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Comparative chloroplast genomics of six Bupleurum (Apiaceae) accessions: candidate barcodes, phylogeny based on available plastomes, and candidate RNA-editing sites.

INTRODUCTION: Bupleurum L. (Apiaceae), a taxonomically intricate genus of about 190 species and a source of Radix Bupleuri (Chai Hu), is difficult to discriminate because of convergent morphology, infraspecific variation, and limited genomic sampling. This study aimed to characterize plastome variation, identify and validate candidate molecular markers, reconstruct plastid phylogenetic relationships, and assess candidate plastid RNA-editing sites in Bupleurum. METHODS: We assembled six plastomes from subgenus Bupleurum, screened 51 Bupleurum plastomes for diagnostic loci, reconstructed whole-plastome and partitioned protein-coding-sequence phylogenies, and predicted plastid C-to-U RNA-editing candidates across the six newly assembled plastomes using a PREP-Cp-compatible workflow. Candidate barcode performance was evaluated against the reference plastome phylogenies, and codon-based models were used to test for positive selection. RESULTS: The plastomes were 154,496-155,778 bp with the canonical quadripartite structure and GC contents of 37.67-37.73%. Gene content was stable (131-132 genes; 86-87 protein-coding genes); B. falcatum subsp. cernuum lacked ycf15 but contained an additional inverted-repeat-associated ycf1 annotation. A/U-ending synonymous codons were favoured. Finite pairwise Ka/Ks estimates were below 1 for most genes, and site-specific codon models detected no positive selection. Each plastome contained 55-61 pure microsatellites, dominated by A/T mononucleotide motifs. MarkerSeek ranked 265 features and identified atpF-atpH, petA-psbJ, rpl32-trnL-UAG, and ycf1 as leading candidate barcodes. ycf1 recovered 38 of 41 nodes strongly supported by both reference trees, whereas a partitioned four-locus analysis recovered 40 of 41 and distinguished all 51 accession sequences. However, only one of seven multi-accession operational binomial groups was monophyletic, and only one showed a positive local barcode gap. The whole-plastome phylogeny recovered Bupleurum as monophyletic relative to Chamaesium. The two sampled Penninervia accessions occupied early-diverging positions without forming an exclusive clade. B. falcatum subsp. cernuum was sister to B. ranunculoides, with B. ranunculoides subsp. telonense sister to that pair. A partitioned 74-CDS analysis recovered the same key relationships and 45 of 50 internal bipartitions. Across the six newly assembled plastomes, 57-63 nonsynonymous C-to-U candidates were predicted per accession (367 total) in 21-22 genes; 269 affected the second codon position and 98 the first. DISCUSSION: Bupleurum plastomes are structurally conservative but retain localised divergence useful for marker development. Concordant whole-plastome and CDS genealogies support genus monophyly, whereas sparse Penninervia sampling and maternal plastid inheritance preclude rejecting traditional subgeneric classification. The predicted RNA-editing sites represent candidates for future experimental validation rather than an established Bupleurum editome. These genomic resources support authentication, conservation, and evolutionary research in Bupleurum.

Apiaceae

Plastome evolution and phylogenomic relationships in Ajuga (Lamiaceae, Ajugoideae).

BACKGROUND: Ajuga is currently known to include approximately 69 species, with a combined distribution extending throughout Eurasia, Africa, and Australia. Its popularity and significance are largely based on an extensive history of medicinal and horticultural use. It is divided into two sections based on morphological characters, and this sectional classification is also reflected in pronounced geographic patterns. Although previous studies have largely focused on Ajuga sect. Ajuga in East Asia, A. sect. Chamaepithys, which ranges from the Mediterranean to Central Asia, remains insufficiently sampled, thereby limiting a comprehensive understanding of infrageneric sectional relationships within the genus. Here, we generated complete plastid genomes for 12 species representing both sections of the genus and used these data to characterize plastome structure and infer evolutionary relationships. RESULTS: In this study, 21 Ajuga plastomes were analyzed, including 12 newly sequenced plastomes and 9 previously published plastomes representing 19 species. Comparative analyses showed that all plastomes exhibited a highly conserved quadripartite structure, with genome sizes ranging from 149,963 to 150,740 bp and GC contents varying from 38.2% to 38.3%. Each plastome contained 133 genes, including 88 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. The boundaries between the inverted repeat (IR) and single-copy (SC) regions were also highly conserved across species. In addition, 796 simple sequence repeats (SSRs), 874 long repeat sequences (LRSs), and 12 highly variable regions (ccsA-ndhD, ndhF-rpl32, petA-psbJ, rpl32-trnL-UAG, rps2-rpoC2, trnH-GUG-psbA, trnK-UUU-rps16, trnP-UGG-psaJ, trnT-UGU-trnL-UAA, ycf15-trnL-CAA, ndhF, and ycf1) were identified among the 21 plastomes. Phylogenetic analyses based on four datasets and conducted using Maximum Likelihood and Bayesian Inference recovered two major clades corresponding to the traditionally recognized sectional classification, with one distributed from the Mediterranean to Central Asia and the other in East Asia. CONCLUSION: This study represents the most comprehensive plastome-based sampling of Ajuga to date, including representative species from the Mediterranean, Central Asia, and East Asia. Our results have significantly enhanced our understanding of its infrageneric relationships. The plastome resources generated in this study provide a valuable foundation for future research on species delimitation, phylogeny, and the evolutionary history of Ajuga.

Phylogeny

Genetic diversity, phylogenetic relationships, and marker development between Hydrangea serrata and H. macrophylla based on plastome and 45S nrDNA.

Ornamental hydrangeas (genus Hydrangea) are cultivated worldwide for their diverse flower colors and attractive morphology. Here, we assembled the complete plastid genome (plastome) and 45S nuclear ribosomal DNA (45S nrDNA) sequences of 22 individuals representing H. serrata, H. macrophylla, and related species (H. arborescens, H. paniculata, H. petiolaris, and H. hydrangeoides). The plastomes contained up to 2,344 single-nucleotide polymorphisms (SNPs) and 367 insertions/deletions (InDels) within the genus, whereas the assembled 45S nrDNA sequences showed 119 SNPs and 10 InDels. Phylogenetic analyses based on plastome and 45S nrDNA sequences clearly separated H. serrata and H. macrophylla from the other Hydrangea species. In the plastome-based tree, H. petiolaris was placed in the same clade as H. arborescens, whereas in the 45S nrDNA-based tree it showed a close relationship to H. hydrangeoides. The H. serrata and H. macrophylla samples were not always separated according to their species boundaries, as observed in samples Hse8-Hse12. Notably, one H. serrata sample (Hse8), collected from a wild mountainous region of Japan, exhibited a closer genetic relationship to H. macrophylla samples, indicating that cultivated hydrangeas may have originated from a specific wild lineage of H. serrata adapted to mountainous habitats. Using plastome-derived molecular markers, 66 Hydrangea samples were further classified into five groups, with Group II comprising both cultivated H. macrophylla and a subset of wild H. serrata samples, suggesting a close genetic affinity between this group and the ancestral gene pool of cultivated H. macrophylla. Based on these genomic resources, eight plastome-derived molecular markers were developed to differentiate cultivated hydrangeas from wild genotypes and to assess genetic diversity within H. serrata and H. macrophylla, providing practical tools for germplasm identification, breeding, and genetic resource management of Hydrangea species.

hydrangea

Comprehensive plastome variation and RNA editing in Mentha: insights into phylogenetic relationships and candidate DNA barcodes.

INTRODUCTION: Mentha is an economically and medicinally important genus in Lamiaceae, but its taxonomy and species delimitation remain challenging because of frequent hybridization, polyploidy, and marked morphological plasticity. METHODS: In this study, we comparatively analyzed 12 plastomes representing major Mentha species, hybrid taxa, and unresolved accessions, including four newly assembled genomes, to characterize plastome structure, repeat composition, sequence divergence, phylogenetic relationships, and plastid RNA editing. The M. arvensis plastome and RNA-seq datasets originated from independent Swiss and Indian accessions, respectively. RESULTS: The plastomes were highly conserved in overall organization, ranging from 151,824 to 152,154 bp and displaying the typical quadripartite structure. Gene content and order were largely stable across taxa, with only minor variation likely associated with annotation differences at IR/SC boundary regions. Codon usage analysis revealed a clear bias toward A/U-ending synonymous codons, and most shared protein-coding genes showed low Ka/Ks ratios, indicating predominant purifying selection. Repeat analyses showed that simple sequence repeats were mainly composed of A/T-rich mononucleotide motifs, whereas long repeats were concentrated in the 30-40 bp size class. Comparative analyses identified six hypervariable regions, namely ccsA-ndhD, ycf1, ndhD, rpl32-trnL-UAG, rbcL-accD, and petA-psbJ, which represent promising candidate plastid markers for species discrimination. Phylogenetic analysis based on complete plastomes provided strong support for relationships among the sampled taxa and recovered a close affinity among M. aquatica, M. arvensis, and M. canadensis. In addition, RNA-seq analysis of M. arvensis identified 17 candidate plastid RNA editing sites, most of which were C-to-U conversions and nonsynonymous events. DISCUSSION: Together, these results expand plastid genomic resources for Mentha and provide a useful framework for phylogenetic inference, species identification, and future germplasm utilization.

RNA editing

The plastid genome of the critically endangered Valeriana trinervis (= Centranthus trinervis) and insights from comparison with other Valeriana plastomes (Caprifoliaceae).

The first complete plastid genome of the critically endangered species Valeriana trinervis was sequenced, assembled and compared with other published Valeriana plastomes. In this study, we assembled the plastid genome of the critically endangered, endemic species Valeriana trinervis (= Centranthus trinervis) and compare it with all published plastomes of Valeriana. We found not only differences in the inverted repeats boundaries, in the type and abundance of repeats, but also similarities in codon usage and microsatellite numbers. We detected non-canonical start codons in several genes and identified variation in several regions that could be useful for phylogenetic and phylogeographic studies. The phylogenetic tree inference based on both full plastomes and coding sequence data indicated that V. trinervis is sister to all Eurasian Valeriana accessions confirming the phylogenetic position recently investigated. This is the first plastome available for a species of the Mediterranean clade of Valeriana previously known as Centranthus, and it adds further data to understand the evolution and diversification of this systematically debated genus.

Genome, Plastid

Complete plastid genome of Iris orchioides and comparative analysis with 19 Iris plastomes.

Iris is a cosmopolitan genus comprising approximately 280 species distributed throughout the Northern Hemisphere. Although Iris is the most diverse group in the Iridaceae, the number of taxa is debatable owing to various taxonomic issues. Plastid genomes have been widely used for phylogenetic research in plants; however, only limited number of plastid DNA markers are available for phylogenetic study of the Iris. To understand the genomic features of plastids within the genus, including its structural and genetic variation, we newly sequenced and analyzed the complete plastid genome of I. orchioides and compared it with those of 19 other Iris taxa. Potential plastid markers for phylogenetic research were identified by computing the sequence divergence and phylogenetic informativeness. We then tested the utility of the markers with the phylogenies inferred from the markers and whole-plastome data. The average size of the plastid genome was 152,926 bp, and the overall genomic content and organization were nearly identical among the 20 Iris taxa, except for minor variations in the inverted repeats. We identified 10 highly informative regions (matK, ndhF, rpoC2, ycf1, ycf2, rps15-ycf, rpoB-trnC, petA-psbJ, ndhG-ndhI and psbK-trnQ) and inferred a phylogeny from each region individually, as well as from their concatenated data. Remarkably, the phylogeny reconstructed from the concatenated data comprising three selected regions (rpoC2, ycf1 and ycf2) exhibited the highest congruence with the phylogeny derived from the entire plastome dataset. The result suggests that this subset of data could serve as a viable alternative to the complete plastome data, especially for molecular diagnoses among closely related Iris taxa, and at a lower cost.

Iris Plant

Use of plastome and nuclear mutants of higher plants to study the genetic control of plastid formation and function.

We conducted comparative biochemical and electron-microscopic studies of several types of plastome and nuclear mutants of Antirrhinum majus and Pelargonium zonale. It was shown that specific blocking of the photosynthetic reaction occurs in plastome mutants of A. majus; Photosystem II was found to be damaged in the en:alba-1 mutant and photo-system I was affected in the en:viridis-1 mutant. The plastid mutations in these mutants caused loss of certain soluble lamellar proteins and pigment--protein complexes or a reduction in their content, which led to disappearance of photosynthetic activity. When the content of high-molecular ribosomal RNA in the leaves of normal and mutant P. zonale plants was compared, the normal plants were found to have four types of RNA: two types of cytoplasmic-ribosome RNA and two types of plastid-ribosome RNA. No plastid-ribosome RNA was detected in the mutant. These results were confirmed by electron-microscopic examination: no ribosomes were detected in the mutant plastids. Thus, use of plastome mutants made it possible to establish that the genetic information concentrated in the plastid DNA controls formation of ribosomes and lamellae in the chloroplasts and thus affects chloroplast photosynthetic function.

Cell Nucleus

Ribulose-1,5-biphosphate carboxylase-deficient plastome mutants of Oenothera.

In spite of only slightly subnormal pigment contents, two plastome mutants of Oenothera (Valpha, Isigma) were practically incapable of photosynthetic CO2 fixation and another one exhibited considerably reduced photosynthesis (IVbeta). While other photosynthetic enzymes were present as far as investigated, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity was very low or missing altogether. As shown by gel electrophoresis, mutant IVbeta contained some, though little, fraction I protein. In the other two mutants fraction I protein could not be detected. Also, neither the small nor the large subunit of ribulose-1,5-biphosphate carboxylase could be found in these mutants. In immunodiffusion experiments with a monospecific antiserum against rye ribulose-1,5-bisphosphate carboxylase, only extracts from wild-type Oenothera produced visible precipitation lines. Still, the presence of very low levels of immunochemically reactive antigen was indicated for all three mutants. The highest level was observed in mutant IVbeta. The behaviour of the mutant extracts suggested that the antigens of mutant and wild type leaves reacting with the antiserum were not identical. All mutants appeared to have a coupled electron transport system as shown by ATP measurements, light scattering and 515 nm absorption changes. Linear electron transport was possible in the mutants. Still, the photoresponse of cytochrome f and fluorescence measurements suggested altered electron transport properties in the mutants. These are interpreted to be secondary lesions of the photosynthetic apparatus caused by primary deficiency in ribulose-1,5-bisphosphate carboxylase activity. From the absence in two mutants (Valpha, Isigna) of the small subunit of ribulose-1,5-bisphosphate carboxylase, which is known to be coded for by nuclear DNA and to be synthesized on cytoplasmic ribosomes, it appears that the genetic system of the plastids is capable of interfering with the genome-controlled synthesis of plastid components.

Carboxy-Lyases

The evolution of the plastid genomes in the holoparasitic Balanophoraceae.

The independent transition to a heterotrophic lifestyle in plants drove remarkably convergent evolutionary trajectories, characterized by morphological modifications and reductions in their plastomes. The characteristics of the minimum plastome required for survival, if they exist, remain a topic of debate. The holoparasitic family Balanophoraceae was initially presumed to have entirely lost their plastids, however, recent reports revealed the presence of reduced and aberrant plastids with odd genomes. Among the outstanding features of these genomes are the highest nucleotide composition bias across the tree of life and the only two genetic code changes ever recorded among plants. In this study, we assembled the plastomes from five genera, four of which had never been studied. Major common features include extremely high AT content, the lack of a typical quadripartite structure and extensive size reduction due to gene elimination and genome compaction. The family exhibits multiple gene and intron losses, and a broad range of scenarios regarding the evolution of the plastid trnE, a gene considered essential because of its dual function in tetrapyrrole biosynthesis and translation within the plastid. In addition, phylogenetic analyses suggest that the genus Scybalium is not monophyletic. An evolutionary model for the plastomes of the Balanophoraceae is proposed.

Genome, Plastid

Unequally Abundant Chromosomes and Unusual Collections of Transferred Sequences Characterize Mitochondrial Genomes of Gastrodia (Orchidaceae), One of the Largest Mycoheterotrophic Plant Genera.

The mystery of genomic alternations in heterotrophic plants is among the most intriguing in evolutionary biology. Compared to plastid genomes (plastomes) with parallel size reduction and gene loss, mitochondrial genome (mitogenome) variation in heterotrophic plants remains underexplored in many aspects. To further unravel the evolutionary outcomes of heterotrophy, we present a comparative mitogenomic study with 13 de novo assemblies of Gastrodia (Orchidaceae), one of the largest fully mycoheterotrophic plant genera, and its relatives. Analyzed Gastrodia mitogenomes range from 0.56 to 2.1 Mb, each consisting of numerous, unequally abundant chromosomes or contigs. Size variation might have evolved through chromosome rearrangements followed by stochastic loss of "dispensable" chromosomes, with deletion-biased mutations. The discovery of a hyper-abundant (∼15 times intragenomic average) chromosome in two assemblies represents the hitherto most extreme copy number variation in any mitogenomes, with similar architectures discovered in two metazoan lineages. Transferred sequence contents highlight asymmetric evolutionary consequences of heterotrophy: despite drastically reduced intracellular plastome transfers convergent across heterotrophic plants, their rarity of horizontally acquired sequences sharply contrasts parasitic plants, where massive transfers from their hosts prevail. Rates of sequence evolution are markedly elevated but not explained by copy number variation, extending prior findings of accelerated molecular evolution from parasitic to heterotrophic plants. Putative evolutionary scenarios for these mitogenomic convergence and divergence fit well with the common (e.g. plastome contraction) and specific (e.g. host identity) aspects of the two heterotrophic types. These idiosyncratic mycoheterotrophs expand known architectural variability of plant mitogenomes and provide mechanistic insights into their content and size variation.

Genome, Mitochondrial

Insights into phylogenetic relationships of Veronica species (Plantaginaceae) based on comparative chloroplast genomics.

INTRODUCTION: Veronica L. is one of the most species-rich genera in Plantaginaceae and several species have medicinal, horticultural, or ecological value. METHODS: In this study, the complete chloroplast genomes of three Veronica species were assembled and annotated using Illumina sequencing data. RESULTS: The plastomes exhibited a typical quadripartite structures, with total lengths of 150,202 bp for Veronica biloba L., 151,159 bp for Veronica ciliata Fisch. and 151,098 bp for Veronica vandellioides Maxim. Each genome contained 130-132 unique genes, including 86-87 protein-coding genes, 36-37 tRNA genes, and 8 rRNA genes. Comparative analyses of 24 Veronica plastomes indicated that the IR/SC junctions were largely conserved, although slight boundary shifts occurred around rps19, ndhF, and ycf1. Forward, palindromic, complement, and reverse repeats were detected, and A/T mononucleotide repeats were the dominant SSR type. Nucleotide diversity analysis identified rpl32-trnL, trnK-rps16, rpl32, ycf1, ndhF, accD, matK, and rpoB as highly variable regions. Phylogenetic analyses recovered Veronica as a well-supported monophyletic lineage and clarified the plastid positions of the three newly sequenced species. Divergence time estimation suggested that the estimation suggested of Veronica was around 14.9 Ma, with V. biloba, V. ciliata and V. vandellioides diverging approximately 3.9 Ma, 0.6 Ma, and 6.9 Ma, respectively. DISCUSSION: Because the analyses were based on plastid genomes, the inferred topology should be interpreted as chloroplast phylogenetic evidence rather than a complete species-history reconstruction. These results provide plastome resources and molecular evidence for taxonomy, species identification, and future evolutionary studies of Veronica.

Plantaginaceae

Assembling genomes of non-model plants: A case study with evolutionary insights from Ranunculus (Ranunculaceae).

Whereas genome sequencing and assembly technologies are improving, cost can still be prohibitive for plant species with large, complex genomes. As a consequence, genomics work on some taxa in evolutionarily pivotal positions in the vascular plant tree of life has been hampered. The species-rich genus Ranunculus (Ranunculaceae) is an important angiosperm group for the study of polyploidy, apomixis, and reticulate evolution. However, neither mitochondrial nor high-quality nuclear genome sequences are available. This limits phylogenomic, functional, and taxonomic analyses thus far. Here, we tested Illumina short-read, Oxford Nanopore Technology (ONT) and PacBio (HiFi) long-read, and hybrid-read assembly strategies. We sequenced the diploid progenitor species R. cassubicifolius (R. auricomus species complex) and selected the best assemblies in terms of completeness, contiguity, and quality scores. We first assembled the plastome (156 kbp, 85 genes) and mitogenome (1.18 Mbp, 40 genes) sequences using Illumina and Illumina-PacBio-hybrid strategies, respectively. We also present an updated plastome and the first mitogenome phylogeny of Ranunculaceae, including studies of gene loss (e.g., infA, ycf15, or rps) with evolutionary implications. For the nuclear genome sequence, we favored a PacBio-based assembly polished three times with filtered short reads and subsequently scaffolded into eight pseudochromosomes by chromatin conformation data (Hi-C). We obtained a haploid genome sequence of 2.69 Gbp, with 94.1% complete BUSCO genes found and 35 482 annotated genes, and inferred ancient gene duplications compared to existing Ranunculales genomes. The genomic information presented here will enable advanced evolutionary-functional analyses for the species complex, but also for the genus and beyond Ranunculaceae.

Ranunculus

Complete chloroplast genomes of endemic Astragalus and Oxytropis species from Uzbekistan.

Chloroplast genomes provide important insights into plant phylogeny, genome evolution, and molecular marker development. In this study, we sequenced, assembled, and analyzed the complete chloroplast genomes of two endemic species from Uzbekistan, Astragalus nuratensis and Oxytropis pseudorosea. Genome skimming generated high-quality paired-end reads, enabling the recovery of complete plastomes with mean sequencing depths of 638× and 1,725×, respectively. The chloroplast genomes were 122,316 bp in A. nuratensis and 122,708 bp in O. pseudorosea. Both genomes encoded 110 unique genes, including 76 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Consistent with members of the inverted repeat-lacking clade of Fabaceae, both species lacked the typical inverted repeat regions, resulting in a single-copy genome structure. Phylogenetic analysis based on 119 complete chloroplast genomes resolved major lineages within Astragalus and related genera with strong support. Astragalus nuratensis was placed within the Phaca clade, while Oxytropis pseudorosea formed part of a distinct Oxytropis lineage. These results provide new genomic resources for understanding evolutionary relationships and plastome evolution in Central Asian legumes.

Genome, Chloroplast

Genetic and metabolite diversity of Sundaland Heptapleurum (Araliaceae) insight into evolutionary and specialized metabolite.

BACKGROUND: The genus Heptapleurum Gaertn (previously treated as Schefflera J.R.Forst. & G.Forst.) within the Araliaceae family is recognized for its significant medicinal value and complex taxonomy. However, an integrated understanding of its evolutionary and metabolite diversity remains unexplored, especially in the Sundaland region (i.e., Java and Sumatra). Here, we integrate genomics and metabolomics to unravel the evolutionary relationships and metabolite diversity of 10 Heptapleurum species from Sundaland. RESULTS: We assembled 10 new complete plastid genomes (plastomes) and 45S nuclear ribosomal DNA (nrDNA) sequences, identifying significant variation and potential key molecular markers. Metabolomics identified 152 metabolites, mainly phenolics and terpenoids. Metabolite profiles of H. rhynchocarpum and H. capituliferum were more correlated with phylogeny than with geography; these two species were separate from the main Heptapleurum clade. Four species, H. farinosum, H. longifolium, H. rigidum, and H. fastigiatum, have almost identical plastomes and 45S nrDNA structures, suggesting they may represent closely related species with different phenotypes, as evidenced by distinctive metabolite compositions. CONCLUSIONS: Crucially, there is an incongruence between the genetic and chemical phylogenies, underscoring that while chemotaxonomy reflects functional diversity, genetic data remains the definitive standard for evolutionary inference, with the potential for reclassifying H. rhynchocarpum and H. capituliferum. This study provides a foundation for future taxonomic revisions, conservation, and drug discovery of Heptapleurum.

Phylogeny

The complete Chloroplast Genome of Dianthus Helenae, an Endemic Species with Medicinal Potential from the Nuratau Mountains, Uzbekistan.

Dianthus helenae Vved. is an endemic medicinal species of the Nuratau Mountains, Uzbekistan, and its genomic resources have remained largely unavailable. In this study, we sequenced, assembled, and characterized the complete chloroplast genome of D. helenae and evaluated its phylogenetic position within Dianthus. The plastome exhibited a typical circular quadripartite structure with a total length of 149,567 bp, comprising a large single-copy (LSC) region of 82,856 bp, a small single-copy (SSC) region of 17,105 bp, and a pair of inverted repeats (IRs) of 24,803 bp each. The genome contained the typical set of chloroplast genes, including protein-coding genes, transfer RNAs, and ribosomal RNAs, with duplicated genes located in the IR regions. Phylogenetic analysis based on complete chloroplast genome sequences strongly supported the placement of D. helenae within Dianthus and recovered it as a distinct lineage relative to other sampled species. Sliding window analysis of nucleotide diversity revealed uneven sequence variation across the plastome, with higher variability in the SSC and LSC regions than in the IRs. Several highly variable loci, including trnK-UUU , rps16-trnQ-UUG , rpl32, ycf1, and ndh-associated regions, were identified as potential molecular markers. These results provide an important genomic resource for Dianthus and establish a foundation for future phylogenetic, taxonomic, conservation, and molecular identification studies of this endemic Central Asian species.

Genome, Chloroplast

Plastid genome evolution and phylogenomics with broad taxon sampling: insights into intrafamilial classification of Hamamelidaceae.

Hamamelidaceae, within the order Saxifragales, comprises 27 genera and approximately 120 species. The family has a pantropical and temperate distribution across the Americas, Asia, Africa, and Australia. Previous molecular investigations, constrained by limited taxon sampling and inadequate genetic markers, supported a five-subfamily classification system. However, these studies predominantly focused on Asian taxa, resulting in poor resolution of the evolutionary relationships among American, African, and Australian genera. To address these sampling gaps, we employed near-complete generic sampling (26 of 27 genera) to investigate plastome architecture, structural variation, and phylogenetic relationships. We newly sequenced and assembled 15 plastid genomes representing geographically and taxonomically underrepresented genera and analyzed them alongside 59 publicly available plastomes retrieved from GenBank. Plastid genomes exhibited conserved quadripartite architecture with sizes ranging from 158, 076 bp to 160, 814 bp, minimal structural variation, consistent GC content (37.7-38.2%), and identical gene order. Inverted repeat (IR) regions had limited size variation (26, 211-26, 429 bp). Simple sequence repeat (SSR) distribution (2, 219 loci) showed no clear correlation with the genus-level phylogenetic relationships. We identified ten hypervariable regions, including coding sequences (accD, ycf1, clpP, ndhF, and rpl22) and intergenic spacers (rpl33-rps18, the trnG-UCC intron, trnH-GUG-psbA, accD-psaI, and petA-psbJ), as promising candidate regions for future applications in species delimitation and phylogenetic studies. Phylogenetic analyses revealed largely congruent topologies across datasets and methods, providing improved resolution and strong support for most subfamilial and tribal relationships compared with previous studies. This study highlights the utility of plastid genome data for resolving deep-level phylogenetic relationships within Hamamelidaceae. The genome architecture reflects the high conservation of plastid genomes, while the identified mutation hotspots represent potential resources for future taxonomic and phylogenetic studies. Our results support the existing subfamily classification while improving geographical coverage and generic representation, providing a robust framework for future taxonomic and evolutionary studies of this globally distributed and taxonomically complex family.

Hamamelidaceae

Plant species identification by genome skimming across the vascular plant tree of life.

Accurate species identification is essential for biodiversity conservation and sustainable use, yet standard plant DNA barcoding often fails to achieve species-level resolution. We present a large-scale empirical evaluation of genome skimming as a tool to improve plant species discrimination. Using standardised data from 1969 individuals representing 475 species from 32 genera across major lineages of the vascular plant tree of life, we compare conventional plastid + internal transcribed spacer (ITS) barcodes with genome skimming approaches. Standard barcoding using rbcL, matK, trnH-psbA and ITS resolved about half of species (49.3%), with six genera showing <&#x2009;25% species discrimination. By contrast, genome skimming enabled the recovery of complete plastid genomes, yielding 57.6% species discrimination. It also generated sufficient nuclear genomic data for additional resolution from k-mer analysis, achieving 66.8% species discrimination - an average gain of 17.5% over standard barcodes - while eliminating cases of extreme failure (<&#x2009;25% resolution). The recovery of complete plastomes and ribosomal DNAs from genome skims also ensures backward compatibility with existing barcode datasets. Our results demonstrate that genome skimming provides data that substantially improves species-level resolution across diverse plant lineages and offers a scalable, high-throughput approach for building comprehensive reference resources to support global biodiversity initiatives.

DNA Barcoding, Taxonomic

Beyond species trees: pervasive gene flow limits phylogenomic resolution in the diversification of Juniperus from the Qinghai-Tibet Plateau.

Understanding how lineages diversify despite persistent ancestral polymorphism and recurrent gene flow remains a central challenge in evolutionary biology. Juniperus distributed across the Qinghai-Tibet Plateau provide an ideal system for addressing this question because repeated geological uplift and climatic oscillations have likely promoted cycles of lineage divergence, range shifts, and secondary contact. Here, we combined approximately 1.08 million genome-wide SNPs from 164 individuals representing thirteen Juniperus lineages with phylogenomic datasets comprising 3,381 nuclear single-copy genes and nearly complete plastomes. We detected extensive phylogenomic discordance and cytonuclear incongruence across genomic datasets. Topology weighting, coalescent simulations, quartet-based tests, and analyses of gene flow and reticulation collectively support the interpretation that these patterns were shaped by the combined effects of prolonged incomplete lineage sorting and gene flow during lineage diversification. Ecological niche analyses further provide a spatial and climatic context in which environmentally similar lineages may have had greater opportunities for secondary contact during historical range shifts. Collectively, our results reveal that the evolutionary history of Qinghai-Tibet Plateau Juniperus is characterized by reticulate diversification rather than strictly bifurcating evolution, and demonstrate how genome-wide discordance can provide biological insights into the evolutionary processes underlying lineage diversification.

Gene Flow