Genomic insights into the clonal reproductive Opuntia cochenillifera: mitochondrial and chloroplast genomes of the cochineal cactus for enhanced understanding of structural dynamics and evolutionary implications.

Background: The cochineal cactus (Opuntia cochenillifera), notable for its substantial agricultural and industrial applications, predominantly undergoes clonal reproduction, which presents significant challenges in breeding and germplasm innovation. Recent developments in mitochondrial genome engineering offer promising avenues for introducing heritable mutations, potentially facilitating selective sexual reproduction through the creation of cytoplasmic male sterile genotypes. However, the lack of comprehensive mitochondrial genome information for Opuntia species hinders these efforts. Here, we intended to sequence and characterize its mitochondrial genome to maximize the potential of its genomes for evolutionary studies, molecular breeding, and molecular marker developments. Results: We sequenced the total DNA of the O. cochenillifera using DNBSEQ and Nanopore platforms. The mitochondrial genome was then assembled using a hybrid assembly strategy using Unicycler software. We found that the mitochondrial genome of O. cochenillifera has a length of 1,156,235 bp, a GC content of 43.06%, and contains 54 unique protein-coding genes and 346 simple repeats. Comparative genomic analysis revealed 48 homologous fragments shared between mitochondrial and chloroplast genomes, with a total length of 47,935 bp. Additionally, the comparison of mitochondrial genomes from four Cactaceae species highlighted their dynamic nature and frequent mitogenomic reorganizations. Conclusion: Our study provides a new perspective on the evolution of the organelle genome and its potential application in genetic breeding. These findings offer valuable insights into the mitochondrial genetics of Cactaceae, potentially facilitating future research and breeding programs aimed at enhancing the genetic diversity and adaptability of O. cochenillifera by leveraging its unique mitochondrial genome characteristics.

Salvia guidongensis sp. nov.: unraveling a critical evolutionary link in East Asian Salvia from Central China integrating morphology, phylogeny, and plastid genomics.

Introduction: Salvia L., representing the largest genus within the mint family, is noted for its global distribution of approximately 1000 species, with East Asia, and particularly China, recognized as a critical center of diversity for the genus. Methods: Our research was conducted through extensive fieldwork in Guidong County, Hunan Province, China, where we identified a previously undescribed species of Salvia. The identification process involved detailed morphological observations, phylogenetic analyses, and plastid genomics. Results: The newly discovered species, Salvia guidongensis, exhibits unique characteristics not commonly observed in the East Asian lineage of Salvia, including dual floral colors within natural populations-either pale purple or pale yellow. Morphologically, while it shares similarities with members of sect. Glutinaria, S. guidongensis is distinct in its floral morphology, stature, and specific foliar traits. Phylogenetic analysis places S. guidongensis in a unique clade within the East Asian lineage of Salvia, suggesting it may serve as an important evolutionary link. Additionally, we explored the plastome features of S. guidongensis, comparing them with those of closely related species. Discussion: The discovery of S. guidongensis not only entriches the taxonomic tapestry of Salvia but also provides critical insights into the biogeography and evolutionary pathways of the genus in East Asia. By integrating morphological and molecular data, we validate the novel status of S. guidongensis and highlight its significance in bridging taxonomic and evolutionary gaps within Sect. Glutinaria of Salvia.

Deciphering the Plastomic Code of Chinese Hog-Peanut (Amphicarpaea edgeworthii Benth., Leguminosae): Comparative Genomics and Evolutionary Insights within the Phaseoleae Tribe.

The classification and phylogenetic relationships within the Phaseoleae tribe (Leguminosae) have consistently posed challenges to botanists. This study addresses these taxonomic intricacies, with a specific focus on the Glycininae subtribe, by conducting a comprehensive analysis of the highly conserved plastome in Amphicarpaea edgeworthii Benth., a critical species within this subtribe. Through meticulous genomic sequencing, we identified a plastome size of 148,650 bp, composed of 128 genes, including 84 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. Comparative genomic analysis across seven Glycininae species illuminated a universally conserved circular and quadripartite structure, with nine genes exhibiting notable nucleotide diversity, signifying a remarkable genomic variability. Phylogenetic reconstruction of 35 Phaseoleae species underscores the affinity of Amphicarpaea with Glycine, placing Apios as a sister lineage to all other Phaseoleae species, excluding Clitorinae and Diocleinae subtribes. Intriguingly, Apios, Butea, Erythrina, and Spatholobus, traditionally clumped together in the Erythrininae subtribe, display paraphyletic divergence, thereby contesting their taxonomic coherence. The pronounced structural differences in the quadripartite boundary genes among taxa with unresolved subtribal affiliations demand a reevaluation of Erythrininae's taxonomic classification, potentially refining the phylogenetic contours of the tribe.

Evolution and chemical diversity of the volatile compounds in Salvia species.

INTRODUCTION: The plant essential oils are composed of volatile compounds and have significant value in preventing and treating neurological diseases, anxiety, depression, among others. The genus Salvia has been shown to be an important medicinal resource, especially the aerial parts of genus Salvia, which are rich in volatile compounds; however, the chemical diversity and distribution patterns of volatile compounds in Salvia species are still unknown. OBJECTIVE: The work is performed to analyse the chemical diversity and distribution patterns of volatile compounds in genus Salvia. METHODS: The genomic single nucleotide polymorphisms (SNPs) combined with gas chromatography-mass spectrometry (GC-MS) were used to explore the evolution and chemical diversity of Salvia volatile compounds. Initially, the genetic relationship of genus Salvia was revealed by phylogenetic tree that was constructed based on SNPs. And then, GC-MS was applied to explore the chemical diversity of volatile compounds. RESULTS: The results indicated that the volatile compounds were mainly monoterpenoids, sesquiterpenoids, and aliphatic compounds. The genomic SNPs divided species involved in this work into four branches. The volatile compounds in the first and second branches were mainly sesquiterpenoids and monoterpenoids, respectively. Species in the third branch contained more aliphatic compounds and sesquiterpenoids. And those in the fourth branch were also rich in monoterpenoids but had relatively simple chemical compositions. CONCLUSION: This study offered new insights into the phylogenetic relationships besides chemistry diversity and distribution pattern of volatile compounds of genus Salvia, providing theoretical guidance for the investigations and development of secondary metabolites.

Unveiling the spatial distribution and molecular mechanisms of terpenoid biosynthesis in Salvia miltiorrhiza and S. grandifolia using multi-omics and DESI-MSI.

Salvia miltiorrhiza and S. grandifolia are rich in diterpenoids and have therapeutic effects on cardiovascular diseases. In this study, the spatial distribution of diterpenoids in both species was analyzed by a combination of metabolomics and mass spectrometry imaging techniques. The results indicated that diterpenoids in S. miltiorrhiza were mainly abietane-type norditerpenoid quinones with a furan or dihydrofuran D-ring and were mainly distributed in the periderm of the roots, e.g. cryptotanshinone and tanshinone IIA. The compounds in S. grandifolia were mainly phenolic abietane-type tricyclic diterpenoids with six- or seven-membered C-rings, and were widely distributed in the periderm, phloem, and xylem of the roots, e.g. 11-hydroxy-sugiol, 11,20-dihydroxy-sugiol, and 11,20-dihydroxy-ferruginol. In addition, the leaves of S. grandifolia were rich in tanshinone biosynthesis precursors, such as 11-hydroxy-sugiol, while those of S. miltiorrhiza were rich in phenolic acids. Genes in the upstream pathway of tanshinone biosynthesis were highly expressed in the root of S. grandifolia, and genes in the downstream pathway were highly expressed in the root of S. miltiorrhiza. Here, we describe the specific tissue distributions and mechanisms of diterpenoids in two Salvia species, which will facilitate further investigations of the biosynthesis of diterpenoids in plant synthetic biology.

Comparative and Phylogenetic Analyses of Complete Chloroplast Genomes of Scrophularia incisa Complex (Scrophulariaceae).

The Scrophularia incisa complex is a group of closely related desert and steppe subshrubs that includes S. incisa, S. kiriloviana and S. dentata, which are the only S. sect. Caninae components found in Northwest China. Based on earlier molecular evidence, the species boundaries and phylogenetic relationships within this complex remain poorly resolved. Here, we characterized seven complete chloroplast genomes encompassing the representatives of the three taxa in the complex and one closely related species, S. integrifolia, as well as three other species of Scrophularia. Comparative genomic analyses indicated that the genomic structure, gene order and content were highly conserved among these eleven plastomes. Highly variable plastid regions and simple sequence repeats (SSRs) were identified. The robust and consistent phylogenetic relationships of the S. incisa complex were firstly constructed based on a total of 26 plastid genomes from Scrophulariaceae. Within the monophyletic complex, a S. kiriloviana individual from Pamirs Plateau was identified as the earliest diverging clade, followed by S. dentata from Tibet, while the remaining individuals of S. kiriloviana from the Tianshan Mountains and S. incisa from Qinghai-Gansu were clustered into sister clades. Our results evidently demonstrate the capability of plastid genomes to improve phylogenetic resolution and species delimitation, particularly among closely related species, and will promote the understanding of plastome evolution in Scrophularia.

Evolutionary Comparison of the Complete Chloroplast Genomes in Convallaria Species and Phylogenetic Study of Asparagaceae.

The genus Convallaria (Asparagaceae) comprises three herbaceous perennial species that are widely distributed in the understory of temperate deciduous forests in the Northern Hemisphere. Although Convallaria species have high medicinal and horticultural values, studies related to the phylogenetic analysis of this genus are few. In the present study, we assembled and reported five complete chloroplast (cp) sequences of three Convallaria species (two of C. keiskei Miq., two of C. majalis L., and one of C. montana Raf.) using Illumina paired-end sequencing data. The cp genomes were highly similar in overall size (161,365-162,972 bp), and all consisted of a pair of inverted repeats (IR) regions (29,140-29,486 bp) separated by a large single-copy (LSC) (85,183-85,521 bp) and a small single-copy (SSC) region (17,877-18,502 bp). Each cp genome contained the same 113 unique genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Gene content, gene order, AT content and IR/SC boundary structure were nearly identical among all of the Convallaria cp genomes. However, their lengths varied due to contraction/expansion at the IR/LSC borders. Simple sequence repeat (SSR) analyses indicated that the richest SSRs are A/T mononucleotides. Three highly variable regions (petA-psbJ, psbI-trnS and ccsA-ndhD) were identified as valuable molecular markers. Phylogenetic analysis of the family Asparagaceae using 48 cp genome sequences supported the monophyly of Convallaria, which formed a sister clade to the genus Rohdea. Our study provides a robust phylogeny of the Asparagaceae family. The complete cp genome sequences will contribute to further studies in the molecular identification, genetic diversity, and phylogeny of Convallaria.

Plio-Pleistocene climatic change drives allopatric speciation and population divergence within the Scrophularia incisa complex (Scrophulariaceae) of desert and steppe subshrubs in Northwest China.

Numerous temperate plants and animals on the Qinghai-Tibet Plateau (QTP) are hypothesized to have differentiated due to vicariant allopatric speciation associated with the geologic uplifts. However, this hypothesis has rarely been tested through a phylogeographic study of relative species in a broader geographic context, including the QTP, Tianshan Mountains, Mongolian Plateau, and surrounding regions. To understand the speciation and diversification process of plants across this wide area, phylogeographic analysis were examined from Scrophularia incisa and two other closely relative species comprising S. kiriloviana and S. dentata. Thirty-two populations of the three close relatives were genotyped using chloroplast DNA fragments and nuclear microsatellite loci to assess population structure and diversity, supplemented by phylogenetic dating, ancestral area reconstructions and species distribution modelings, as well as niche identity tests. Our chloroplast DNA (cpDNA) phylogeny showed that this monophyletic group of desert and steppe semi-shrub is derived from a Middle Pliocene ancestor of the Central Asia. Lineages in Central Asia vs. China diverged through climate/tectonic-induced vicariance during Middle Pliocene. Genetic and ENM data in conjunction with niche differentiation analyses support that the divergence of S. incisa, S. dentata and S. kiriloviana in China lineage proceeded through allopatric speciation, might triggered by early Pleistocene climate change of increase of aridification and enlargement of deserts, while subsequent climate-induced cycles of range contractions/expansions enhanced the geographical isolation and habit fragmentation of these taxa. These findings highlight the importance of the Plio-Pleistocene climate change in shaping genetic diversity and driving speciation in temperate steppes and deserts of Northwestern China.

The complete chloroplast genome sequence of Elatostema stewardii Merr. (Urticaceae).

Elatostema stewardii is an important medicinal plant endemic to China. In this study, the complete chloroplast genome of E. stewardii was sequenced and assembled using next-generation sequencing technology. The complete chloroplast genome length of E. stewardii was 150,263 bp, including two inverted repeats (IRs) of 24,681 bp, which are separated by LSC and SSC of 83,791 bp and 17,110 bp, respectively. A total of 129 genes were included in the genome, consisting 85 protein-coding genes, eight rRNA genes, and 36 tRNA genes, the overall GC content of this genome was 36.3%. There are few studies on the genus Elatostema of Urticaceae, this chloroplast genome sequence will provide useful data for further research on solving the generic and familial relationships in Urticaceae.

An overlooked dispersal route of Cardueae (Asteraceae) from the Mediterranean to East Asia revealed by phylogenomic and biogeographical analyses of Atractylodes.

BACKGROUND AND AIMS: The East Asian-Tethyan disjunction pattern and its mechanisms of formation have long been of interest to researchers. Here, we studied the biogeographical history of Asteraceae tribe Cardueae, with a particular focus on the temperate East Asian genus Atractylodes DC., to understand the role of tectonic and climatic events in driving the diversification and disjunctions of the genus. METHODS: A total of 76 samples of Atractylodes from 36 locations were collected for RAD-sequencing. Three single nucleotide polymorphism (SNP) datasets based on different filtering strategies were used for phylogenetic analyses. Molecular dating and ancestral distribution reconstruction were performed using both chloroplast DNA sequences (127 Cardueae samples) and SNP (36 Atractylodes samples) datasets. KEY RESULTS: Six species of Atractylodes were well resolved as individually monophyletic, although some introgression was identified among accessions of A. chinensis, A. lancea and A. koreana. Dispersal of the subtribe Carlininae from the Mediterranean to East Asia occurred after divergence between Atractylodes and Carlina L. + Atractylis L. + Thevenotia DC. at ~31.57 Ma, resulting in an East Asian-Tethyan disjunction. Diversification of Atractylodes in East Asia mainly occurred from the Late Miocene to the Early Pleistocene. CONCLUSIONS: Aridification of Asia and the closure of the Turgai Strait in the Late Oligocene promoted the dispersal of Cardueae from the Mediterranean to East China. Subsequent uplift of the Qinghai-Tibet Plateau as well as changes in Asian monsoon systems resulted in an East Asian-Tethyan disjunction between Atractylodes and Carlina + Atractylis + Thevenotia. In addition, Late Miocene to Quaternary climates and sea level fluctuations played major roles in the diversification of Atractylodes. Through this study of different taxonomic levels using genomic data, we have revealed an overlooked dispersal route between the Mediterranean and far East Asia (Japan/Korea) via Central Asia and East China.

Smilax weniae, a New Species of Smilacaceae from Limestone Areas Bordering Guizhou and Guangxi, China.

A new species, Smilax weniae (Smilacaceae), from Southwest China, is described and illustrated. The new species bears peltate leaves, which was previously a unique feature of S. luei. However, it differs from the latter by having a broad ovate leaf blade, longer peduncle, and sexual dimorphic flowers. Further phylogenetic analyses revealed that the new species were placed in a unique position in a subclade of Old World Smilax based on ptDNA and nrITS sequences. Combining detailed morphological comparisons and molecular evidence, we validated that S. weniae is an undescribed new species. Moreover, the plastome characteristics of S. weniae are reported.

The complete chloroplast genome sequence of Japanese buttercup Ranunculus japonicus Thunb.

Ranunculus japonicus is an important medicinal herb widely used in East Asia. In this study, we report the first complete chloroplast genome sequence of Ranunculus japonicus using next-generation sequencing technology. The chloroplast genome size of R. japonicus was 156,981 bp. A total of 129 genes were included, consisting 84 protein-coding genes, eight rRNA genes, and 37 tRNA genes. Thirteen protein-coding genes had intron (ycf3 gene, rps12 gene, rps12 gene, clpP gene contained two introns). A further phylogenomic analysis of Ranunculaceae, including 10 taxa, was conducted for assessing the placement of R. japonicus. It will provide valuable genetic information for this medicinally important species.

Characterization of the complete chloroplast genome of Sparganium glomeratum (Typhaceae) from Jilin Province, China and phylogenetic analysis.

The complete chloroplast genome of Sparganium glomeratum was sequenced and assembled in this study. The circular genome is 160,391 bp in length and exhibits a typical quadripartite structure with a large single-copy (LSC, 87,660 bp) and small single-copy (SSC, 18,721 bp) regions, separated by a pair of inverted repeats (IRs, 27,005 bp). The cp genome contains 113 unique genes, including 79 protein-coding, 30 tRNA, and four rRNA genes. The phylogenetic analysis within the Poales showed that Sparganium is monophyletic and most closely related to Typha. Within Sparganium, S. glomeratum is sister to the clade of S. stoloniferum and S. euricarpum. The work reported here will provide useful information for the evolutionary studies on the genus of Sparganium.

The complete chloroplast genome sequence of spleen amaranth (Amaranthus dubius Mart. ex Thell., Amaranthaceae).

Amaranthus dubius is a leafy vegetable widely cultivated in Asia and Africa. The complete chloroplast genome of Amaranthus dubius was sequenced and assembled in this study. The complete chloroplast genome is 150,520 bp. A total of 130 genes were identified, including 85 protein-coding genes, eight rRNA genes, and 37 tRNA genes. The overall GC content of this genome was 36.6%. The phylogenetic tree based on 10 chloroplast genomes in Amaranthaceae supports that A. dubius is sister to A. hypochondriacus and A. caudatus.

The complete chloroplast genome sequence of wild Japanese pepper Tubocapsicum anomalum Makino (Solanaceae).

As an important medicinal herb, no complete organelle molecular data has been reported for Tubocapsicum anomalum. In this study, the first complete chloroplast genome of Tubocapsicum anomalum Makino was sequenced and assembled. The genome is 155,802 bp in length and contained 124 encoded genes in total, including 75 protein-coding genes, 10 ribosomal RNA genes, and 39 transfer RNA genes. The phylogenomic analysis showed that Tubocapsicum anomalum was closely related to Withania somnifera according the current sampling extent.

The complete chloroplast genome of balsam aster (Aster ageratoides Turcz. var. scaberulus (Miq.) Ling., Asteraceae).

The first complete chloroplast genome of Aster ageratoides Turcz. var. scaberulus (Miq.) Ling. is reported in this study. The total chloroplast genome size of A. ageratoides var. scaberulus was 153,071 bp and comprised of a large single-copy region (LSC with 84,896 bp), a small single-copy region (SSC with 18,269 bp), and two inverted repeat regions (IR with 24,953 bp). A total of 122 genes were included in the genome, including 83 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. Eleven protein-coding genes had intron (ycf3, clpP and rps12 gene contained two introns. Further phylogenomic analysis of Asteraceae, including 13 taxa, was conducted for the placement of A. ageratoides var. scaberulus.

Conservation Genomics of Wild Red Sage (Salvia miltiorrhiza) and Its Endangered Relatives in China: Population Structure and Interspecific Relationships Revealed From 2b-RAD Data.

Red sage (Salvia miltiorrhiza) is a widely used medicinal plant for treatment of cardiovascular and cerebrovascular diseases. Because of excessive excavation by huge market demand and habitat loss by human activities, the wild population resources of S. miltiorrhiza have reduced drastically in recent years. Meanwhile, population status of two closely related species S. bowleyana and S. paramiltiorrhiza were in a trend of decreasing due to their potential replacement of S. miltiorrhiza. Particularly, S. paramiltiorrhiza was threatened and endemic to a small region in eastern China. However, to date there has been no conservation genetic research reported for wild S. miltiorrhiza population and its endangered relatives. Assess the wild germplasm diversity for S. miltiorrhiza and its related species would provide fundamental genetic background for cultivation and molecular breeding of this medicinally important species. In the present study, we investigated the genetic diversity, population structure, and intra/inter-specific differentiation of S. miltiorrhiza and above two relatives using 2b-RAD genome-wide genotyping method. By investigating 81 individuals of S. miltiorrhiza, 55 individuals of S. bowleyana and 15 individuals of S. paramiltiorrhiza from 23 locations in China, we obtained 23,928 SNPs in total. A comparatively high genetic diversity was observed in S. miltiorrhiza (π = 0.0788, H e = 0.0783 ± 0.0007). The observed and expected heterozygosity in populations of these three species ranged from 0.0297 to 0.1481 and 0.0251 to 0.831, respectively. Two major lineage groups were detected in the examined S. miltiorrhiza populations. The results indicated that Dabie Mountain as a genetic diversity center of S. miltiorrhiza and possible complex inter-specific genetic exchange/hybridization occurred between S. miltiorrhiza and the two relatives. We suggest that strategic conservation and germplasm preservation should be considered not only for wild populations of S. miltiorrhiza, but also for its related S. bowleyana and S. paramiltiorrhiza.

The complete chloroplast genome sequences of Hippuris vulgaris (Plantaginaceae).

Two complete chloroplast genomes of Hippuris vulgaris (H. vulgaris_A and H. vulgaris_B), representing two distinct clades in China, were sequenced and assembled in this study. The circular genomes were 152,763 and 152,713 bp in length and exhibit a typical quadripartite structure of the large single-copy (LSC, 82,983/82,949 bp) and small single-copy (SSC, 18,294/18,278 bp) regions, separated by a pair of inverted repeats (IRs, both 25,743 bp). Both two cp genomes identically contain 133 genes, including 88 protein-coding genes, 37 tRNA, and eight rRNA genes. The phylogenetic analysis within Plantaginaceae demonstrated Hippuris an independent clade included in the expanded Plantaginaceae.

Integrated Transcriptomics and Proteomics to Reveal Regulation Mechanism and Evolution of SmWRKY61 on Tanshinone Biosynthesis in Salvia miltiorrhiza and Salvia castanea.

Tanshinones found in Salvia species are the main active compounds for the treatment of cardiovascular and cerebrovascular diseases, but their contents are hugely different in different species. For example, tanshinone IIA content in Salvia castanea Diels f. tomentosa Stib. is about 49 times higher than that in Salvia miltiorrhiza Bunge. The molecular mechanism responsible for this phenomenon remains largely unknown. To address this, we performed comparative transcriptomic and proteomic analyses of S. miltiorrhiza and S. castanea. A total of 296 genes in S. castanea and 125 genes in S. miltiorrhiza were highly expressed at both the transcriptional and proteome levels, including hormone signal regulation, fungus response genes, transcription factors, and CYP450. Among these differentially expressed genes, the expression of SmWRKY61 was particularly high in S. castanea. Overexpression of SmWRKY61 in S. miltiorrhiza could significantly increase the content of tanshinone I and tanshinone IIA, which were 11.09 and 33.37 times of the control, respectively. Moreover, SmWRKY61 had a strong regulatory effect, elevating the expression levels of tanshinone pathway genes such as DXS2, CMK, HMGS2, 1, KSL1, KSL2, CYP76AH1, and CYP76AK3. For the WRKY family, 79 SmWRKYs were originally obtained and classified into three main groups. Collinearity analysis indicated a more specific extension of WRKY gene family in Salvia genus. In 55 Salvia species, only 37 species contained the WRKY61 sequence, and high SmWRKY61 expression in some Salvia L. species was often accompanied by high tanshinone accumulation. The above results suggest that SmWRKY61 is a highly effective regulator of tanshinone accumulation and may be a key factor resulting in high tanshinone accumulation in S. castanea.

Radiation history of Asian Asarum (sect. Heterotropa, Aristolochiaceae) resolved using a phylogenomic approach based on double-digested RAD-seq data.

BACKGROUND AND AIMS: The genus Asarum sect. Heterotropa (Aristolochiaceae) probably experienced rapid diversification into 62 species centred on the Japanese Archipelago and Taiwan, providing an ideal model for studying island adaptive radiation. However, resolving the phylogeny of this plant group using Sanger sequencing-based approaches has been challenging. To uncover the radiation history of Heterotropa, we employed a phylogenomic approach using double-digested RAD-seq (ddRAD-seq) to yield a sufficient number of phylogenetic signals and compared its utility with that of the Sanger sequencing-based approach. METHODS: We first compared the performance of phylogenetic analysis based on the plastid matK and trnL-F regions and nuclear ribosomal internal transcribed spacer (nrITS), and phylogenomic analysis based on ddRAD-seq using a reduced set of the plant materials (83 plant accessions consisting of 50 species, one subspecies and six varieties). We also conducted more thorough phylogenomic analyses including the reconstruction of biogeographic history using comprehensive samples of 135 plant accessions consisting of 54 species, one subspecies, nine varieties of Heterotropa and six outgroup species. KEY RESULTS: Phylogenomic analyses of Heterotropa based on ddRAD-seq were superior to Sanger sequencing-based approaches and resulted in a fully resolved phylogenetic tree with strong support for 72.0-84.8 % (depending on the tree reconstruction methods) of the branches. We clarified the history of Heterotropa radiation and found that A. forbesii, the only deciduous Heterotropa species native to mainland China, is sister to the evergreen species (core Heterotropa) mostly distributed across the Japanese Archipelago and Taiwan. CONCLUSIONS: The core Heterotropa group was divided into nine subclades, each of which had a narrow geographic distribution. Moreover, most estimated dispersal events (22 out of 24) were between adjacent areas, indicating that the range expansion has been geographically restricted throughout the radiation history. The findings enhance our understanding of the remarkable diversification of plant lineages in the Japanese Archipelago and Taiwan.