, for which gut dysbiosis may be implicated. Our previous study showed that treatment with Pseudostellaria heterophylla aqueous extract and one of its cyclopeptides, heterophyllin B, attenuate memory deficits via immunomodulation and neurite regeneration. However, whether Pseudostellaria heterophylla polysaccharide (PH-PS) exerts neuroprotective effects against AD and its underlying mechanisms remain unclear. The infrared spectrum, molecular weight, and carbohydrate composition of the PH-PS were determined. The results showed that PH-PS (Mw 8.771 kDa) was composed of glucose (57.78 %), galactose (41.52 %), and arabinose (0.70 %). PH-PS treatment ameliorated learning and spatial memory deficits, reduced amyloid ß build-up, and suppressed reactive glia and astrocytes in 5 × FAD mice. 16S rRNA sequencing further showed that PH-PS remodelled the intestinal flora composition by promoting probiotic microbiota, such as Lactobacillus, Muribaculum, Monoglobus, and [Eubacterium]_siraeum_group, and suppressing inflammation-related UCG-009 and Blautia. Additionally, PH-PS restored intestinal barrier function; ameliorated peripheral inflammation by reducing the secretion of inflammatory cytokines, thereby converting M1 microglia and A1 astrocyte toward beneficial M2 and A2 phenotypes; and contributed to Aß plaques clearance by upregulation of insulin degradation enzyme and neprilysin. Collectively, our findings demonstrate that PH-PS may prevent the progression of AD via modulation of the gut microbiota and regulation of glial polarisation, which could provide evidence to design a potential diet therapy for preventing or curing AD.

Chromosome-level genome assembly of Platycarya strobilacea.

Platycarya strobilacea belongs to the walnut family (Juglandaceae), is commonly known as species endemic to East Asia, and is an ecologically important, wind pollinated, woody deciduous tree. To facilitate this ancient tree for the ecological value and conservation of this ancient tree, we report a new high-quality genome assembly of P. strobilacea. The genome size was 677.30 Mb, with a scaffold N50 size of 45,791,698 bp, and 98.43% of the assembly was anchored to 15 chromosomes. We annotated 32,246 protein-coding genes in the genome, of which 96.30% were functionally annotated in six databases. This new high-quality assembly of P. strobilacea provide valuable resource for the phylogenetic and evolutionary analysis of the walnut family and angiosperm.

Spatial metabolome of biosynthesis and metabolism in Cyclocarya paliurus leaves.

A large number of plant metabolites were discovered, but their biosynthetic and metabolic pathways are still largely unknown. However, the spatial distribution of metabolites and their changes in metabolic pathways can be supplemented by mass spectrometry imaging (MSI) techniques. For this purpose, the combination of desorption electrospray ionization (DESI)-MSI and non-targeted metabolomics was used to obtain the spatial distribution information of metabolites in the leaves of Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus). The sample pretreatment method was optimized to have higher detection sensitivity in DESI. The changes of metabolites in C. paliurus were analyzed in depth with the integration of the spatial distribution information of metabolites. The main pathways for biosynthesis of flavonoid precursor and the effect of changes in compound structure on the spatial distribution were found. Spatial metabolomics can provide more metabolite information and a platform for the in-depth understanding of the biosynthesis and metabolism in plants.

Insights into the Hormone-Regulating Mechanism of Adventitious Root Formation in Softwood Cuttings of Cyclocarya paliurus and Optimization of the Hormone-Based Formula for Promoting Rooting.

Adventitious root (AR) formation is vital for successful cutting propagation in plants, while the dynamic regulation of phytohormones is viewed as one of the most important factors affecting AR formation. Cyclocarya paliurus, a hard-to-root plant, is faced with the bottleneck of cloning its superior varieties in practice. In this study, ten treatments were designed to figure out the best hormone-based formula for promoting AR formation in softwood cuttings and explore their hormone-regulating mechanisms. Both the rooting process and the rooting parameters of the softwood cuttings were significantly affected by different hormone-based formulas (p < 0.05), while the greatest rooting rate (93%) and root quality index were achieved in the H3 formula (SR3:IR3 = 1:1). Significant differences in the measured phytohormone concentrations, as well as in their ratios, were detected among the cuttings sampled at various AR formation stages (p < 0.05), whereas the dynamics for each phytohormone varied greatly during AR formation. The transcriptome analysis showed 12,028 differentially expressed genes (DEGs) identified during the rooting process of C. paliurus cuttings, while the KEGG enrichment analysis indicated that a total of 20 KEGG terms were significantly enriched in all the comparison samples, with 253 DEGs detected in signal transduction. Furthermore, 19 genes with vital functions in regulating the hormone signaling pathway were identified by means of a WGCNA analysis. Our results not only optimize a hormone-based formula for improving the rooting of C. paliurus cuttings but also provide an insight into the hormonal regulatory network during AR formation in softwood C. paliurus cuttings.

Cyclosorus Terminans Extract Alleviates Neuroinflammation in Insulin Resistant Rats.

High-fat diet consumption for an extended period causes obesity, systemic metabolic disturbance, and brain insulin resistance, resulting in neuroinflammation. Although the beneficial effect of Cyclosorus terminans extract on obesity-related insulin resistance has been demonstrated, little is known about how it affects neuroinflammation and brain insulin resistance in obese rats. Male Wistar rats were given either a normal diet (ND, n = 6) or a high-fat diet (HFD, n = 24) for a total of 14 weeks. At the beginning of the week, 13 rats in the ND group were given vehicle orally for 2 weeks, while rats on HFD diets were randomized to one of four groups and given either vehicle, 100 mg/kg/day of Cyclosorus terminans extract, 200 mg/kg/day of Cyclosorus terminans extract, or 20 mg/kg/day of pioglitazone orally for 2 weeks. After the experimental period, blood and brain samples were taken to assess metabolic and brain parameters. HFD-fed rats had obesity, systemic and brain insulin resistance, brain inflammation, microglial and astrocyte hyperactivity, and brain necroptosis. Treatment with 200 mg/kg/day of Cyclosorus terminans extract and pioglitazone equally attenuated obesity, insulin resistance, brain insulin dysfunction, and neuroinflammation in insulin resistant rats. Our findings suggest that Cyclosorus terminans extract may hold promise as a therapeutic agent for insulin resistance and neuroinflammation in obese conditions.

Triterpenoid Compounds from Cyclocarya paliurus: A Review of Their Phytochemistry, Quality Control, Pharmacology, and Structure-Activity Relationship.

Cyclocarya paliurus (Batalin) Iljinskaja (C. paliurus) is a single species of Cyclocarya paliurus in Juglandaceae. It is a unique rare medicinal plant resource in China that is mainly distributed in the south of China. The leaves of C. paliurus, as a new food ingredient, are processed into tea products in daily life. Triterpenoids are the main active ingredient in C. paliurus. So far, 164 triterpenoid compounds have been isolated and identified from C. paliurus, which are included 3,4-seco-dammaranes, dammaranes, oleanane, ursane, lupinanes, taraxeranes, and norceanothanes. Modern pharmacological studies manifested that these ingredients have a wide range of pharmacological activities both in vitro and in vivo, such as reducing blood sugar, lowering blood lipids, and anti-tumor, anti-inflammatory, anti-oxidant, and other activities. In addition, current results indicate that the pharmacological mechanisms of triterpenoids were closely related to their chemical structure, molecular signaling pathways, and the expression of related proteins. In order to further study C. paliurus based on the current research situation, this review summarizes the prospect and systematic summary of the triterpenes of C. paliurus from the aspects of structural characteristics, quality control, biological activity, and the structure-activity relationship, which provide a reference for further research and application of the triterpenoids from C. paliurus in the field of functional food and medicine.

Identification and Analysis of Protein Family Associated with RNA Interference Pathway in Juglandaceae.

BACKGROUND: One of the crucial processes for small RNA synthesis and plant disease resistance is RNA interference (RNAi). Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA binding (DRB), and Argonaute are important proteins implicated in RNAi (AGO). Numerous significant woody plants belong to the Juglandaceae; walnut is one of the four groups of woody plants on earth and one of the four groups of dried fruits. METHODS: In order to correlate walnuts and their homologues, this work integrated numerous web resources from structural analysis and transcriptome data collected from gene families in order to elucidate the evolution and functional differentiation of RNA-related proteins in the walnut (Juglans rega) genome. RESULTS: 5 DCL genes, 13 RDR genes, 15 DRB genes, and 15 AGO genes are found in the walnut genome and encode conserved protein domains and motifs with similar subcellular distribution.There are three classes and seven subclasses of walnut AGO proteins. RDRS are primarily split into four categories, whereas DRBs can be divided into six. DCLs are separated into four groups. The walnut RDR1 copy number of 9 is the exception, with 7 of those copies being dispersed in clusters on chromosome 16. Proteins are susceptible to various levels of purification selection, but in walnut, purification selection drives gene creation. These findings also indicated some resemblance in other plants belonging to the walnut family. Under various tissues and stresses, many RNA-related genes in walnut produced abundant, selective expression. CONCLUSIONS: In this study, the genome of the Juglandaceae's DCL, RDR, DRB, and AGO gene families were discovered and analysed for the first time. The evolution, structure, and expression characteristics of these families were also preliminary studied, offering a foundation for the development and breeding of the walnut RNAi pathway.

Genome assembly of two diploid and one auto-tetraploid Cyclocarya paliurus genomes.

Cyclocarya paliurus, an endemic species in the genus Juglandaceae with the character of heterodichogamy, is one of triterpene-rich medicinal plants in China. To uncover the genetic mechanisms behind the special characteristics, we sequenced the genomes of two diploid (protandry, PA-dip and protogyny, PG-dip) and one auto-tetraploid (PA-tetra) C. paliurus genomes. Based on 134.9 (~225x), 75.5 (~125x) and 271.8 Gb (~226x) subreads of PacBio platform sequencing data, we assembled 586.62 Mb (contig N50 = 1.9 Mb), 583.45 Mb (contig N50 = 1.4 Mb), and 2.38 Gb (contig N50 = 430.9 kb) for PA-dip, PG-dip and PA-tetra genome, respectively. Furthermore, 543.53, 553.87, and 2168.65 Mb in PA-dip, PG-dip, and PA-tetra, were respectively anchored to 16, 16, and 64 pseudo-chromosomes using over 65.4 Gb (~109x), 68 Gb (~113x), and 264 (~220x) Hi-C sequencing data. Annotation of PA-dip, PG-dip, and PA-tetra genome assembly identified 34,699, 35,221, and 34,633 protein-coding genes (90,752 gene models) or allele-defined genes, respectively. In addition, 45 accessions from nine locations were re-sequenced, and more than 10 × coverage reads were generated.

Genomic Analysis of Plastid-Nuclear Interactions and Differential Evolution Rates in Coevolved Genes across Juglandaceae Species.

The interaction between the nuclear and chloroplast genomes in plants is crucial for preserving essential cellular functions in the face of varying rates of mutation, levels of selection, and modes of transmission. Despite this, identifying nuclear genes that coevolve with chloroplast genomes at a genome-wide level has remained a challenge. In this study, we conducted an evolutionary rate covariation analysis to identify candidate nuclear genes coevolving with chloroplast genomes in Juglandaceae. Our analysis was based on 4,894 orthologous nuclear genes and 76 genes across seven chloroplast partitions in nine Juglandaceae species. Our results indicated that 1,369 (27.97%) of the nuclear genes demonstrated signatures of coevolution, with the Ycf1/2 partition yielding the largest number of hits (765) and the ClpP1 partition yielding the fewest (13). These hits were found to be significantly enriched in biological processes related to leaf development, photoperiodism, and response to abiotic stress. Among the seven partitions, AccD, ClpP1, MatK, and RNA polymerase partitions and their respective hits exhibited a narrow range, characterized by dN/dS values below 1. In contrast, the Ribosomal, Photosynthesis, Ycf1/2 partitions and their corresponding hits, displayed a broader range of dN/dS values, with certain values exceeding 1. Our findings highlight the differences in the number of candidate nuclear genes coevolving with the seven chloroplast partitions in Juglandaceae species and the correlation between the evolution rates of these genes and their corresponding chloroplast partitions.

Molecular mechanism of thiamine in mitigating drought stress in Chinese wingnut (Pterocarya stenoptera): Insights from transcriptomics.

Urban garden plants are frequently affected by drought, which can hinder their growth, development, and greening effect. Previous studies have indicated that Chinese wingnut (Pterocarya stenoptera) responds to drought stress by increasing the expression of thiamine synthesis genes. In this study, it was found that exogenous thiamine can effectively alleviate the negative effects of drought stress on plants. Forward transcriptome sequencing and physiological tests were further conducted to reveal the molecular mechanism of thiamine in alleviating drought stress. Results showed that exogenous thiamine activated the expression of eight chlorophyll synthesis genes in Chinese wingnut under drought stress. Moreover, physiological indicators proved that chlorophyll content increased in leaves of Chinese wingnut with thiamine treatment under drought stress. Photosynthesis genes were also activated in Chinese wingnut treated with exogenous thiamine under drought stress, as supported by photosynthetic indicators PIabs and PItotal. Additionally, exogenous thiamine stimulated the expression of genes in the auxin-activated signaling pathway, thus attenuating the effects of drought stress. This study demonstrates the molecular mechanism of thiamine in mitigating the effects of drought stress on non-model woody plants lacking transgenic systems. This study also provides an effective method to mitigate the negative impacts of drought stress on plants.

Integration of transcriptomic and metabolomic analysis unveils the response mechanism of sugar metabolism in Cyclocarya paliurus seedlings subjected to PEG-induced drought stress.

Cyclocarya paliurus (Batal.) Iljinskaja is a multiple function tree species used for functional food and valued timber production. Carbohydrates, especially water-soluble carbohydrates, play an important role in osmotic protection, signal transduction and carbon storage. Under the circumstance of global climate change the abiotic stress would restrict the development of C. paliurus plantation, whereas there is few knowledge on the regulatory mechanisms of sugar metabolism under drought stress in C. paliurus. To investigate the drought response of C. paliurus at molecular level, we conducted an integrated analysis of transcriptomic and metabolomic of C. paliurus at three PEG-induced drought stress levels (0%: control; 15%: moderate drought; 25%: severe drought) in short term. Both moderate and severe drought treatments activated the chemical defense with lowering relative water content, and enhancing the contents of soluble protein, proline and malondialdehyde in the leaves. Meanwhile, alterations in the expression of differentially expressed genes and carbohydrate metabolism profiles were observed among the treatments. Weighted gene co-expression network analysis (WGCNA) showed 3 key modules, 8 structural genes (such as genes encoding beta-fructofuranosidase (INV), sucrose synthase (SUS), raffinose synthase (RS)) and 14 regulatory transcription factors were closely linked to sugar metabolism. Our results provided the foundation to understand the response mechanism of sugar metabolism in C. paliurus under drought stress, and would drive progress in breeding of drought-tolerant varieties and plantation development of the species.

Genomic Insights into Adaptation to Karst Limestone and Incipient Speciation in East Asian Platycarya spp. (Juglandaceae).

When challenged by similar environmental conditions, phylogenetically distant taxa often independently evolve similar traits (convergent evolution). Meanwhile, adaptation to extreme habitats might lead to divergence between taxa that are otherwise closely related. These processes have long existed in the conceptual sphere, yet molecular evidence, especially for woody perennials, is scarce. The karst endemic Platycarya longipes, and its only congeneric species, P. strobilacea, which is widely distributed in the mountains in East Asia, provide an ideal model for examining the molecular basis of both convergent evolution and speciation. Using chromosome-level genome assemblies of both species, and whole genome resequencing data from 207 individuals spanning their entire distribution range, we demonstrate that P. longipes and P. strobilacea form two species-specific clades, which diverged around 2.09 million years ago. We find an excess of genomic regions exhibiting extreme interspecific differentiation, potentially due to long-term selection in P. longipes, likely contributing to the incipient speciation of the genus Platycarya. Interestingly, our results unveil underlying karst adaptation in both copies of the calcium influx channel gene TPC1 in P. longipes. TPC1 has previously been identified as a selective target in certain karst-endemic herbs, indicating a convergent adaptation to high calcium stress among karst-endemic species. Our study reveals the genic convergence of TPC1 among karst endemics, and the driving forces underneath the incipient speciation of the two Platycarya lineages.

Genomic Insights into Adaptation to Karst Limestone and Incipient Speciation in East Asian Platycarya spp. (Juglandaceae).

When challenged by similar environmental conditions, phylogenetically distant taxa often independently evolve similar traits (convergent evolution). Meanwhile, adaptation to extreme habitats might lead to divergence between taxa that are otherwise closely related. These processes have long existed in the conceptual sphere, yet molecular evidence, especially for woody perennials, is scarce. The karst endemic Platycarya longipes and its only congeneric species, Platycarya strobilacea, which is widely distributed in the mountains in East Asia, provide an ideal model for examining the molecular basis of both convergent evolution and speciation. Using chromosome-level genome assemblies of both species, and whole-genome resequencing data from 207 individuals spanning their entire distribution range, we demonstrate that P. longipes and P. strobilacea form two species-specific clades, which diverged around 2.09 million years ago. We find an excess of genomic regions exhibiting extreme interspecific differentiation, potentially due to long-term selection in P. longipes, likely contributing to the incipient speciation of the genus Platycarya. Interestingly, our results unveil underlying karst adaptation in both copies of the calcium influx channel gene TPC1 in P. longipes. TPC1 has previously been identified as a selective target in certain karst-endemic herbs, indicating a convergent adaptation to high calcium stress among karst-endemic species. Our study reveals the genic convergence of TPC1 among karst endemics and the driving forces underneath the incipient speciation of the two Platycarya lineages.