Subgenome-aware analyses reveal the genomic consequences of ancient allopolyploid hybridizations throughout the cotton family.

Malvaceae comprise some 4,225 species in 243 genera and nine subfamilies and include economically important species, such as cacao, cotton, durian, and jute, with cotton an important model system for studying the domestication of polyploids. Here, we use chromosome-level genome assemblies from representatives of five or six subfamilies (depending on the placement of Ochroma) to differentiate coexisting subgenomes and their evolution during the family's deep history. The results reveal that the allohexaploid Helicteroideae partially derive from an allotetraploid Sterculioideae and also form a component of the allodecaploid Bombacoideae and Malvoideae. The ancestral Malvaceae karyotype consists of 11 protochromosomes. Four subfamilies share a unique reciprocal chromosome translocation, and two other subfamilies share a chromosome fusion. DNA alignments of single-copy nuclear genes do not yield the same relationships as inferred from chromosome structural traits, probably because of genes originating from different ancestral subgenomes. These results illustrate how chromosome-structural data can unravel the evolutionary history of groups with ancient hybrid genomes.

Scattered differentiation of unlinked loci across the genome underlines ecological divergence of the selfing grass Brachypodium stacei.

Ecological divergence without geographic isolation, as an early speciation process that may lead finally to reproductive isolation through natural selection, remains a captivating topic in evolutionary biology. However, the pattern of genetic divergence underlying this process across the genome may vary between species and mating systems. Here, we present evidence that Brachypodium stacei, an annual and highly selfing grass model species, has undergone sympatric ecological divergence without geographic isolation. Genomic, transcriptomic, and metabolomic analyses together with lab experiments mimicking the two opposite environmental conditions suggest that diploid B. stacei populations have diverged sympatrically in two slopes characterized by distinct biomes at Evolution Canyon I (ECI), Mount Carmel, Israel. Despite ongoing gene flow, primarily facilitated by seed dispersal, the level of gene flow has progressively decreased over time. This local adaptation involves the scattered divergence of many unlinked loci across the total genome that include both coding genes and noncoding regions. Additionally, we have identified significant differential expressions of genes related to the ABA signaling pathway and contrasting metabolome composition between the arid- vs. forest-adapted B. stacei populations in ECI. These results suggest that multiple small loci involved in environmental responses act additively to account for ecological adaptations by this selfing species in contrasting environments.

ERGIC2 and ERGIC3 regulate the ER-to-Golgi transport of gap junction proteins in metazoans.

The extremely dynamic life cycle of gap junction connections requires highly efficient intracellular trafficking system especially designed for gap junction proteins, but the underlying mechanisms are largely unknown. Here, we identified that the COPII-associated proteins ERGIC2 (ER-Golgi intermediate compartment) and ERGIC3 are specifically required for the efficient intracellular transport of gap junction proteins in both Caenorhabditis elegans and mice. In the absence of Ergic2 or Ergic3, gap junction proteins accumulate in the ER and Golgi apparatus and the size of endogenous gap junction plaques is reduced. Knocking out the Ergic2 or Ergic3 in mice results in heart enlargement and cardiac malfunction accompanied by reduced number and size of connexin 43 (Cx43) gap junctions. Invertebrates' gap junction protein innexins share no sequence similarity with vertebrates' connexins. However, ERGIC2 and ERGIC3 could bind to gap junction proteins in both worms and mice. Characterization of the highly specialized roles of ERGIC2 and ERGIC3 in metazoans reveals how the early secretory pathway could be adapted to facilitate the efficient transport for gap junction proteins in vivo.

Subgenomic Stability of Progenitor Genomes During Repeated Allotetraploid Origins of the Same Grass Brachypodium hybridum.

Both homeologous exchanges and homeologous expression bias are generally found in most allopolyploid species. Whether homeologous exchanges and homeologous expression bias differ between repeated allopolyploid speciation events from the same progenitor species remains unknown. Here, we detected a third independent and recent allotetraploid origin for the model grass Brachypodium hybridum. Our homeologous exchange with replacement analyses indicated the absence of significant homeologous exchanges in any of the three types of wild allotetraploids, supporting the integrity of their progenitor subgenomes and the immediate creation of the amphidiploids. Further homeologous expression bias tests did not uncover significant subgenomic dominance in different tissues and conditions of the allotetraploids. This suggests a balanced expression of homeologs under similar or dissimilar ecological conditions in their natural habitats. We observed that the density of transposons around genes was not associated with the initial establishment of subgenome dominance; rather, this feature is inherited from the progenitor genome. We found that drought response genes were highly induced in the two subgenomes, likely contributing to the local adaptation of this species to arid habitats in the third allotetraploid event. These findings provide evidence for the consistency of subgenomic stability of parental genomes across multiple allopolyploidization events that led to the same species at different periods. Our study emphasizes the importance of selecting closely related progenitor species genomes to accurately assess homeologous exchange with replacement in allopolyploids, thereby avoiding the detection of false homeologous exchanges when using less related progenitor species genomes.

Integrated metagenomic and metabolomic analysis reveals distinct gut-microbiome-derived phenotypes in early-onset colorectal cancer.

OBJECTIVE: The incidence of early-onset colorectal cancer (EO-CRC) is steadily increasing. Here, we aimed to characterise the interactions between gut microbiome, metabolites and microbial enzymes in EO-CRC patients and evaluate their potential as non-invasive biomarkers for EO-CRC. DESIGN: We performed metagenomic and metabolomic analyses, identified multiomics markers and constructed CRC classifiers for the discovery cohort with 130 late-onset CRC (LO-CRC), 114 EO-CRC subjects and age-matched healthy controls (97 LO-Control and 100 EO-Control). An independent cohort of 38 LO-CRC, 24 EO-CRC, 22 LO-Controls and 24 EO-Controls was analysed to validate the results. RESULTS: Compared with controls, reduced alpha-diversity was apparent in both, LO-CRC and EO-CRC subjects. Although common variations existed, integrative analyses identified distinct microbiome-metabolome associations in LO-CRC and EO-CRC. Fusobacterium nucleatum enrichment and short-chain fatty acid depletion, including reduced microbial GABA biosynthesis and a shift in acetate/acetaldehyde metabolism towards acetyl-CoA production characterises LO-CRC. In comparison, multiomics signatures of EO-CRC tended to be associated with enriched Flavonifractor plauti and increased tryptophan, bile acid and choline metabolism. Notably, elevated red meat intake-related species, choline metabolites and KEGG orthology (KO) pldB and cbh gene axis may be potential tumour stimulators in EO-CRC. The predictive model based on metagenomic, metabolomic and KO gene markers achieved a powerful classification performance for distinguishing EO-CRC from controls. CONCLUSION: Our large-sample multiomics data suggest that altered microbiome-metabolome interplay helps explain the pathogenesis of EO-CRC and LO-CRC. The potential of microbiome-derived biomarkers as promising non-invasive tools could be used for the accurate detection and distinction of individuals with EO-CRC.

Function and mechanism of MCM8 in the development and progression of colorectal cancer.

Colorectal cancer (CRC) has become a global health problem which has almost highest morbidity and mortality in all types of cancers. This study aimed to uncover the biological functions and underlying mechanism of MCM8 in the development and progression of CRC. The expression level of MCM8 was found to be upregulated in CRC tissues and significantly associated with tumor grade and patients' survival. Knocking down MCM8 expression in CRC cells could restrain cell growth and cell motility while promoting cell apoptosis in vitro, as well as inhibit tumor growth in xenograft mice model. Based on the RNA screening performing on CRC cells with or without MCM8 knockdown and the following IPA analysis, CHSY1 was identified as a potential target of MCM8 in CRC, whose expression was also found to be higher in tumor tissues than in normal tissues. Moreover, it was demonstrated that MCM8 may regulate the expression of CHSY1 through affecting its NEDD4-mediated ubiquitination, both of which synergistically execute tumor promotion effects on CRC. In conclusion, the outcomes of our study showed the first evidence that MCM8 act as a tumor promotor in CRC, and may be a promising therapeutic target of CRC treatment.

Multi-omics reveal differentiation and maintenance of dimorphic flowers in an alpine plant on the Qinghai-Tibet Plateau.

Dimorphic flowers growing on a single individual plant play a critical role in extreme adaption and reproductive assurance in plants and have high ecological and evolutionary significance. However, the omics bases underlying such a differentiation and maintenance remain largely unknown. We aimed to investigate this through genomic, transcriptome and metabolomic analyses of dimorphic flowers in an alpine biennial, Sinoswertia tetraptera (Gentianaceae). A high-quality chromosome-level genome sequence (903 Mb) was first assembled for S. tetraptera with 31,359 protein-coding genes annotated. Two rounds of recent independent whole-genome duplication (WGD) were revealed. Numerous genes from the recent species-specific WGD were found to be differentially expressed in the two types of flowers, and this may have helped contribute to the origin of this innovative trait. The genes with contrasting expressions between flowers were related to biosynthesis of hormones, floral pigments (carotenoids and flavonoids) and iridoid compounds, which are involved in both flower development and colour. Metabolomic analyses similarly suggested differential concentrations of these chemicals in the two types of flowers. The expression interactions between multiple genes may together lead to contrasting morphology and chemical concentration and open versus closed pollination of the dimorphic flowers in this species for reproductive assurance.

Turfgrass-dependent mycotrophic change enhances soil deterioration in dry, cold and high-alkali environments.

AIMS: Soil quality is undergoing severe degradation under anthropogenic effects. Different methods of land management have been implemented for soil reclamation, such as turfing. Although widely accepted to improve soil quality, turfing in specific environments may also culminate in soil deterioration. We aim to know how turfing impacts soils by changing mycobiomes. METHODS AND RESULTS: The soil physicochemical properties and ITS metabarcoding were used to investigate mycobiome diversity and eco-function differences between the eudicot Dianthus plumarius and the monocot Poa pratensis in dry, cold, and high-alkali soil. The effects of plantation and the rhizosphere (e.g. root exudates) were tested. We showed that the change in soil mycobiomes in different planted bulk soils and rhizospheres could mainly be attributed to species turnover, with minor nestedness. Unexpectedly, the soil deteriorates more following turfing. The increasing saprotrophs in planted bulk soil were more marked in the monocot than in the eudicot, even the rhizosphere effect alleviated saprotrophic risks in the rhizosphere. CONCLUSIONS: Turfing deteriorates the health of high-alkali soil by reducing nitrification, and upshift the soil saprotrophs in a dry and cold environment.

Alleviating chronic ER stress by p38-Ire1-Xbp1 pathway and insulin-associated autophagy in C. elegans neurons.

ER stress occurs in many physiological and pathological conditions. However, how chronic ER stress is alleviated in specific cells in an intact organism is an outstanding question. Here, overexpressing the gap junction protein UNC-9 (Uncoordinated) in C. elegans neurons triggers the Ire1-Xbp1-mediated stress response in an age-dependent and cell-autonomous manner. The p38 MAPK PMK-3 regulates the chronic stress through IRE-1 phosphorylation. Overexpressing gap junction protein also activates autophagy. The insulin pathway functions through autophagy, but not the transcription of genes encoding ER chaperones, to counteract the p38-Ire1-Xbp1-mediated stress response. Together, these results reveal an intricate cellular regulatory network in response to chronic stress in a subset of cells in multicellular organism.

A high-quality Buxus austro-yunnanensis (Buxales) genome provides new insights into karyotype evolution in early eudicots.

BACKGROUND: Eudicots are the most diverse group of flowering plants that compromise five well-defined lineages: core eudicots, Ranunculales, Proteales, Trochodendrales, and Buxales. However, the phylogenetic relationships between these five lineages and their chromosomal evolutions remain unclear, and a lack of high-quality genome analyses for Buxales has hindered many efforts to address this knowledge gap. RESULTS: Here, we present a high-quality chromosome-level genome of Buxus austro-yunnanensis (Buxales). Our phylogenomic analyses revealed that Buxales and Trochodendrales are genetically similar and classified as sisters. Additionally, both are sisters to the core eudicots, while Ranunculales was found to be the first lineage to diverge from these groups. Incomplete lineage sorting and hybridization were identified as the main contributors to phylogenetic discordance (34.33%) between the lineages. In fact, B. austro-yunnanensis underwent only one whole-genome duplication event, and collinear gene phylogeny analyses suggested that separate independent polyploidizations occurred in the five eudicot lineages. Using representative genomes from these five lineages, we reconstructed the ancestral eudicot karyotype (AEK) and generated a nearly gapless karyotype projection for each eudicot species. Within core eudicots, we recovered one common chromosome fusion event in asterids and malvids, respectively. Further, we also found that the previously reported fused AEKs in Aquilegia (Ranunculales) and Vitis (core eudicots) have different fusion positions, which indicates that these two species have different karyotype evolution histories. CONCLUSIONS: Based on our phylogenomic and karyotype evolution analyses, we revealed the likely relationships and evolutionary histories of early eudicots. Ultimately, our study expands genomic resources for early-diverging eudicots.

Genome-scale angiosperm phylogenies based on nuclear, plastome, and mitochondrial datasets.

Angiosperms dominate the Earth's ecosystems and provide most of the basic necessities for human life. The major angiosperm clades comprise 64 orders, as recognized by the APG IV classification. However, the phylogenetic relationships of angiosperms remain unclear, as phylogenetic trees with different topologies have been reconstructed depending on the sequence datasets utilized, from targeted genes to transcriptomes. Here, we used currently available de novo genome data to reconstruct the phylogenies of 366 angiosperm species from 241 genera belonging to 97 families across 43 of the 64 orders based on orthologous genes from the nuclear, plastid, and mitochondrial genomes of the same species with compatible datasets. The phylogenetic relationships were largely consistent with previously constructed phylogenies based on sequence variations in each genome type. However, there were major inconsistencies in the phylogenetic relationships of the five Mesangiospermae lineages when different genomes were examined. We discuss ways to address these inconsistencies, which could ultimately lead to the reconstruction of a comprehensive angiosperm tree of life. The angiosperm phylogenies presented here provide a basic framework for further updates and comparisons. These phylogenies can also be used as guides to examine the evolutionary trajectories among the three genome types during lineage radiation.

Transcriptome analysis reveals multiple effects of nitrogen accumulation and metabolism in the roots, shoots, and leaves of potato (Solanum tuberosum L.).

BACKGROUND: Nitrogen (N) is a major element and fundamental constituent of grain yield. N fertilizer plays an essential role in the roots, shoots, and leaves of crop plants. Here, we obtained two N-sensitive potato cultivars. RESULTS: The plants were cultivated in the pots using N-deficient and N-sufficient conditions. Crop height, leaf chlorophyll content, dry matter, and N-accumulation significantly decreased under N-deficient conditions. Furthermore, we performed a comprehensive analysis of the phenotype and transcriptome, GO terms, and KEGG pathways. We used WGCNA of co-expressed genes, and 116 differentially expressed hub genes involved in photosynthesis, nitrogen metabolism, and secondary metabolites to generate 23 modules. Among those modules, six NRT gene families, four pigment genes, two auxin-related genes, and two energy-related genes were selected for qRT-PCR validation. CONCLUSIONS: Overall, our study demonstrates the co-expressed genes and potential pathways associated with N transport and accumulation in potato cultivars' roots, shoots, and leaves under N-deficient conditions. Therefore, this study provides new ideas to conduct further research on improving nitrogen use efficiency in potatoes.

Genomic divergence of Stellera chamaejasme through local selection across the Qinghai-Tibet plateau and northern China.

Understanding how populations diverge and new species arise is a central question in evolutionary biology. "Allopatric" divergence through geographical isolation is considered to be the commonest mechanism generating species biodiversity in mountain ecosystems. However, the underlying genomic dynamics, especially genomic islands of elevated divergence and genes that are highly diverged as a result of lineage-specific selection, remain poorly understood. Stellera chamaejasme has a wide geographical range across the Qinghai-Tibet Plateau and northern China, making it a good model with which to explore genomic divergence during speciation in mountain ecosystems. We assembled a high-quality, chromosome-level genome for this species and resequenced the genomes of 24 populations across its major distribution. Our population genomic analyses recovered four distinct genetic lineages corresponding to geographical distributions with contrasting environments. However, we revealed continuous gene flow during the historical divergences of these four lineages. Interlineage hybrids and plastome introgressions were frequently found in regions of contact, which further increased gene flow between two contacting lineages in the recent past. The elevated divergences were highly heterogeneous across the genome and selection of ancestral polymorphisims and divergence hitchhiking contributed greatly to the formation of genomic islands. The highly diverged and lineage-specific positively selected genes within and outside genomic islands were annotated to be mainly involved in local adaptation. Our results suggest that genomic divergence in S. chamaejasme is likely to have been triggered and maintained by local selection together with geographical isolation.

Physicochemical and Biotic Changes and the Phylogenetic Evenness of Microbial Community in Soil Subjected to Phytoreclamation.

Phytoreclamation is the intervention of plants to improve degraded soil quality, changing soil biotic and abiotic properties. Many studies have focused on microbial composition and bioactivity, but few explored the changes in phylogenetic assemblages of soil microbiota after phytoreclamation. This study compared microbiomes of bare land to those of planted soils and investigated how the rhizosphere environment affects microbial assemblages from monocot Poa pratensis and eudicot Dianthus plumarius plantings using 16S rRNA metabarcoding. The results showed that the biotic susceptibility of soil to the rhizosphere environment was higher than that of the abiotic. A noticeable change was in some soil physicochemical properties like Na, P, Zn, Cu, C, and sand-to-silt proportion before and after phytoreclamation, but not between the rhizosphere and bulk soil of plantings. Contrastingly, microbial composition and diversity were significantly affected by both turfing and rhizosphere effects and were more susceptible to differences in turfing or not than in planting species. In the turfgrass, the microbiome differences between plants were greater in the rhizosphere than in the surrounding bulk soil, indicating the proximal influence of root exudates. We also found that the main abiotic factors that influenced microbial composition were Na, Zn, NOx, N, and S; as for the phylogenetic assemblages, were by K levels and the increase of silt. Turfgrass decomposes soil aggregates and changes the physicochemical properties, thereby evens the phylogenetic clustering of the soil microbial community. We demonstrated that the deterministic process affects the microbial assemblage and acts as a selective agent of the soil microbiota in fundamental and realized niches. Phytoreclamation may lead to abiotic soil changes that reallocate resources to microbes. This could affect the phylogeny of the microbial assemblages and increase microbial richness.

A novel epithelial-mesenchymal transition molecular signature predicts the oncological outcomes in colorectal cancer.

Epithelial-mesenchymal transition (EMT), a biological process involving the transformation of epithelial cells into mesenchymal cells, promotes tumour initiation and metastasis. The aim of this study was to construct an EMT molecular signature for predicting colorectal cancer (CRC) prognosis and evaluate the efficacy of the model. The risk scoring system, constructed by log-rank test and multivariate Cox regression analysis according to EMT-related gene expression in CRC patients from TCGA database, demonstrated the highest correlation with prognosis compared with other parameters in CRC patients. The risk scores were significantly correlated with more lymph node metastasis, distal metastasis and advanced clinical stage of CRC. The model was further successfully validated in two independent external cohorts from GEO database. Furthermore, we developed a nomogram to integrate the EMT signature with the pathological stage of CRC, which was found to perform well in predicting the overall survival. Additionally, this risk scoring model was found to be associated with immune cell infiltration, implying a potential role of EMT involved in immunity regulation in tumour microenvironment. Taken together, our novel EMT molecular model may be useful in identifying high-risk patients who need an intensive follow-up and more aggressive therapy, finally contributing to more precise individualized therapeutic strategies.

Survival in the Tropics despite isolation, inbreeding and asexual reproduction: insights from the genome of the world's southernmost poplar (Populus ilicifolia).

Species are becoming extinct at unprecedented rates as a consequence of human activity. Hence it is important to understand the evolutionary dynamics of species with already small population sizes. Populus ilicifolia is a vulnerable poplar species that is isolated from other poplar species and is uniquely adapted to the Tropics. It has a very limited size, reproduces partly clonally and is therefore an excellent case study for conservation genomics. We present here the first annotated draft genome of P. ilicifolia, characterize genome-wide patterns of polymorphisms and compare those to other poplar species with larger natural ranges. P. ilicifolia experienced a more prolonged and severe decline of effective population size (Ne ) and signs of genetic erosion than any other poplar species with which it was compared. At present, the species has the lowest genome-wide genetic diversity, the highest abundance of long runs of homozygosity, high inbreeding levels as well as a high overall accumulation of deleterious variants. However, more effective purging of severely deleterious variants and adaptation to the Tropics may have contributed to its survival. Hence, in spite of its limited genetic variation, it is certainly worth pursuing the conservation efforts of this unique species.

Evolutionary origin of a tetraploid Allium species on the Qinghai-Tibet Plateau.

Extinct taxa may be detectable if they were ancestors to extant hybrid species, which retain their genetic signature. In this study, we combined phylogenomics, population genetics and fluorescence in situ hybridization (GISH and FISH) analyses to trace the origin of the alpine tetraploid Allium tetraploideum (2n = 4x = 32), one of the five known members in the subgenus Cyathophora. We found that A. tetraploideum was an obvious allotetrapoploid derived from ancestors including at least two closely related diploid species, A. farreri and A. cyathophorum, from which it differs by multiple ecological and genomic attributes. However, these two species cannot account for the full genome of A. tetraploideum, indicating that at least one extinct diploid is also involved in its ancestry. Furthermore, A. tetraploideum appears to have arisen via homoploid hybrid speciation (HHS) from two extinct allotetraploid parents, which derived in turn from the aforementioned diploids. Other modes of origin were possible, but all were even more complex and involved additional extinct ancestors. Our study together highlights how some polyploid species might have very complex origins, involving both HHS and polyploid speciation and also extinct ancestors.

Species delimitation and hybridization history of a hazel species complex.

BACKGROUND AND AIMS: Hybridization increases species adaptation and biodiversity but also obscures species boundaries. In this study, species delimitation and hybridization history were examined within one Chinese hazel species complex (Corylus chinensis-Corylus fargesii). Two species including four varieties have already been described for this complex, with overlapping distributions. METHODS: A total of 322 trees from 44 populations of these four varieties across their ranges were sampled for morphological and molecular analyses. Climatic datasets based on 108 geographical locations were used to evaluate their niche differentiations. Flowering phenology was also observed for two co-occurring species or varieties. KEY RESULTS: Four statistically different phenotypic clusters were revealed, but these clusters were highly inconsistent with the traditional taxonomic groups. All the clusters showed statistically distinct niches, with complete or partial geographical isolation. Only two clusters displayed a distributional overlap, but they had distinct flowering phenologies at the site where they co-occurred. Population-level evidence based on the genotypes of ten simple sequence repeat loci supported four phenotypic clusters. In addition, one cluster was shown to have an admixed genetic composition derived from the other three clusters through repeated historical hybridizations. CONCLUSIONS: Based on our new evidence, it is better to treat the four clusters identified here as four independent species. One of them was shown to have an admixed genetic composition derived from the other three through repeated historical hybridizations. This study highlights the importance of applying integrative and statistical methods to infer species delimitations and hybridization history. Such a protocol should be adopted widely for future taxonomic studies.

WGDdetector: a pipeline for detecting whole genome duplication events using the genome or transcriptome annotations.

BACKGROUND: With the availability of well-assembled genomes of a growing number of organisms, identifying the bioinformatic basis of whole genome duplication (WGD) is a growing field of genomics. The most extant software for detecting footprints of WGDs has been restricted to a well-assembled genome. However, the massive poor quality genomes and the more accessible transcriptomes have been largely ignored, and in theoretically they are also likely to contribute to detect WGD using dS based method. Here, to resolve these problems, we have designed a universal and simple technical tool WGDdetector for detecting WGDs using either genome or transcriptome annotations in different organisms based on the widely used dS based method. RESULTS: We have constructed WGDdetector pipeline that integrates all analyses including gene family constructing, dS estimating and phasing, and outputting the dS values of each paralogs pairs processed with only one command. We further chose four species (Arabidopsis thaliana, Juglans regia, Populus trichocarpa and Xenopus laevis) representing herb, wood and animal, to test its practicability. Our final results showed a high degree of accuracy with the previous studies using both genome and transcriptome data. CONCLUSION: WGDdetector is not only reliable and stable for genome data, but also a new way to using the transcriptome data to obtain the correct dS distribution for detecting WGD. The source code is freely available, and is implemented in Windows and Linux operation system.

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.