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Sambucus L. is found in the family Viburnaceae (syn. Adoxaceae) and encompasses approximately 29 accepted species. The complex morphology of these species has caused continued confusion concerning their nomenclature, classification, and identification. Despite previous attempts to resolve taxonomic complexities in the Sambucus genus, there are still unclear phylogenetic relationships among several species. In this study, the newly obtained plastome of Sambucus williamsii Hance. as well as the populations of Sambucus canadensis L., Sambucus javanica Blume, and Sambucus adnata Wall. ex DC were sequenced, and their sizes, structural similarity, gene order, gene number, and guanine-cytosine (GC) contents were analyzed. The phylogenetic analyses were conducted using the whole chloroplast genomes and protein-coding genes (PCGs). The findings revealed that the chloroplast genomes of Sambucus species exhibited typical quadripartite double-stranded DNA molecules. Their lengths ranged from 158,012 base pairs (bp) ( S. javanica ) to 158,716 bp ( S. canadensis L). Each genome comprised a pair of inverted repeats (IRs), which separated the large single-copy (LSC) and small single-copy (SSC) regions. In addition, the plastomes contained 132 genes, encompassing 87 protein-coding, 37 tRNA, and four rRNA genes. In the simple sequence repeat (SSR) analysis, A/T mononucleotides had the highest proportion, with the most repetitive sequences observed in S. williamsii . The comparative genome analyses showed high similarities in structure, order, and gene contents. The hypervariable regions in the studied chloroplast genomes were trnT-GGU , trnF-GAA , psaJ , trnL-UAG , ndhF , and ndhE , which may be used as candidate barcodes for species discrimination in Sambucus genus. Phylogenetic analyses supported the monophyly of Sambucus and revealed the separation of S. javanica and S. adnata populations. Sambucus chinensis Lindl. was nested within S. javanica in the same clade, collaborating their conspecific treatment. These outcomes indicate that the chloroplast genome of Sambucus plants is a valuable genetic resource for resolving taxonomic discrepancies at the lower taxonomic levels and can be applied in molecular evolutionary studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Waswa, Mkala, Odago, Amenu, Mutinda, Muthui, Ding, Hu and Wang.)

Recently, the systematic status of Fortunella Swingle and its taxonomy has attracted much attention. Flora of China incorporates Fortunella into Citrus Linn. and treats all species of the traditional Fortunella as one species, namely Citrus japonica (Thunb.) Swingle. Furthermore, F. venosa (Champ. ex Benth.) C. C. Huang and F. hindsii (Champ. ex Benth.) Swingle are currently considered as synonyms of C. japonica . In this paper, morphological, palynological, and phylogenetic analyses were used to systematically explore the taxonomic status of traditional Fortunella . The key morphological features that differed among the Fortunella species were the leaf and the petiole hence could be key in its taxonomic classification of the species. Additionally, pollen morphological analysis based on the pollen size, germination grooves, polar, and equatorial axes also supported the separation of the species. The results of the phylogenetic analysis showed that each of the three species clustered separately, hence strongly supporting the conclusion of independent species. In addition, the phylogenetic analysis showed that the two genera clustered closely together hence our results support the incorporation of Fortunella into Citrus . Based on the above, this article has revised the classification of the traditional Fortunella and determined that this genus has three species, namely; F. venosa , F. hindsii , and F. japonica . F. venosa and F. hindsii are placed in the Citrus as separate species, and their species names still use the previous specific epithet. The revised scientific names of the new combinations of F. venosa and F. hindsii are as follows: Citrus venosa (Champ. ex Benth.) K. M. Liu, X. Z. Cai, and G. W. Hu, comb. nov . and Citrus hindsii (Champ. ex Benth.) K. M. Liu, G. W. Hu, and X. Z. Cai, comb. nov . F. venosa is the original species of Fortunella , F. venosa and F. hindsii are both listed as the second-class key protected wild plants in China. Therefore, the establishment of the taxonomic status of F. venosa and F. hindsii not only deepens our understanding, importance, and the complexity of the systematic classification of Fortunella , but is also significant for global biodiversity conservation, genetic resources for breeding purposes, and population genetics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Reviewer W-BY declared a shared parent affiliation with JR, VW, and FM at the time of the review., (Copyright © 2022 Wang, Chen, Shu, You, Liang, Li, Ren, Wanga, Mutie, Cai, Liu and Hu.)

Coleanthus subtilis (Tratt.) Seidel (Poaceae) is an ephemeral grass from the monotypic genus Coleanthus Seidl, which grows on wet muddy areas such as fishponds or reservoirs. As a rare species with strict habitat requirements, it is protected at international and national levels. In this study, we sequenced its whole chloroplast genome for the first time using the next-generation sequencing (NGS) technology on the Illumina platform, and performed a comparative and phylogenetic analysis with the related species in Poaceae. The complete chloroplast genome of C . subtilis is 135,915 bp in length, with a quadripartite structure having two 21,529 bp inverted repeat regions (IRs) dividing the entire circular genome into a large single copy region (LSC) of 80,100 bp and a small single copy region (SSC) of 12,757 bp. The overall GC content is 38.3%, while the GC contents in LSC, SSC, and IR regions are 36.3%, 32.4%, and 43.9%, respectively. A total of 129 genes were annotated in the chloroplast genome, including 83 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The accD gene and the introns of both clpP and rpoC1 genes were missing. In addition, the ycf1 , ycf2 , ycf15 , and ycf68 were pseudogenes. Although the chloroplast genome structure of C . subtilis was found to be conserved and stable in general, 26 SSRs and 13 highly variable loci were detected, these regions have the potential to be developed as important molecular markers for the subfamily Pooideae. Phylogenetic analysis with species in Poaceae indicated that Coleanthus and Phippsia were sister groups, and provided new insights into the relationship between Coleanthus , Zingeria , and Colpodium . This study presents the initial chloroplast genome report of C. subtilis , which provides an essential data reference for further research on its origin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ren, Tian, Jiang, Zhu, Mutie, Wanga, Ding, Yang, Dong, Chen, Cai and Hu.)

Impatiens is one of the most species-rich genera of angiosperms, with more than 1,000 species distributed in Eurasia and Africa. Previous studies have shown that seeds of Impatiens display enormous morphological diversity, and seed micromorphology may contribute to the classification of those species. However, the micromorphological seed coat characteristics of Impatiens seeds have not been systematically studied. In this study, we examined 117 Impatiens seeds from two subgenera and seven sections, and analyzed the seed characters of shape, primary ornamentation, secondary ornamentation, anticlinal cell wall, and periclinal cell wall. We discovered that, according to the different shapes of ornamentation, seed coat can be divided into three types, viz, reticulate type, protrusive type, appendicular type, and that they can be further subdivided into 10 subtypes. In addition, the characteristics of seed coat ornamentation with taxonomic significance in this genus are discussed. We reconstructed the ancestral states of the above seed characters of the Impatiens using the maximum likelihood approach based on the reconstructed phylogenetic framework. The seed character reconstruction showed that the seed shape, primary ornamentation, and anticlinal cell wall could be identified as unambiguous, while other characters were ambiguous in the last common ancestor of Impatiens . Reconstruction of important seed morphological characters showed that the secondary ornamentation possesses high plasticity, and the primary ornamentation has high homology. In addition, we inferred the evolutionary trends of seed ornamentation and found that the seed coat ornamentation of Impatiens experienced a complex evolutionary process from a reticulate type into more complex types. We also discussed the relationship between seed coat diversity vs. environmental adaptation and seed dispersal in Impatiens ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Song, Peng, Mutie, Jiang, Ren, Cong and Hu.)

Hoya is a genus in Apocynaceae-Asclepiadoideae, known for its showy wax flowers, making it a popular ornamental plant. However, phylogenetic relationships among most Hoya species are not yet fully resolved. In this study, we sequenced 31 plastomes of Hoya group species using genome skimming data and carried out multiple analyses to understand genome variation to resolve the phylogenetic positions of some newly sequenced Chinese endemic species. We also screened possible hotspots, trnT-trnL-trnF, psba-trnH , and trnG-UCC , ndhF , ycf1, matK, rps16 , and accD genes that could be used as molecular markers for DNA barcoding and species identification. Using maximum likelihood (ML) and Bayesian Inference (BI), a species phylogeny was constructed. The newly assembled plastomes genomes showed the quasi-tripartite structure characteristic for Hoya and Dischidia with a reduced small single copy (SSC) and extremely enlarged inverted repeats (IR). The lengths ranged from 175,404 bp in Hoya lacunosa to 179,069 bp in H. ariadna . The large single copy (LSC) regions ranged from 80,795 bp ( Hoya liangii ) to 92,072 bp ( Hoya_ sp2 _ ZCF6006). The massively expanded IR regions were relatively conserved in length, with the small single-copy region reduced to a single gene, ndhF . We identified 235 long dispersed repeats (LDRs) and ten highly divergent hotspots in the 31 Hoya plastomes, which can be used as DNA barcodes for species identification. The phylogeny supports Clemensiella as a distinct genus. Hoya ignorata is resolved as a relative to Clade VI species. This study discloses the advantages of using Plastome genome data to study phylogenetic relationships., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Odago, Waswa, Nanjala, Mutinda, Wanga, Mkala, Oulo, Wang, Zhang, Hu and Wang.)

With c. 2,000 species, Euphorbia is one of the largest angiosperm genera, yet a lack of chloroplast genome (plastome) resources impedes a better understanding of its evolution. In this study, we assembled and annotated 28 plastomes from Euphorbiaceae, of which 15 were newly sequenced. Phylogenomic and comparative analyses of 22 plastome sequences from all four recognized subgenera within Euphorbia revealed that plastome length in Euphorbia is labile, presenting a range of variation c. 42 kb. Large-scale expansions of the inverted repeat (IR) region were identified, and at the extreme opposite, the near-complete loss of the IR region (with only 355 bp left) was detected for the first time in Euphorbiaceae. Other structural variations, including gene inversion and duplication, and gene loss/pseudogenization, were also observed. We screened the most promising molecular markers from both intergenic and coding regions for phylogeny-based utilities, and estimated maximum likelihood and Bayesian phylogenies from four datasets including whole plastome sequences. The monophyly of Euphorbia is supported, and its four subgenera are recovered in a successive sister relationship. Our study constitutes the first comprehensive investigation on the plastome structural variation in Euphorbia and it provides resources for phylogenetic research in the genus, facilitating further studies on its taxonomy, evolution, and conservation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wei, Pérez-Escobar, Musili, Huang, Yang, Hu, Hu, Grace and Wang.)

Background: Somatosensory impairments and pain are common symptoms following stroke. However, the condition of perception and pain threshold for pressure stimuli and the factors that can influence this in individuals with stroke are still unclear. This study aimed to investigate the gender differences in pressure pain threshold (PPT) and positive somatosensory signs for pressure stimuli, and explore the effects of joint pain, motor function, and activities of daily living (ADL) on pain threshold in post-stroke patients., Design: A cross-sectional study., Methods: A total of 60 participants with stroke were recruited, and their pain condition, motor functions, and ADL were evaluated by the Fugl-Meyer assessment of joint pain scale, motor function scale, and Barthel index, respectively. PPTs in eight tested points at the affected and unaffected sides were assessed., Results: Significant differences in PPTs were found between male and female patients in all measured muscles ( p < 0.05). Positive somatosensory signs for pressure stimuli, including hypoalgesia and hyperalgesia, were frequently found at the affected side, particularly in the extremity muscles, but such signs were not significantly influenced by gender ( p > 0.05). More equal PPTs between both sides and relatively lower PPTs at the affected side in the trunk and medial gastrocnemius muscles ( p < 0.05) were observed in patients with less pain, better motor functions, and ADL., Conclusion: Gender differences widely exist in post-stroke survivors either at the affected or unaffected side, which are multifactorial. Sensory loss and central and/or peripheral sensitization, such as hypoalgesia and hyperalgesia for pressure stimuli, caused by a brain lesion are common signs in male and female stroke patients. Moreover, patients who are in a better condition show a more symmetrical pain sensitivity between both sides in the trunk and in female lower extremities, indicating the bidirectional improvement of somatosensory abnormalities caused by a possible neural plasticity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Wang, Hu, Ding, Shen, Yang, Rong and Wang.)

Acanthochlamys P.C. Kao is a Chinese endemic monotypic genus, whereas Xerophyta Juss . is a genus endemic to Africa mainland, Arabian Peninsula and Madagascar with ca.70 species. In this recent study, the complete chloroplast genome of Acanthochlamys bracteata was sequenced and its genome structure compared with two African Xerophyta species ( Xerophyta spekei and Xerophyta viscosa ) present in the NCBI database. The genomes showed a quadripartite structure with their sizes ranging from 153,843 bp to 155,498 bp, having large single-copy (LSC) and small single-copy (SSC) regions divided by a pair of inverted repeats (IR regions). The total number of genes found in A. bracteata , X. spekei and X. viscosa cp genomes are 129, 130, and 132, respectively. About 50, 29, 28 palindromic, forward and reverse repeats and 90, 59, 53 simple sequence repeats (SSRs) were found in the A. bracteata , X. spekei , and X. viscosa cp genome, respectively. Nucleotide diversity analysis in all species was 0.03501, Ka/Ks ratio average score was calculated to be 0.26, and intergeneric K2P value within the Order Pandanales was averaged to be 0.0831. Genomic characterization was undertaken by comparing the genomes of the three species of Velloziaceae and it revealed that the coding regions were more conserved than the non-coding regions. However, key variations were noted mostly at the junctions of IRs/SSC regions. Phylogenetic analysis suggests that A. bracteata species has a closer genetic relationship to the genus Xerophyta . The present study reveals the complete chloroplast genome of A. bracteata and gives a genomic comparative analysis with the African species of Xerophyta . Thus, can be useful in developing DNA markers for use in the study of genetic variabilities and evolutionary studies in Velloziaceae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wanga, Dong, Oulo, Mkala, Yang, Onjalalaina, Gichua, Kirika, Gituru, Hu and Wang.)

The genus Alchemilla L., known for its medicinal and ornamental value, is widely distributed in the Holarctic regions with a few species found in Asia and Africa. Delimitation of species within Alchemilla is difficult due to hybridization, autonomous apomixes, and polyploidy, necessitating efficient molecular-based characterization. Herein, we report the initial complete chloroplast (cp) genomes of Alchemilla . The cp genomes of two African ( Afromilla ) species Alchemilla pedata and Alchemilla argyrophylla were sequenced, and phylogenetic and comparative analyses were conducted in the family Rosaceae. The cp genomes mapped a typical circular quadripartite structure of lengths 152,438 and 152,427 base pairs (bp) in A. pedata and A. argyrophylla , respectively. Alchemilla cp genomes were composed of a pair of inverted repeat regions (IRa/IRb) of length 25,923 and 25,915 bp, separating the small single copy (SSC) region of 17,980 and 17,981 bp and a large single copy (LSC) region of 82,612 and 82,616 bp in A. pedata and A. argyrophylla , respectively. The cp genomes encoded 114 unique genes including 88 protein-coding genes, 37 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Additionally, 88 and 95 simple sequence repeats (SSRs) and 37 and 40 tandem repeats were identified in A. pedata and A. argyrophylla , respectively. Significantly, the loss of group II intron in atpF gene in Alchemilla species was detected. Phylogenetic analysis based on 26 whole cp genome sequences and 78 protein-coding gene sequences of 27 Rosaceae species revealed a monophyletic clustering of Alchemilla nested within subfamily Rosoideae. Based on a protein-coding region, negative selective pressure (Ka/Ks < 1) was detected with an average Ka/Ks value of 0.1322 in A. argyrophylla and 0.1418 in A. pedata . The availability of complete cp genome in the genus Alchemilla will contribute to species delineation and further phylogenetic and evolutionary studies in the family Rosaceae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Rono, Dong, Yang, Mutie, Oulo, Malombe, Kirika, Hu and Wang.)

The giant lobelias in East African mountains are good models for studying molecular mechanisms of adaptation to different altitudes. In this study, we generated RNA-seq data of a middle-altitude species Lobelia aberdarica and a high-altitude species L. telekii, followed by selective pressure estimation of their orthologous genes. Our aim was to explore the important genes potentially involved in adaptation to different altitudes. About 9.3 Gb of clean nucleotides, 167,929-170,534 unigenes with total lengths of 159,762,099-171,138,936 bp for each of the two species were generated. OrthoMCL method identified 3,049 1:1 orthologous genes (each species was represented by one ortholog). Estimations of non-synonymous to synonymous rate were performed using an approximate method and a maximum likelihood method in PAML. Eighty-five orthologous genes were under positive selection. At least 8 of these genes are possibly involved in DNA repair, response to DNA damage and temperature stimulus, and regulation of gene expression, which hints on how giant lobelias adapt to high altitudinal environment that characterized by cold, low oxygen, and strong ultraviolet radiation. The negatively selected genes are over-represented in Gene Ontology terms of hydrolase, macromolecular complex assembly among others. This study sheds light on understanding the molecular mechanism of adaptation to different altitudes, and provides genomic resources for further studies of giant lobelias.