Comparative transcriptome provides new insights to the molecular regulation of olive trees to chilling stress.

Olive (Olea europaea. L), an economically important oil-producing crop, is sensitive to low temperature, which severely limits its productivity and geographical distribution. However, the underlying mechanism of cold tolerance in olive remains elusive. In this study, a chilling experiment (4 °C) on the living saplings of two olive cultivars revealed that O. europaea cv. Arbequina showed stronger cold tolerance with greater photosynthetic activity compared to O. europaea cv. Leccino. Transcriptome analyses revealed that early light-inducible protein 1 (ELIP1), the main regulator for chlorophyll synthesis, is dramatically induced to protect the photosynthesis at low temperatures. Furthermore, weighted gene co-expression network analysis (WGCNA), yeast one-hybrid (Y1H), and luciferase (LUC) assays demonstrated that transcription factor bHLH66 serves as an important regulator of ELIP1 transcription by binding to the G-box motif in the promoter. Taken together, our research revealed a novel transcriptional module consisting of bHLH66- ELIP1 in the adaptation of olive trees to cold stress.

An alternative 3' splice site of PeuHKT1;3 improves the response to salt stress through enhancing affinity to K+ in Populus.

Alternative splicing (AS) serves as a crucial post-transcriptional regulator in plants that contributes to the resistance to salt stress. However, the underlying mechanism is largely unknown. In this research, we identified an important AS transcript in Populus euphratica, PeuHKT1:3a, generated by alternative 3' splice site splicing mode that resulted in the removal of 252 bases at the 5' end of the first exon in PeuHKT1:3. Protein sequence comparison showed that the site of AS occurred in PeuHKT1:3 is located at a crucial Ser residue within the first pore-loop domain, which leads to inefficient K+ transport in HKT I-type transporters. Expressing PeuHKT1;3a in an axt3 mutant yeast strain can effectively compensate for the lack of intracellular K+, whereas the expression of PeuHKT1;3 cannot yield the effect. Furthermore, in transgenic Arabidopsis and poplar plants, it was observed that lines expressing PeuHKT1;3a exhibited greater salt tolerance compared to those expressing the PeuHKT1;3 strain. Analysis of ion content and flux demonstrated that the transgenic PeuHKT1;3a line exhibited significantly higher K+ content compared to the PeuHKT1;3 line, while there was no significant difference in Na+ content. In conclusion, our findings revealed that AS can give rise to novel variants of HKT I-type proteins in P. euphratica with modified K+ selectivity to keep a higher K+/Na+ ratio to enhanced salt tolerance.

Evolution of the thermostability of actin-depolymerizing factors enhances the adaptation of pollen germination to high temperature.

Double fertilization in many flowering plants (angiosperms) often occurs during the hot summer season, but the mechanisms that enable angiosperms to adapt specifically to high temperatures are largely unknown. The actin cytoskeleton is essential for pollen germination and the polarized growth of pollen tubes, yet how this process responds to high temperatures remains unclear. Here, we reveal that the high thermal stability of 11 Arabidopsis (Arabidopsis thaliana) actin-depolymerizing factors (ADFs) is significantly different: ADFs that specifically accumulate in tip-growing cells (pollen and root hairs) exhibit high thermal stability. Through ancestral protein reconstruction, we found that subclass II ADFs (expressed specifically in pollen) have undergone a dynamic wave-like evolution of the retention, loss, and regeneration of thermostable sites. Additionally, the sites of AtADF7 with high thermal stability are conserved in ADFs specific to angiosperm pollen. Moreover, the high thermal stability of ADFs is required to regulate actin dynamics and turnover at high temperatures to promote pollen germination. Collectively, these findings suggest strategies for the adaptation of sexual reproduction to high temperature in angiosperms at the cell biology level.

Whole-genome resequencing and transcriptome analyses of four generation mutants to reveal spur-type and skin-color related genes in apple (Malus domestica Borkh. Cv. Red delicious).

BACKGROUND: Bud sport is a kind of somatic mutation that usually occurred in apple. 'Red Delicious' is considered to be a special plant material of bud sport, whereas the genetic basis of plant mutants is still unknown. In this study, we used whole-genome resequencing and transcriptome sequencing to identify genes related to spur-type and skin-color in the 'Red Delicious' (G0) and its four generation mutants including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee Spur' (G4). RESULTS: The number of single nucleotide polymorphisms (SNPs), insertions and deletions (InDels) and structural variations (SVs) were decreased in four generation mutants compared to G0, and the number of unique SNPs and InDels were over 9-fold and 4-fold higher in G1 versus (vs.) G2 and G2 vs. G3, respectively. Chromosomes 2, 5, 11 and 15 carried the most SNPs, InDels and SVs, while chromosomes 1 and 6 carried the least. Meanwhile, we identified 4,356 variation genes by whole-genome resequencing and transcriptome, and obtained 13 and 16 differentially expressed genes (DEGs) related to spur-type and skin-color by gene expression levels. Among them, DELLA and 4CL7 were the potential genes that regulate the difference of spur-type and skin-color characters, respectively. CONCLUSIONS: Our study identified potential genes associated with spur-type and skin-color differences in 'Red Delicious' and its four generation mutants, which provides a theoretical foundation for the mechanism of the apple bud sport.

Multi-omics data provide insight into the adaptation of the glasshouse plant Rheum nobile to the alpine subnival zone.

Subnival glasshouse plants provide a text-book example of high-altitude adaptation with reproductive organs enclosed in specialized semi-translucent bracts, monocarpic reproduction and continuous survival under stress. Here, we present genomic, transcriptomic and metabolomic analyses for one such plant, the Noble rhubarb (Rheum nobile). Comparative genomic analyses show that an expanded number of genes and retained genes from two recent whole-genome duplication events are both relevant to subnival adaptation of this species. Most photosynthesis genes are downregulated within bracts compared to within leaves, and indeed bracts exhibit a sharp reduction in photosynthetic pigments, indicating that the bracts no longer perform photosynthesis. Contrastingly, genes related to flavonol synthesis are upregulated, providing enhanced defense against UV irradiation damage. Additionally, anatomically abnormal mesophyll combined with the downregulation of genes related to mesophyll differentiation in bracts illustrates the innovation and specification of the glass-like bracts. We further detect substantial accumulation of antifreeze proteins (e.g. AFPs, LEAs) and various metabolites (e.g. Proline, Protective sugars, procyanidins) in over-wintering roots. These findings provide new insights into subnival adaptation and the evolution of glasshouse alpine plants.

A chromosome-scale Rhubarb (Rheum tanguticum) genome assembly provides insights into the evolution of anthraquinone biosynthesis.

Rhubarb is the collective name for various perennial plants from the genus Rheum L. and the Polygonaceae family. They are one of the most ancient, commonly used, and important herbs in traditional Chinese medicine. Rhubarb is a major source of anthraquinones, but how they are synthesized remains largely unknown. Here, we generate a genome sequence assembly of one important medicinal rhubarb R. tanguticum at the chromosome level, with 2.76 Gb assembled into 11 chromosomes. The genome is shaped by two recent whole-genome duplication events and recent bursts of retrotransposons. Metabolic analyses show that the major anthraquinones are mainly synthesized in its roots. Transcriptomic analysis reveals a co-expression module with a high correlation to anthraquinone biosynthesis that includes key chalcone synthase genes. One CHS, four CYP450 and two BGL genes involved in secondary metabolism show significantly upregulated expression levels in roots compared with other tissues and clustered in the co-expression module, which implies that they may also act as candidate genes for anthraquinone biosynthesis. This study provides valuable insights into the genetic bases of anthraquinone biosynthesis that will facilitate improved breeding practices and agronomic properties for rhubarb in the future.

LncRNA expression analysis by comparative transcriptomics among closely related poplars and their regulatory roles in response to salt stress.

Long noncoding ribonucleic acids (lncRNAs) play crucial roles in regulating key biological processes; however, our knowledge of lncRNAs' roles in plant adaptive evolution is still limited. Here, we determined the divergence of conserved lncRNAs in closely related poplar species that were either tolerant or sensitive to salt stress by comparative transcriptome analysis. Among the 34,363 identified lncRNAs, ~3% were shared among poplar species with conserved sequences but diversified in their function, copy number, originating genomic region and expression patterns. Further cluster analysis revealed that the conserved lncRNAs showed more similar expression patterns within salt-tolerant poplars (Populus euphratica and P. pruinosa) than between salt-tolerant and salt-sensitive poplars. Among these lncRNAs, the antisense lncRNA lncERF024 was induced by salt and the differentiated expression between salt-sensitive and salt-tolerant poplars. The overexpression of lncERF024 in P. alba var. pyramidalis enhanced poplar tolerance to salt stress. Furthermore, RNA pull-down and RNA-seq analysis showed that numerous candidate genes or proteins associated with stress response and photosynthesis might be involved in salt resistance in PeulncERF024-OE poplars. Altogether, our study provided a novel insight into how the diversification of lncRNA expression contributes to plant adaptation traits and showed that lncERF024 may be involved in the regulation of both gene expression and protein function conferring salt tolerance in Populus.

The C2 H2 -type zinc finger transcription factor OSIC1 positively regulates stomatal closure under osmotic stress in poplar.

Salt and drought impair plant osmotic homeostasis and greatly limit plant growth and development. Plants decrease stomatal aperture to reduce water loss and maintain osmotic homeostasis, leading to improved stress tolerance. Herein, we identified the C2 H2 transcription factor gene OSMOTIC STRESS INDUCED C2 H2 1 (OSIC1) from Populus alba var. pyramidalis to be induced by salt, drought, polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA). Overexpression of OSIC1 conferred transgenic poplar more tolerance to high salinity, drought and PEG6000 treatment by reducing stomatal aperture, while its mutant generated by the CRISPR/Cas9 system showed the opposite phenotype. Furthermore, OSIC1 directly up-regulates PalCuAOζ in vitro and in vivo, encoding a copper-containing polyamine oxidase, to enhance H2 O2 accumulation in guard cells and thus modulates stomatal closure when stresses occur. Additionally, ABA-, drought- and salt-induced PalMPK3 phosphorylates OSIC1 to increase its transcriptional activity to PalCuAOζ. This regulation of OSIC1 at the transcriptional and protein levels guarantees rapid stomatal closure when poplar responds to osmotic stress. Our results revealed a novel transcriptional regulatory mechanism of H2 O2 production in guard cells mediated by the OSIC1-PalCuAOζ module. These findings deepen our understanding of how perennial woody plants, like poplar, respond to osmotic stress caused by salt and drought and provide potential targets for breeding.

Somatic mutations during rapid clonal domestication of Populus alba var. pyramidalis.

For many clonally propagated species, the accumulation of somatic mutations is the principal driver of declines in yield and quality. However, somatic mutations may also promote genetic diversification. Thus, elucidating somatic mutation rates and patterns is important to understand the genetic basis undergirding the emergence of commercially valuable traits and developmental processes. In this study, we studied the effect of short-time clonal domestication of Populus alba var. pyramidalis, a species that has been propagated by cutting for the last 67 years. We found that: (1) the somatic mutation rate for P. alba var. pyramidalis is 9.24 × 10-9, which is higher than rates observed in related species; (2) there were more mutations near heterozygous regions, and a larger proportion of CpG and CHG sites were associated with somatic mutations, which may be related to the blocking of DNA repair by methylation; and (3) deleterious mutations were not shared by multiple individuals, and all occurred in heterozygous states, demonstrating the strong selective pressures that act against deleterious mutations. Taken together, the results of our study provide a global view of somatic mutation that will aid efforts to understand the genetic basis of commercially valuable traits and to improve clonally breeding species.

Effects of biodegradable film mulching on bacterial diversity in soils.

The spread of biodegradable plastic films (BDFs) not only increases grain yield but also reduces environmental pollution from plastic film to a large extent. Soil microbes are considered to be involved in biodegradation processes. However, the study of microbe diversity in soil mulched with biodegradable plastic film remains limited. Here, we compared the diversity of microbes between soils with biodegradable film and nonbiodegradable film (NBDF) mulch. The results showed that BDFs affected total C, P and NH4+-N, especially organism C content, as well as microbe species richness (ACE; Chao1) and diversity (Simpson index; Shannon index). In terms of dominant phyla and genera, BDFs and NBDF can influence the abundance of disparate species. Furthermore, BDFs could also contribute to improving the richness of the important functional bacterial groups in soil, e.g., Pedomicrobium and Comamonas, both of which are involved in the degradation of plastic residues in soil. Finally, we found that BDFs improved the transformation of nitrogen by significantly increasing the abundances of Nitrobacter and Nitrospira. Our results highlight the impact of BDF mulch on the abundance of functional bacteria in the soil.

A CLE-BAM-CIK signalling module controls root protophloem differentiation in Arabidopsis.

Exogenous application of CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE) peptides suppresses protophloem differentiation and leads to the consumption of the proximal root meristem. However, the exact CLE peptides and the corresponding receptor complex regulating protophloem differentiation have not yet been clarified. Through expression pattern and phylogenetic analyses, CLE25/26/45 were identified as candidate peptides. Further genetic analyses, physiological assays and specific protophloem marker observations indicated that CLE25/26/45, BARELY ANY MERISTEM1/3 (BAM1/3) and CLV3 INSENSITIVE KINASEs (CIKs) are involved in regulating protophloem differentiation. The cle25 26 45 and cik2 3 4 5 6 mutation can greatly rescue the root defects of brevis radix (brx) and octopus (ops) mutants. The protophloem differentiation and proximal root meristem consumption of clv1 bam1 3 and cik2 3 4 5 6 were insensitive to CLE25/26/45 treatments. cle25 26 45, clv1 bam1 3 and cik2 3 4 5 6 displayed similar premature protophloem. In addition, CLE25/26/45 induced the interactions between BAMs and CIKs in vivo. Furthermore, CLE25/26/45 enhanced the phosphorylation levels of CIKs, which were greatly impaired in clv1 bam1 3 mutant. Our work clarifies that the CLE25/26/45-BAM1/3-CIK2/3/4/5/6 signalling module genetically acts downstream of BRX and OPS to suppress protophloem differentiation.

Evolution of RGF/GLV/CLEL Peptide Hormones and Their Roles in Land Plant Growth and Regulation.

Rooting is a key innovation during plant terrestrialization. RGFs/GLVs/CLELs are a family of secreted peptides, playing key roles in root stem cell niche maintenance and pattern formation. The origin of this peptide family is not well characterized. RGFs and their receptor genes, RGIs, were investigated comprehensively using phylogenetic and genetic analyses. We identified 203 RGF genes from 24 plant species, representing a variety of land plant lineages. We found that the RGF genes originate from land plants and expand via multiple duplication events. The lineage-specific RGF duplicates are retained due to their regulatory divergence, while a majority of RGFs experienced strong purifying selection in most land plants. Functional analysis indicated that RGFs and their receptor genes, RGIs, isolated from liverwort, tomato, and maize possess similar biological functions with their counterparts from Arabidopsis in root development. RGFs and RGIs are likely coevolved in land plants. Our studies shed light on the origin and functional conservation of this important peptide family in plant root development.

Genome evolution of the psammophyte Pugionium for desert adaptation and further speciation.

Deserts exert strong selection pressures on plants, but the underlying genomic drivers of ecological adaptation and subsequent speciation remain largely unknown. Here, we generated de novo genome assemblies and conducted population genomic analyses of the psammophytic genus Pugionium (Brassicaceae). Our results indicated that this bispecific genus had undergone an allopolyploid event, and the two parental genomes were derived from two ancestral lineages with different chromosome numbers and structures. The postpolyploid expansion of gene families related to abiotic stress responses and lignin biosynthesis facilitated environmental adaptations of the genus to desert habitats. Population genomic analyses of both species further revealed their recent divergence with continuous gene flow, and the most divergent regions were found to be centered on three highly structurally reshuffled chromosomes. Genes under selection in these regions, which were mainly located in one of the two subgenomes, contributed greatly to the interspecific divergence in microhabitat adaptation.

A gene that underwent adaptive evolution, LAC2 (LACCASE), in Populus euphratica improves drought tolerance by improving water transport capacity.

Drought severely limits plant development and growth; accordingly, plants have evolved strategies to prevent water loss and adapt to water deficit conditions. However, experimental cases that corroborate these evolutionary processes are limited. The LACCASEs (LACs) family is involved in various plant development and growth processes. Here, we performed an evolutionary analysis of LACs from Populus euphratica and characterized the functions of LACs in Arabidopsis and poplar. The results showed that in PeuLACs, multiple gene duplications led to apparent functional redundancy as the result of various selective pressures. Among them, PeuLAC2 underwent strong positive selection. Heterologous expression analyses showed that the overexpression of PeuLAC2 alters the xylem structure of plants, including thickening the secondary cell wall (SCW) and increasing the fiber cell length and stem tensile strength. Altogether, these changes improve the water transport capacity of plants. The analysis of the physiological experimental results showed that PeuLAC2-OE lines exhibited a stronger antioxidant response and greater drought tolerance than WT. Three genes screened by transcriptome analysis, NAC025, BG1, and UGT, that are associated with SCW synthesis and drought stress were all upregulated in the PeuLAC2-OE lines, implying that the overexpression of PeuLAC2 thickened the SCW, improved the water transport capacity of the plant, and further enhanced its drought tolerance. Our study highlights that genes that have undergone adaptive evolution may participate in the development of adaptive traits in P. euphratica and that PeuLAC2 could be a candidate gene for molecular genetic breeding in trees.

Analysis of Alternative Splicing and Alternative Polyadenylation in Populus alba var. pyramidalis by Single-Molecular Long-Read Sequencing.

Poplars are worldwidely cultivated with ecologically and economically important value. Populus alba var. pyramidalis (= P. bolleana) is a main tree of the farmland shelter-belt system in the arid region of Northwest China due to its rapid growth, erect stems, and high biomass production. However, the full-length messenger RNA (mRNA) sequences and complete structure of P. alba var. pyramidalis remain unclear. In this study, using single-molecular real-time (SMRT) and next-generation high-throughput sequencing (NGS) platform, we sequenced transcripts from leaf, root, xylem, and phloem of P. alba var. pyramidalis, to obtain the full-length mRNA transcripts and annotate the complete structure. In total, 86,327 mapped full-length non-chimeric (FLNC) reads were identified, with 705 previously unannotated loci and 3,410 long noncoding RNAs (lncRNAs) and 174 fusion genes found. Alternative spicing (AS) events were detected in 7,536 genes, of which 4,652 genes had multiple AS events. A total of 10,213 alternative polyadenylation (APA) sites were identified, with two or more APA sites observed in 2,212 genes. Our transcriptome data provided the full-length sequences and gene isoforms of transcripts for P. alba var. pyramidalis, which will be helpful in improving our understanding for the genome annotation and gene structures of P. alba var. pyramidalis.

Improved genome assembly provides new insights into genome evolution in a desert poplar (Populus euphratica).

Populus euphratica is well adapted to extreme desert environments and is an important model species for elucidating the mechanisms of abiotic stress resistance in trees. The current assembly of P. euphratica genome is highly fragmented with many gaps and errors, thereby impeding downstream applications. Here, we report an improved chromosome-level reference genome of P. euphratica (v2.0) using single-molecule sequencing and chromosome conformation capture (Hi-C) technologies. Relative to the previous reference genome, our assembly represents a nearly 60-fold improvement in contiguity, with a scaffold N50 size of 28.59 Mb. Using this genome, we have found that extensive expansion of Gypsy elements in P. euphratica led to its rapid increase in genome size compared to any other Salicaceae species studied to date, and potentially contributed to adaptive divergence driven by insertions near genes involved in stress tolerance. We also detected a wide range of unique structural rearrangements in P. euphratica, including 2,549 translocations, 454 inversions, 121 tandem and 14 segmental duplications. Several key genes likely to be involved in tolerance to abiotic stress were identified within these regions. This high-quality genome represents a valuable resource for poplar breeding and genetic improvement in the future, as well as comparative genomic analysis with other Salicaceae species.

A Novel Gene Coding γ-Aminobutyric Acid Transporter May Improve the Tolerance of Populus euphratica to Adverse Environments.

Novel genes provide important genetic resource for organism innovation. However, the evidence from genetic experiment is limited. In plants, γ-aminobutyric acid (GABA) transporters (GATs) primarily transport GABA and further involve in plant growth, development, and response to various stresses. In this study, we have identified the GATs family in Populus species and characterized their functional evolution and divergence in a desert poplar species (Populus euphratica). We found that the GATs underwent genus-specific expansion via multiple whole-genome duplications in Populus species. The purifying selection were identified across those GATs evolution and divergence in poplar diversity, except two paralogous PeuGAT2 and PeuGAT3 from P. euphratica. The both genes arose from a tandem duplication event about 49 million years ago and have experienced strong positive selection, suggesting that the divergence in PeuGAT3 protein function/structure might define gene function better than in expression pattern. Both PeuGAT genes were functionally characterized in Arabidopsis and poplar, respectively. The overexpression of PeuGAT3 increased the thickness of xylem cells walls in both Arabidopsis and poplar and enhanced the lignin content of xylem tissues and the proline accumulation in poplar leaves, all of which may improve tolerance of salt/drought stress in desert poplars. Our findings help clarify the genetic mechanisms underpinning high tolerance in desert poplars and suggest that PeuGAT3 could be an attractive candidate gene for engineering trees with improved brown-rot resistance.

Genome-Wide Identification of Long Noncoding RNAs and Their Responses to Salt Stress in Two Closely Related Poplars.

Long noncoding RNAs (lncRNAs) are involved in various biological regulatory processes, but their roles in plants resistance to salt stress remain largely unknown. To systematically explore the characteristics of lncRNAs and their roles in plant salt responses, we conducted strand-specific RNA-sequencing of four tissue types with salt treatments in two closely related poplars (Populus euphratica and Populus alba var. pyramidalis), and a total of 10,646 and 10,531 lncRNAs were identified, respectively. These lncRNAs showed significantly lower values in terms of length, expression, and expression correction than with mRNA. We further found that about 40% and 60% of these identified lncRNAs responded to salt stress with tissue-specific expression patterns across the two poplars. Furthermore, lncRNAs showed weak evolutionary conservation in sequences and exhibited diverse regulatory styles; in particular, tissue- and species-specific responses to salt stress varied greatly in two poplars, for example, 322 lncRNAs were found highly expressed in P. euphratica but not in P. alba var. pyramidalis and 3,425 lncRNAs were identified to be species-specific in P. euphratica in response to salt stress. Moreover, tissue-specific expression of lncRNAs in two poplars were identified with predicted target genes included Aux/IAA, NAC, MYB, involved in regulating plant growth and the plant stress response. Taken together, the systematic analysis of lncRNAs between sister species enhances our understanding of the characteristics of lncRNAs and their roles in plant growth and salt response.

Transcriptomic Analysis of Seed Germination Under Salt Stress in Two Desert Sister Species (Populus euphratica and P. pruinosa).

As a major abiotic stress, soil salinity limits seed germination and plant growth, development and production. Seed germination is highly related not only to the seedlings survival rate but also subsequent vegetative growth. Populus euphratica and P. pruinosa are closely related species that show a distinguished adaptability to salinity stress. In this study, we performed an integrative transcriptome analyses of three seed germination phases from P. euphratica and P. pruinosa under salt stress. A two-dimensional data set of this study provides a comprehensive view of the dynamic biochemical processes that underpin seed germination and salt tolerance. Our analysis identified 12831 differentially expressed genes (DEGs) for seed germination processes and 8071 DEGs for salt tolerance in the two species. Furthermore, we identified the expression profiles and main pathways in each growth phase. For seed germination, a large number of DEGs, including those involved in energy production and hormonal regulation pathways, were transiently and specifically induced in the late phase. In the comparison of salt tolerance between the two species, the flavonoid and brassinosteroid pathways were significantly enriched. More specifically, in the flavonoid pathway, FLS and F3'5'H exhibited significant differential expression. In the brassinosteroid pathway, the expression levels of DWF4, BR6OX2 and ROT3 were notably higher in P. pruinosa than in P. euphratica. Our results describe transcript dynamics and highlight secondary metabolite pathways involved in the response to salt stress during the seed germination of two desert poplars.

Interspecific delimitation and relationships among four Ostrya species based on plastomes.

BACKGROUND: The genus Ostrya (Betulaceae) contains eight species and four of them are distributed in China. However, studies based on limited informative sites of several chloroplast markers failed to resolve interspecific delimitation and relationships among the four Chinese species. In this study, we aimed to use the whole chloroplast genomes to address these two issues. RESULTS: We assembled and annotated 33 complete chloroplast genomes (plastomes) of the four Chinese species, representing 17 populations across most of their geographical distributions. Each species contained samples of several individuals that cover most of geographic distributions of the species. All plastomes are highly conserved in genome structure and gene order, with a total length of 158-159 kb and 122 genes. Phylogenetic analyses of whole plastomes, non-coding regions and protein-coding genes produced almost the same topological relationships. In contrast to the well-delimitated species boundary inferred from the nuclear ITS sequence variations, three of the four species are non-monophyletic in the plastome trees, which is consistent with previous studies based on a few chloroplast markers. CONCLUSIONS: The high incongruence between the ITS and plastome trees may suggest the widespread occurrences of hybrid introgression and incomplete lineage sorting during the divergence of these species. In addition, the plastomes with more informative sites compared with a few chloroplast markers still failed to resolve the phylogenetic relationships of the four species, and further studies involving population genomic data may be needed to better understand their evolutionary histories.