Species-Specific miRNAs Contribute to the Divergence between Deciduous and Evergreen Species in Ilex.

MicroRNAs (miRNAs) are pivotal regulators of gene expression, playing crucial roles in plant developmental processes and environmental responses. However, the function of miRNAs in influencing deciduous traits has been little explored. Here, we utilized sRNA-seq on two deciduous species, Ilex polyneura (Hand.-Mazz.) S. Y. Hu and Ilex asprella Champ. ex Benth., along with an evergreen species, Ilex latifolia Thunb., to identify and annotate miRNAs within these species. Our analysis revealed 162 species-specific miRNAs (termed SS-miRNAs) from 120 families, underscoring the fundamental roles and potential influence of SS-miRNAs on plant phenotypic diversity and adaptation. Notably, three SS-miRNAs in I. latifolia were found to target crucial genes within the abscission signaling pathway. Analysis of cis-regulatory elements suggested a novel regulatory relationship that may contribute to the evergreen phenotype of I. latifolia by modulating the abscission process in a light-independent manner. These findings propose a potential mechanism by which SS-miRNAs can influence the conserved abscission pathway, contributing to the phenotypic divergence between deciduous and evergreen species within the genus Ilex.

Landform and lithospheric development contribute to the assembly of mountain floras in China.

Although it is well documented that mountains tend to exhibit high biodiversity, how geological processes affect the assemblage of montane floras is a matter of ongoing research. Here, we explore landform-specific differences among montane floras based on a dataset comprising 17,576 angiosperm species representing 140 Chinese mountain floras, which we define as the collection of all angiosperm species growing on a specific mountain. Our results show that igneous bedrock (granitic and karst-granitic landforms) is correlated with higher species richness and phylogenetic overdispersion, while the opposite is true for sedimentary bedrock (karst, Danxia, and desert landforms), which is correlated with phylogenetic clustering. Furthermore, we show that landform type was the primary determinant of the assembly of evolutionarily older species within floras, while climate was a greater determinant for younger species. Our study indicates that landform type not only affects montane species richness, but also contributes to the composition of montane floras. To explain the assembly and differentiation of mountain floras, we propose the 'floristic geo-lithology hypothesis', which highlights the role of bedrock and landform processes in montane floristic assembly and provides insights for future research on speciation, migration, and biodiversity in montane regions.

Aspleniumguodanum (Aspleniaceae), a distinct new fern species from northern Guangdong, China, based on morphological data and molecular phylogeny.

A new spleenwort species, Aspleniumguodanum, was found and described from Danxia landform region in Guangdong, China. The new species has close resemblance to A.subcrenatum Ching ex S.H.Wu in morphology, but can be distinguished by having plants small, stipes and rachises not covered with fibrous scales, relatively fewer pairs of pinnae, pinnae short, pinna margin weakly biserrate, pinna apex acute and lower pinnae obviously reduced. Phylogenetic analyses, based on six plastid markers (atpB, rbcL, rps4 & rps4-trnS and trnL & trnL-F) of the new species and its relatives, support a close relationship between A.guodanum and A.subcrenatum. Only one population with no more than 50 individuals were found and, therefore, it is recommended to be classified as Critically Endangered (CR) following IUCN Red List Criteria.

PMAT: an efficient plant mitogenome assembly toolkit using low-coverage HiFi sequencing data.

Complete mitochondrial genomes (mitogenomes) of plants are valuable resources for nucleocytoplasmic interactions, plant evolution, and plant cytoplasmic male sterile line breeding. However, the complete assembly of plant mitogenomes is challenging due to frequent recombination events and horizontal gene transfers. Previous studies have adopted Illumina, PacBio, and Nanopore sequencing data to assemble plant mitogenomes, but the poor assembly completeness, low sequencing accuracy, and high cost limit the sampling capacity. Here, we present an efficient assembly toolkit (PMAT) for de novo assembly of plant mitogenomes using low-coverage HiFi sequencing data. PMAT has been applied to the de novo assembly of 13 broadly representative plant mitogenomes, outperforming existing organelle genome assemblers in terms of assembly accuracy and completeness. By evaluating the assembly of plant mitogenomes from different sequencing data, it was confirmed that PMAT only requires 1× HiFi sequencing data to obtain a complete plant mitogenome. The source code for PMAT is available at https://github.com/bichangwei/PMAT. The developed PMAT toolkit will indeed accelerate the understanding of evolutionary variation and breeding application of plant mitogenomes.

The first mitogenome of Lauraceae (Cinnamomum chekiangense).

•The first reported mitochondrial genome (Cinnamomum chekiangense) of the Lauraceae family.•The mitogenome of C. chekiangense retains almost all of the ancestral protein-coding genes and has the highest RNA editing number in angiosperms.•Both of the plastid and mitochondrial phylogenetic trees support the magnoliids as a sister group of monocots and eudicots.

The chromosome-level holly (Ilex latifolia) genome reveals key enzymes in triterpenoid saponin biosynthesis and fruit color change.

The Ilex L. (hollies) genus of Aquifoliaceae shows high species diversity in tropical and subtropical regions of Asia and South America. Throughout the range of the genus, Ilex species have been widely used in beverage and medicine production and as ornamentals. Here, we assembled a high-quality, chromosome-level genome of Ilex latifolia, which has extremely high economic value because of its useful secondary metabolite production and the high ornamental value of its decorative red berries. The 99.8% genome sequence was anchored to 20 pseudochromosomes, with a total length of 766.02 Mb and a scaffold N50 of 33.45 Mb. Based on the comparative genomic analysis of 14 angiosperm species, we recovered I. latifolia as the sister group to all other campanulids. Two whole-genome duplication (WGD) events were identified in hollies: one shared ancient WGD in the ancestor of all eudicots and a recent and independent WGD in hollies. We performed a genome-wide search to screen candidate genes involved in the biosynthesis of pentacyclic triterpenoid saponins in I. latifolia. Three subfamilies of CYP450 (CYP71A, CYP72A, and CYP716A) appear to have expanded. The transcriptomic analysis of I. latifolia leaves at five developmental stages revealed that two CYP716A genes and one CYP72A gene probably play important roles in this biosynthetic pathway. In addition, we totally identified 12 genes in the biosynthesis pathways of pelargonidin and cyanidin and observed their differential expression in green and red fruit pericarps, suggesting an association between pelargonidin and cyanidin biosynthesis and fruit pericarp color change. The accumulation of pelargonidin and cyanidin is expected to play an important role in the ornamental value of I. latifolia. Altogether, this study elucidated the molecular basis of the medicinal and ornamental value of I. latifolia, providing a data basis and promising clues for further applications.

Molecular phylogenetic analyses based on the complete plastid genomes and nuclear sequences reveal Daphne (Thymelaeaceae) to be non-monophyletic as current circumscription.

The diverse members of the genus Daphne are prized for their fragrant flowers. Despite being promising ornamental plants in many countries, genetic information of Daphne is scarce. In this study, the plastomes of four species and one variety of Daphne were sequenced and analyzed. The plastomes were typical and contained a pair of inverted repeat (IR) regions that separated the large single-copy (LSC) region from the small single-copy (SSC) region. With a length ranging from 132,869 bp (D. genkwa) to 174,773 bp (D. championii), 106 to 141 genes were predicted. Comparative plastome analysis of the newly sequenced plastomes with four publicly available Daphne plastomes identified an expansion of the IRs, sequence variations, and mutational hotspots. Phylogenetic analyses indicated that the genus Daphne in its current circumscription is polyphyletic. Daphne genkwa was nested within the genus Wikstroemia, while D. championii was well resolved as sister to Edgeworthia. These findings concurred with results from our study that used nuclear ribosomal internal transcribed spacer sequence data. The conflicts on the molecular placement of D. championii and D. genkwa and the present taxonomic classification in Daphne suggest that a new intergeneric classification system of Daphneae warrants consideration.

Comparative analysis of complete Ilex (Aquifoliaceae) chloroplast genomes: insights into evolutionary dynamics and phylogenetic relationships.

BACKGROUND: Ilex (Aquifoliaceae) are of great horticultural importance throughout the world for their foliage and decorative berries, yet a dearth of genetic information has hampered our understanding of phylogenetic relationships and evolutionary history. Here, we compare chloroplast genomes from across Ilex and estimate phylogenetic relationships. RESULTS: We sequenced the chloroplast genomes of seven Ilex species and compared them with 34 previously published Ilex plastomes. The length of the seven newly sequenced Ilex chloroplast genomes ranged from 157,182 bp to 158,009 bp, and contained a total of 118 genes, including 83 protein-coding, 31 rRNA, and four tRNA genes. GC content ranged from 37.6 to 37.69%. Comparative analysis showed shared genomic structures and gene rearrangements. Expansion and contraction of the inverted repeat regions at the LSC/IRa and IRa/SSC junctions were observed in 22 and 26 taxa, respectively; in contrast, the IRb boundary was largely invariant. A total of 2146 simple sequence repeats and 2843 large repeats were detected in the 41 Ilex plastomes. Additionally, six genes (psaC, rbcL, trnQ, trnR, trnT, and ycf1) and two intergenic spacer regions (ndhC-trnV and petN-psbM) were identified as hypervariable, and thus potentially useful for future phylogenetic studies and DNA barcoding. We recovered consistent phylogenetic relationships regardless of inference methodology or choice of loci. We recovered five distinct, major clades, which were inconsistent with traditional taxonomic systems. CONCLUSION: Our findings challenge traditional circumscriptions of the genus Ilex and provide new insights into the evolutionary history of this important clade. Furthermore, we detail hypervariable and repetitive regions that will be useful for future phylogenetic and population genetic studies.

When tropical and subtropical congeners met: Multiple ancient hybridization events within Eriobotrya in the Yunnan-Guizhou Plateau, a tropical-subtropical transition area in China.

Global climate changes during the Miocene may have created ample opportunities for hybridization between members of tropical and subtropical biomes at the boundary between these zones. Yet, very few studies have explored this possibility. The Yunnan-Guizhou Plateau (YGP) in Southwest China is a biodiversity hotspot for vascular plants, located in a transitional area between the floristic regions of tropical Southeast Asia and subtropical East Asia. The genus Eriobotrya (Rosaceae) comprises both tropical and subtropical taxa, with 12 species recorded in the YGP, making it a suitable basis for testing the hypothesis of between-biome hybridization. Therefore, we surveyed the evolutionary history of Eriobotrya by examining three chloroplast regions and five nuclear genes for 817 individuals (47 populations) of 23 Eriobotrya species (including 19 populations of 12 species in the YGP), plus genome re-sequencing of 33 representative samples. We concluded that: (1) phylogenetic positions for 16 species exhibited strong cytonuclear conflicts, most probably due to ancient hybridization; (2) the YGP is a hotspot for hybridization, with 11 species showing clear evidence of chloroplast capture; and (3) Eriobotrya probably originated in tropical Asia during the Eocene. From the Miocene onwards, the intensification of the Eastern Asia monsoon and global cooling may have shifted the tropical-subtropical boundary and caused secondary contact between species, thus providing ample opportunity for hybridization and diversification of Eriobotrya, especially in the YGP. Our study highlights the significant role that paleoclimate changes probably played in driving hybridization and generating rich species diversity in climate transition zones.

Hymenaspleniumtholiformis (Aspleniaceae), a new fern species from southeastern Xizang, China based on morphological and molecular evidence.

A new species of Aspleniaceae, Hymenaspleniumtholiformis sp. nov., is described from Medog County in southeastern Xizang, China. The new species is morphologically similar to H.apogamum and H.szechuanense, but the former has ascending pinnae, pinna apex obtuse to rounded, pinna-marginal teeth entire, and veins terminating just below marginal teeth. Phylogenetic analysis based on five plastid markers confirmed that this new species represents a diverging lineage in the H.excisum subclade of Hymenasplenium.

Selaginellawuyishanensis (sect. Tetragonostachyae, Selaginellaceae), a new species from East China and its phylogenetic position based on molecular data.

A new spikemoss species, Selaginellawuyishanensis, is described and illustrated based on materials collected from Fujian Province, East China. The new species can be distinguished from S.lutchuensis Koidzumi and S.albociliata P. S. Wang by its leaves with extremely long cilia (up to 8 mm) and distinctly white margins, ovate ventral sporophylls, and sporophyll-pteryx completely inverted on dorsal sporophylls. In the present work, a molecular phylogeny, taxonomic description, distribution information, line drawing, and photographs of this new species are presented. A morphological comparison is also given to distinguish it from morphologically similar species in Selaginellasect.Tetragonostachyae (Hook. & Grev.) Hieron. & Sadeb.

Molecular, chromosomal, and morphological evidence reveals a new allotetraploid fern species of Asplenium (Aspleniaceae) from southern Jiangxi, China.

Aspleniumjiulianshanense, a new tetraploid fern species of the A.normale complex (Aspleniaceae) from Jiulianshan National Nature Reserve, southern Jiangxi, China is described and illustrated. We inferred the phylogenetic position of the new species based on sequences from seven plastid markers (atpB, rbcL, rps4, rps4-trnS, trnL, trnL-F, and trnG) and one low-copy nuclear gene, pgiC. The plastid phylogeny supported a close relationship among the new species A.jiulianshanense, A.minutifolium, and A.kiangsuense, while the nuclear phylogeny differed in topology from the plastid tree. The new species may be due to hybridization between A.kiangsuense and A.boreale. Morphologically, the new species can easily be distinguished from other members of the A.normale complex by rachises bearing a gemma near the apex, pinna margins entire to sparsely crenate, and (1‒)3‒4(‒6) sori per pinna.

Taxonomic revision of the Asplenium wrightii complex (Aspleniaceae) with reinstatement of A. alatulum and A. subcrenatum.

The Asplenium wrightii complex is morphologically variable and difficult in species delimitation. Owing to lack of comprehensive sampling in phylogenetic studies, the taxonomy of this complex remains unresolved. Based on extensive field observations, specimen examination and our recent molecular data, the present study aims to clarify the identities of three species of Asplenium in this complex from Asia. Our study revealed that A. alatulum and A. subcrenatum, previously treated as synonyms of A. wrightii, should be reinstated. A taxonomic revision of the three species, including their type information, detailed descriptions, voucher specimens, distribution, ecology, as well as taxonomic notes, is carried out.

Phylogenomic analyses based on genome-skimming data reveal cyto-nuclear discordance in the evolutionary history of Cotoneaster (Rosaceae).

As a consequence of hybridization, polyploidization, and apomixis, the genus Cotoneaster (Rosaceae) represents one of the most complicated and controversial lineages in Rosaceae, with ca. 370 species which have been classified into two subgenera and several sections, and is notorious for its taxonomic difficulty. The infrageneric relationships and taxonomy of Cotoneaster have remained poorly understood. Previous studies have focused mainly on natural hybridization involving only several species, and phylogeny based on very limited markers. In the present study, the sequences of complete chloroplast genomes and 204 low-copy nuclear genes of 72 accessions, representing 69 species as ingroups, were used to conduct the most comprehensive phylogenetic analysis so far for Cotoneaster. Based on the sequences of complete chloroplast genomes and many nuclear genes, our analyses yield two robust phylogenetic trees respectively. Chloroplast genome and nuclear data confidently resolved relationships of this genus into two major clades which largely supported current classification based on morphological evidence. However, conflicts between the chloroplast genome and low-copy nuclear phylogenies were observed in both the species level and clade level. Cyto-nuclear discordance in the phylogeny could be caused by frequent hybridization events and incomplete sorting lineage (ILS). In addition, our divergence-time analysis revealed an evolutionary radiation of the genus from late Miocene to date.

The complete chloroplast genome of Edgeworthia chrysantha (Thymelaeaceae).

Edgeworthia chrysantha is a subtropical plant with significant medicinal value. Herein, we assembled and characterized the complete chloroplast genome of E. chrysantha as genomic resource for future study. The genome consisted a total length of 172,446 bp, comprising of a large single-copy (LSC) region of 85,527 bp, a small single-copy (SSC) region of 2871 bp, separated by two inverted repeat (IR) regions of 42,024 bp each; a total of 139 genes were predicted for the chloroplast genome. Phylogenetic analysis showed that E. chrysantha is placed at the base of the Daphne group of tribe Daphneae, within the family Thymelaeaceae.

A global plastid phylogeny of the fern genus Asplenium (Aspleniaceae).

The infrageneric relationships and taxonomy of the largest fern genus, Asplenium (Aspleniaceae), have remained poorly understood. Previous studies have focused mainly on specific species complexes involving a few or dozens of species only, or have achieved a large taxon sampling but only one plastid marker was used. In the present study, DNA sequences from six plastid markers (atpB, rbcL, rps4, rps4-trnS, trnL and trnL-F) of 1030 accessions (616 of them newly sequenced here) representing c. 420 species of Asplenium (60% of estimated species diversity), 16 species of Hymenasplenium, three Diplaziopsidaceae, and four Rhachidosoraceae were used to produce the largest genus-level phylogeny yet for ferns. Our major results include: (i) Asplenium as broadly circumscribed is monophyletic based on our inclusion of representatives of 32 of 38 named segregate genera; (ii) 11 major clades in Asplenium are identified, and their relationships are mostly well-resolved and strongly supported; (iii) numerous species, unsampled in previous studies, suggest new relationships and numerous cryptic species and species complexes in Asplenium; and (iv) the accrued molecular evidence provides an essential foundation for further investigations of complex patterns of geographical diversification, speciation and reticulate evolution in this family.

A global plastid phylogeny uncovers extensive cryptic speciation in the fern genus Hymenasplenium (Aspleniaceae).

The fern genus Hymenasplenium (Aspleniaceae) is one of the two genera in the family. It is generally recognized among modern pteridologists. However, its infrageneric relationships and species diversity have been unclear and controversial. The molecular studies so far have had small taxon and character sampling. In the present study, DNA sequences of six plastid markers of 158 accessions representing ca. 40 out of ca. 50 known species of Hymenasplenium, and 16 species of Asplenium were used to infer a phylogeny with maximum likelihood, Bayesian inference, and maximum parsimony approaches. Our major results include: (1) Hymenasplenium as currently defined is strongly supported as monophyletic; (2) three major clades representing early splits in Hymenasplenium are identified, with the Old World species being strongly supported as monophyletic; it is ambiguous if the New World species are monophyletic; (3) extensive cryptic speciation in the Old World is discovered demonstrating the complexity of evolution of the genus; and (4) six strongly or moderately supported subclades in the Old World clade are revealed, differing from one another in molecular, morphological, and geographical features.