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Arabidopsis thaliana, particularly the ecotype Columbia-0 (Col-0), has been extensively employed in the study of genetics of the nuclear genome. However, the difficulty of modifying the plastid genome of Col-0, the most widely used ecotype, has hindered investigation of the functional interactions between nuclear-encoded and plastid-encoded genes in this ecotype. Recently, we achieved targeted base editing, substituting a specific C:G pair with a T:A pair in the plastid genome of Col-0 through the application of genome-editing technology. This article introduces the method employed to accomplish this targeted base editing. The process involves four steps: (i) designing and constructing a binary vector encoding the genome-editing enzyme, (ii) introducing the binary vector into the nuclear genome of Col-0 via floral dipping, (iii) identifying base-edited plants, and (iv) verifying inheritance of the edited base(s) by the next generation. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design and construction of a binary vector encoding ptpTALECD or ptpTALECD_v2 Basic Protocol 2: Agrobacterium-mediated introduction of a binary vector into the Arabidopsis nuclear genome Basic Protocol 3: Selection of plants harboring T-DNA in the nucleus and detection of base editing in the plastid genome., (© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

As the Earth warms, understanding the long-term dynamics of forest ecosystems is essential for guiding forest management and biodiversity conservation. Insights from past dynamics may provide valuable lessons for managing today's forests. Here, we investigated the spatiotemporal evolution of global larches to gain further insights into how boreal forests change over time. We first reconstructed a highly resolved and robust phylogeny of Larix covering all widely recognized species, using both transcriptome-based 1,301 orthologous genes (OGs) and plastid genomes. In sharp contrast to previous studies, an unexpected deep split between the circumboreal and Qinghai-Tibetan Plateau (QTP) larches was revealed in our study. Within each lineage, two geographically distinct clades were further resolved. Biogeographical analyses suggest that Larix might have an origin of Eocene in high-latitude uplands, and during the Miocene, all extant species have appeared. Cenozoic climate- and orogeny-triggered vicariance likely played a major role in the divergence of global larches. Our results also demonstrate that the proto-boreal forest biome may have a relatively old origin back to the early Miocene, and significant winnowing and species alteration would have occurred as the climate shifted to much colder and drier conditions during the Neogene. Ecological niche analyses show various responses of the circumboreal and QTP larches under different climate scenarios, but both lineages are negatively impacted by warming climates. These findings have important conservation implications given the sensitivity of boreal forests in the face of global warming. Our work further emphasizes the importance of a solid phylogenetic framework for evolutionary and biogeographical inferences., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plastids represent promising targets in plant genetic engineering for many biotech applications, ranging from their use as bioreactors for the overproduction of valuable molecules to the installation of transgenes for improving plant traits. For over 30 years, routine methods of plastid transformation have relied on homologous recombination integrating vectors. However, nonintegrating episomal plasmids have recently received more attention as an innovative tool for the plastid genetic engineering of plant cells. One of these novel technologies is the mini-synplastome, an episomal plasmid with a chloroplast-specific origin of replication ( ori ) used to express transgenes in plastids. In order to improve episome sequence stability overtime by reducing the frequency of spurious recombination events, an optimized version of mini-synplastome (Gen3) was designed. The innovation in the Gen3 design was to substantially reduce the size of the plastomic sequence containing oris to include only domains involved in replication and to reduce the sequence homology of the whole episome with the endogenous plastome. In this work, we have demonstrated that Gen3 can be used to install a multigene pathway in Solanum tuberosum (potato) chloroplasts, and the episome is stable in a full-length circular form at high copy number throughout all plant developmental stages to anthesis in plants with normal phenotypic parameters. It is anticipated that in the next decade the mini-synplastome will be a valuable tool for installing complex genetic circuits in plastids.

Background: Recent studies have revealed atypical features in the plastomes of the family Cactaceae, the largest lineage of succulent species adapted to arid and semi-arid regions. Most plastomes sequenced to date are from short-globose and cylindrical cacti, while little is known about plastomes of epiphytic cacti. Published cactus plastomes reveal reduction and complete loss of IRs, loss of genes, pseudogenization, and even degeneration of tRNA structures. Aiming to contribute with new insights into the plastid evolution of Cactaceae, particularly within the tribe Rhipsalideae, we de novo assembled and analyzed the plastomes of Lepismium cruciforme and Schlumbergera truncata, two South American epiphytic cacti., Methods and Results: Our data reveal many gene losses in both plastomes and the first loss of functionality of the trnT-GGU gene in Cactaceae. The trnT-GGU is a pseudogene in L. cruciforme plastome and appears to be degenerating in the tribe Rhipsalideae. Although the plastome structure is conserved among the species of the tribe Rhipsalideae, with tribe-specific rearrangements, we mapped around 200 simple sequence repeats and identified nine nucleotide polymorphism hotspots, useful to improve the phylogenetic resolutions of the Rhipsalideae. Furthermore, our analysis indicated high gene divergence and rapid evolution of RNA editing sites in plastid protein-coding genes in Cactaceae., Conclusions: Our findings show that some characteristics of the Rhipsalideae tribe are conserved, such as plastome structure with IRs containing only the ycf2 and two tRNA genes, structural degeneration of the trnT-GGU gene and ndh complex, and lastly, pseudogenization of rpl33 and rpl23 genes, both plastid translation-related genes., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The taxonomic classification of the genera Salsola L., Pyankovia Akhani and Roalson, and Xylosalsola Tzvelev within Chenopodiaceae Vent. (Amaranthaceae s.l.) remains controversial, with the precise number of species within these genera still unresolved. This study presents a comparative analysis of the complete plastid genomes of S. foliosa , S. tragus , P. affinis , and X. richteri species collected in Kazakhstan. The assembled plastid genomes varied in length, ranging from 151,177 bp to 152,969 bp for X. richteri and S. tragus . These genomes contained 133 genes, of which 114 were unique, including 80 protein-coding, 30 tRNA, and 4 rRNA genes. Thirteen regions, including ndhC-ndhD , rps16-psbK , petD , rpoC2 , ndhA , petB , clpP , atpF , ycf3 , accD , ndhF-ndhG , matK , and rpl20-rpl22 , exhibited relatively high levels of nucleotide variation. A total of 987 SSRs were detected across the four analyzed plastid genomes, primarily located in the intergenic spacer regions. Additionally, 254 repeats were identified, including 92 tandem repeats, 88 forward repeats, 100 palindromic repeats, and only one reverse repeat. A phylogenetic analysis revealed clear clustering into four clusters corresponding to the Salsoleae and Caroxyloneae tribe clades. These nucleotide sequences obtained in this study represent a valuable resource for future phylogenetic analyses within the Salsoleae s.l. tribe.

The tribe Collabieae (Epidendroideae, Orchidaceae) comprises approximately 500 species. Generic delimitation within Collabieae are confusing and phylogenetic interrelationships within the Collabieae have not been well resolved. Plastid genomes and nuclear internal transcribed spacer (ITS) sequences were used to estimate the phylogenetic relationships, ancestral ranges, and diversification rates of Collabieae. The results showed that Collabieae was subdivided into nine clades with high support. We proposed to combine Ancistrochilus and Pachystoma into Spathoglottis, merge Collabium and Chrysoglossum into Diglyphosa, and separate Pilophyllum and Hancockia as distinctive genera. The diversification of the nine clades of Collabieae might be associated with the uplift of the Himalayas during the Late Oligocene/Early Miocene. The enhanced East Asian summer monsoon in the Late Miocene may have promoted the rapid diversification of Collabieae at a sustained high diversification rate. The increased size of terrestrial pseudobulbs may be one of the drivers of Collabieae diversification. Our results suggest that the establishment and development of evergreen broadleaved forests facilitated the diversification of Collabieae., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Reevesia is an eastern Asian-eastern North American disjunction genus in the family Malvaceae s.l. and comprises approximately 25 species. The relationships within the genus are not well understood. Here, 15 plastomes representing 12 Reevesia species were compared, with the aim of better understanding the species circumscription and phylogenetic relationships within the genus and among genera in the family Malvaceae s.l. The 11 newly sequenced plastomes range between 161,532 and 161, 945 bp in length. The genomes contain 114 unique genes, 18 of which are duplicated in the inverted repeats (IRs). Gene content of these plastomes is nearly identical. All the protein-coding genes are under purifying selection in the Reevesia plastomes compared. The top ten hypervariable regions, SSRs, and the long repeats identified are potential molecular markers for future population genetic and phylogenetic studies. Phylogenetic analysis based on the whole plastomes confirmed the monophyly of Reevesia and a close relationship with Durio (traditional Bombacaceae) in subfamily Helicteroideae, but not with the morphologically similar genera Pterospermum and Sterculia (both of traditional Sterculiaceae). Phylogenetic relationships within Reevesia suggested that two species, R. pubescens and R. thyrsoidea, as newly defined, are not monophyletic. Six taxa, R. membranacea, R. xuefengensis, R. botingensis, R. lofouensis, R. longipetiolata and R. pycnantha, are suggested to be recognized., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Main Conclusion: Significant past, present, and potential future research into the organellar (plastid and mitochondrial) genomes of gymnosperms that can provide insight into the unknown origin and evolution of plants is highlighted. Gymnosperms are vascular seed plants that predominated the ancient world before their sister clade, angiosperms, took over during the Late Cretaceous. The divergence of gymnosperms and angiosperms took place around 300 Mya, with the latter evolving into the diverse group of flowering plants that dominate the plant kingdom today. Although gymnosperms have reportedly made some evolutionary innovations, the literature on their genome advances, particularly their organellar (plastid and mitochondrial) genomes, is relatively scattered and fragmented. While organellar genomes can shed light on plant origin and evolution, they are frequently overlooked, due in part to their limited contribution to gene expression and lack of evolutionary dynamics when compared to nuclear genomes. A better understanding of gymnosperm organellar genomes is critical because they reveal genetic changes that have contributed to their unique adaptations and ecological success, potentially aiding in plant survival, enhancement, and biodiversity conservation in the face of climate change. This review reveals significant information and gaps in the existing knowledge base of organellar genomes in gymnosperms, as well as the challenges and research needed to unravel their complexity., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The plastid organelle is vital for photosynthesis and energy production. Advances in sequencing technology have enabled the exploration of plastomic resources, offering insights into plant evolution, diversity, and conservation. As an important group of horticultural ornamentals in the Crassulaceae family, Sempervivum plants are known for their unique rosette-like structures and reproduction through offsets. Despite their popularity, the classification status of Sempervivum remains uncertain, with only a single plastome sequence currently available. Furthermore, codon usage bias (CUB) is a widespread phenomenon of the unbalanced usage of synonymous codons in the coding sequence (CDS). However, due to the limited available plastid data, there has been no research that focused on the CUB analysis among Sempervivum until now. To address these gaps, we sequenced and released the plastomes of seven species and one subspecies from Sempervivum , revealing several consistent patterns. These included a shared 110 bp extension of the rps19 gene, 14 hypervariable regions (HVRs) with distinct nucleotide diversity (π: 0.01173 to 0.02702), and evidence of selective pressures shaping codon usage. Notably, phylogenetic analysis robustly divided the monophyletic clade into two sections: Jovibarba and Sempervivum. In conclusion, this comprehensive plastomic resource provides valuable insights into Sempervivum evolution and offers potential molecular markers for DNA barcoding.

Background: Expansion and contraction of inverted repeats can cause considerable variation of plastid genomes (plastomes) in angiosperms. However, little is known about whether structural variations of plastomes are associated with adaptation to or occupancy of new environments. Moreover, adaptive evolution of angiosperm plastid genes remains poorly understood. Here, we sequenced the complete plastomes for four species of xerophytic Ceratocephala and hydrophytic Myosurus, as well as Ficaria verna. By an integration of phylogenomic, comparative genomic, and selection pressure analyses, we investigated evolutionary patterns of plastomes in Ranunculeae and their relationships with adaptation to dry and aquatic habitats., Results: Owing to the significant contraction of the boundary of IR A /LSC towards the IR A , plastome sizes and IR lengths of Myosurus and Ceratocephala are smaller within Ranunculeae. Compared to other Ranunculeae, the Myosurus plastome lost clpP and rps16, one copy of rpl2 and rpl23, and one intron of rpoC1 and rpl16, and the Ceratocephala plastome added an infA gene and lost one copy of rpl2 and two introns of clpP. A total of 11 plastid genes (14%) showed positive selection, two genes common to Myosurus and Ceratocephala, seven in Ceratocephala only, and two in Myosurus only. Four genes showed strong signals of episodic positive selection. The rps7 gene of Ceratocephala and the rpl32 and ycf4 genes of Myosurus showed an increase in the rate of variation close to 3.3 Ma., Conclusions: The plastomic structure variations as well as the positive selection of two plastid genes might be related to the colonization of new environments by the common ancestor of Ceratocephala and Myosurus. The seven and two genes under positive selection might be related to the adaptation to dry and aquatic habitats in Ceratocephala and Myosurus, respectively. Moreover, intensified aridity and frequent sea-level fluctuations, as well as global cooling, might have favored an increased rate of change in some genes at about 3.3 Ma, associated with adaptation to dry and aquatic environments, respectively. These findings suggest that changing environments might have influenced structural variations of plastomes and fixed new mutations arising on some plastid genes owing to adaptation to specific habitats., (© 2024. The Author(s).)

Passiflora tripartita var. mollissima , known locally as poro-poro, is an important native fruit used in traditional Peruvian medicine with relevant agro-industrial and pharmaceutical potential for its antioxidant capacity for human health. However, to date, only a few genetic data are available, which limits exploring its genetic diversity and developing new genetic studies for its improvement. We report the poro-poro plastid genome to expand the knowledge of its molecular markers, evolutionary studies, molecular pathways, and conservation genetics. The complete chloroplast (cp) genome is 163,451 bp in length with a typical quadripartite structure, containing a large single-copy region of 85,525 bp and a small single-copy region of 13,518 bp, separated by a pair of inverted repeat regions (IR) of 32,204 bp, and the overall GC content was 36.87%. This cp genome contains 128 genes (110 genes were unique and 18 genes were found duplicated in each IR region), including 84 protein-coding genes, 36 transfer RNA-coding genes, eight ribosomal RNA-coding genes, and 13 genes with introns (11 genes with one intron and two genes with two introns). The inverted repeat region boundaries among species were similar in organization, gene order, and content, with a few revisions. The phylogenetic tree reconstructed based on single-copy orthologous genes and maximum likelihood analysis demonstrates poro-poro is most closely related to Passiflora menispermifolia and Passiflora oerstedii. In summary, our study constitutes a valuable resource for studying molecular evolution, phylogenetics, and domestication. It also provides a powerful foundation for conservation genetics research and plant breeding programs. To our knowledge, this is the first report on the plastid genome of Passiflora tripartita var. mollissima from Peru., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Aliaga F et al.)

Background: The genus Triplostegia contains two recognized species, T. glandulifera and T. grandiflora, but its phylogenetic position and species delimitation remain controversial. In this study, we assembled plastid genomes and nuclear ribosomal DNA (nrDNA) cistrons sampled from 22 wild Triplostegia individuals, each from a separate population, and examined these with 11 recently published Triplostegia plastomes. Morphological traits were measured from herbarium specimens and wild material, and ecological niche models were constructed., Results: Triplostegia is a monophyletic genus within the subfamily Dipsacoideae comprising three monophyletic species, T. glandulifera, T. grandiflora, and an unrecognized species Triplostegia sp. A, which occupies much higher altitude than the other two. The new species had previously been misidentified as T. glandulifera, but differs in taproot, leaf, and other characters. Triplotegia is an old genus, with stem age 39.96 Ma, and within it T. glandulifera diverged 7.94 Ma. Triplostegia grandiflora and sp. A diverged 1.05 Ma, perhaps in response to Quaternary climate fluctuations. Niche overlap between Triplostegia species was positively correlated with their phylogenetic relatedness., Conclusions: Our results provide new insights into the species delimitation of Triplostegia, and indicate that a taxonomic revision of Triplostegia is needed. We also identified that either rpoB-trnC or ycf1 could serve as a DNA barcode for Triplostegia., (© 2023. The Author(s).)

The tribe Ranunculeae, Ranunculaceae, comprising 19 genera widely distributed all over the world. Although a large number of Sanger sequencing-based molecular phylogenetic studies have been published, very few studies have been performed on using genomic data to infer phylogenetic relationships within Ranunculeae. In this study, the complete plastid genomes of nine species (eleven samples) from Ceratocephala , Halerpestes , and Ranunculus were de novo assembled using a next-generation sequencing method. Previously published plastomes of Oxygraphis and other related genera of the family were downloaded from GenBank for comparative analysis. The complete plastome of each Ranunculeae species has 112 genes in total, including 78 protein-coding genes, 30 transfer RNA genes, and four ribosomal RNA genes. The plastome structure of Ranunculeae samples is conserved in gene order and arrangement. There are no inverted repeat (IR) region expansions and only one IR contraction was found in the tested samples. This study also compared plastome sequences across all the samples in gene collinearity, codon usage, RNA editing sites, nucleotide variability, simple sequence repeats, and positive selection sites. Phylogeny of the available Ranunculeae species was inferred by the plastome data using maximum-likelihood and Bayesian inference methods, and data partitioning strategies were tested. The phylogenetic relationships were better resolved compared to previous studies based on Sanger sequencing methods, showing the potential value of the plastome data in inferring the phylogeny of the tribe.

Background: Plastid genomes (plastomes) have long been recognized as highly conserved in their overall structure, size, gene arrangement and content among land plants. However, recent studies have shown that some lineages present unusual variations in some of these features. Members of the cactus family are one of these lineages, with distinct plastome structures reported across disparate lineages, including gene losses, inversions, boundary movements or loss of the canonical inverted repeat (IR) region. However, only a small fraction of cactus diversity has been analysed so far., Methods: Here, we investigated plastome features of the tribe Opuntieae, the remarkable prickly pear cacti, which represent one of the most diverse and important lineages of Cactaceae. We assembled de novo the plastome of 43 species, representing a comprehensive sampling of the tribe, including all seven genera, and analysed their evolution in a phylogenetic comparative framework. Phylogenomic analyses with different datasets (full plastome sequences and genes only) were performed, followed by congruence analyses to assess signals underlying contentious nodes., Key Results: Plastomes varied considerably in length, from 121 to 162 kbp, with striking differences in the content and size of the IR region (contraction and expansion events), including a lack of the canonical IR in some lineages and the pseudogenization or loss of some genes. Overall, nine different types of plastomes were reported, deviating in the presence of the IR region or the genes contained in the IR. Overall, plastome sequences resolved phylogenetic relationships within major clades of Opuntieae with high bootstrap values but presented some contentious nodes depending on the dataset analysed (e.g. whole plastome vs. genes only). Congruence analyses revealed that most plastidial regions lack phylogenetic resolution, while few markers are supporting the most likely topology. Likewise, alternative topologies are driven by a handful of plastome markers, suggesting recalcitrant nodes in the phylogeny., Conclusions: Our study reveals a dynamic nature of plastome evolution across closely related lineages, shedding light on peculiar features of plastomes. Variation of plastome types across Opuntieae is remarkable in size, structure and content and can be important for the recognition of species in some major clades. Unravelling connections between the causes of plastome variation and the consequences for species biology, physiology, ecology, diversification and adaptation is a promising and ambitious endeavour in cactus research. Although plastome data resolved major phylogenetic relationships, the generation of nuclear genomic data is necessary to confront these hypotheses and assess the recalcitrant nodes further., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Potamogetonaceae are aquatic plants divided into six genera. The largest genus in the family is Potamogeton , which is morphologically diverse with many hybrids and polyploids. Potamogetonaceae plastomes were conserved in genome size (155,863 bp-156,669 bp), gene contents (113 genes in total, comprising 79 protein-coding genes and 30 tRNA and 4 rRNA genes), and GC content (36.5%). However, we detected a duplication of the trnH gene in the IR region of the Potamogeton crispus and P. maakianus plastomes. A comparative analysis of Alismatales indicated that the plastomes of Potamogetonaceae, Cymodaceae, and Ruppiaceae have experienced a 6-kb inversion of the rbcL-trnV region and the ndh complex has been lost in the Najas flexilis plastome. Five divergent hotspots ( rps16-trnQ , atpF intron, rpoB-trnC , trnC-psbM , and ndhF-rpl32 ) were identified among the Potamogeton plastomes, which will be useful for species identification. Phylogenetic analyses showed that the family Potamogetonaceae is a well-defined with 100% bootstrap support and divided into two different clades, Potamogeton and Stuckenia . Compared to the nucleotide substitution rates among Alismatales, we found neutral selection in all plastid genes of Potamogeton species. Our results reveal the complete plastome sequences of Potamogeton species, and will be helpful for taxonomic identification, the elucidation of phylogenetic relationships, and the plastome structural analysis of aquatic plants.

Limestone karsts are renowned for extremely high species richness and endemism. Aspidistra (Asparagaceae) is among the highly diversified genera distributed in karst areas, making it an ideal group for studying the evolutionary mechanisms of karst plants. The taxonomy and identification of Aspidistra species are mainly based on their specialized and diverse floral structures. Aspidistra plants have inconspicuous flowers, and the similarity in vegetative morphology often leads to difficulties in species discrimination. Chloroplast genomes possess variable genetic information and offer the potential for interspecies identification. However, as yet there is little information about the interspecific diversity and evolution of the plastid genomes of Aspidistra . In this study, we reported chloroplast (cp) genomes of seven Aspidistra species ( A. crassifila , A. dolichanthera , A. erecta , A. longgangensis , A. minutiflora , A. nankunshanensis , and A. retusa ). These seven highly-conserved plastid genomes all have a typical quartile structure and include a total of 113 unique genes, comprising 79 protein-coding genes, 4 rRNA genes and 30 tRNA genes. Additionally, we conducted a comprehensive comparative analysis of Aspidistra cp genomes. We identified eight divergent hotspot regions ( trnC -GCA- petN , trnE -UUC- psbD , accD-psaI , petA-psbJ , rpl20-rps12 , rpl36-rps8 , ccsA-ndhD and rps15-ycf1 ) that serve as potential molecular markers. Our newly generated Aspidistra plastomes enrich the resources of plastid genomes of karst plants, and an investigation into the plastome diversity offers novel perspectives on the taxonomy, phylogeny and evolution of Aspidistra species.

The modification of photosynthesis-related genes in plastid genomes may improve crop yields. Recently, we reported that a plastid-targeting base editor named ptpTALECD, in which a cytidine deaminase DddA functions as the catalytic domain, can homoplasmically substitute a targeted C to T in plastid genomes of Arabidopsis thaliana. However, some target Cs were not substituted. In addition, although ptpTALECD could substitute Cs on the 3' side of T and A, it was unclear whether it could also substitute Cs on the 3' side of G and C. In this study, we identified the preferential positions of the substituted Cs in ptpTALECD-targeting sequences in the Arabidopsis plastid genome. We also found that ptpTALECD could substitute Cs on the 3' side of all four bases in plastid genomes of Arabidopsis. More recently, a base editor containing an improved version of DddA (DddA11) was reported to substitute Cs more efficiently, and to substitute Cs on the 3' side of more varieties of bases in human mitochondrial genomes than a base editor containing DddA. Here, we also show that ptpTALECD_v2, in which a modified version of DddA11 functions as the catalytic domain, more frequently substituted Cs than ptpTALECD in the Arabidopsis plastid genome. We also found that ptpTALECD_v2 tended to substitute Cs at more positions than ptpTALECD. Our results reveal that ptpTALECD can cause a greater variety of codon changes and amino acid substitutions than previously thought, and that ptpTALECD and ptpTALECD_v2 are useful tools for the targeted base editing of plastid genomes., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plastid genomes (plastomes) of angiosperms are well known for their relative stability in size, structure, and gene content. However, little is known about their heredity and variations in wide crossing. To such an end, the plastomes of five representative rice backcross inbred lines (BILs) developed from crosses of O. glaberrima / O. sativa were analyzed. We found that the size of all plastomes was about 134,580 bp, with a quadripartite structure that included a pair of inverted repeat (IR) regions, a small single-copy (SSC) region and a large single-copy (LSC) region. They contained 76 protein genes, 4 rRNA genes, and 30 tRNA genes. Although their size, structure, and gene content were stable, repeat-mediated recombination, gene expression, and RNA editing were extensively changed between the maternal line and the BILs. These novel discoveries demonstrate that wide crossing causes not only nuclear genomic recombination, but also plastome variation in plants, and that the plastome plays a critical role in coordinating the nuclear-cytoplasmic interaction.

Premise: Pogoniopsis likely represents an independent photosynthesis loss in orchids. We use phylogenomic data to better identify the phylogenetic placement of this fully mycoheterotrophic taxon, and investigate its molecular evolution., Methods: We performed likelihood analysis of plastid and mitochondrial phylogenomic data to localize the position of Pogoniopsis schenckii in orchid phylogeny, and investigated the evolution of its plastid genome., Results: All analyses place Pogoniopsis in subfamily Epidendroideae, with strongest support from mitochondrial data, which also place it near tribe Sobralieae with moderately strong support. Extreme rate elevation in Pogoniopsis plastid genes broadly depresses branch support; in contrast, mitochondrial genes are only mildly rate elevated and display very modest and localized reductions in bootstrap support. Despite considerable genome reduction, including loss of photosynthesis genes and multiple translation apparatus genes, gene order in Pogoniopsis plastomes is identical to related autotrophs, apart from moderately shifted inverted repeat (IR) boundaries. All cis-spliced introns have been lost in retained genes. Two plastid genes (accD, rpl2) show significant strengthening of purifying selection. A retained plastid tRNA gene (trnE-UUC) of Pogoniopsis lacks an anticodon; we predict that it no longer functions in translation but retains a secondary role in heme biosynthesis., Conclusions: Slowly evolving mitochondrial genes clarify the placement of Pogoniopsis in orchid phylogeny, a strong contrast with analysis of rate-elevated plastome data. We documented the effects of the novel loss of photosynthesis: for example, despite massive gene loss, its plastome is fully colinear with other orchids, and it displays only moderate shifts in selective pressure in retained genes., (© 2022 Botanical Society of America.)

Background and Aims: Rhododendron is a species-rich and taxonomically challenging genus due to recent adaptive radiation and frequent hybridization. A well-resolved phylogenetic tree would help to understand the diverse history of Rhododendron in the Himalaya-Hengduan Mountains where the genus is most diverse., Methods: We reconstructed the phylogeny based on plastid genomes with broad taxon sampling, covering 161 species representing all eight subgenera and all 12 sections, including ~45 % of the Rhododendron species native to the Himalaya-Hengduan Mountains. We compared this phylogeny with nuclear phylogenies to elucidate reticulate evolutionary events and clarify relationships at all levels within the genus. We also estimated the timing and diversification history of Rhododendron, especially the two species-rich subgenera Rhododendron and Hymenanthes that comprise >90 % of Rhododendron species in the Himalaya-Hengduan Mountains., Key Results: The full plastid dataset produced a well-resolved and supported phylogeny of Rhododendron. We identified 13 clades that were almost always monophyletic across all published phylogenies. The conflicts between nuclear and plastid phylogenies suggested strongly that reticulation events may have occurred in the deep lineage history of the genus. Within Rhododendron, subgenus Therorhodion diverged first at 56 Mya, then a burst of diversification occurred from 23.8 to 17.6 Mya, generating ten lineages among the component 12 clades of core Rhododendron. Diversification in subgenus Rhododendron accelerated c. 16.6 Mya and then became fairly continuous. Conversely, Hymenanthes diversification was slow at first, then accelerated very rapidly around 5 Mya. In the Himalaya-Hengduan Mountains, subgenus Rhododendron contained one major clade adapted to high altitudes and another to low altitudes, whereas most clades in Hymenanthes contained both low- and high-altitude species, indicating greater ecological plasticity during its diversification., Conclusions: The 13 clades proposed here may help to identify specific ancient hybridization events. This study will help to establish a stable and reliable taxonomic framework for Rhododendron, and provides insight into what drove its diversification and ecological adaption. Denser sampling of taxa, examining both organelle and nuclear genomes, is needed to better understand the divergence and diversification history of Rhododendron., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Main Conclusion: Members of Apiales are monophyletic and radiated in the Late Cretaceous. Fruit morphologies are critical for Apiales evolution and negative selection and mutation pressure play important roles in environmental adaptation. Apiales include many foods, spices, medicinal, and ornamental plants, but the phylogenetic relationships, origin and divergence, and adaptive evolution remain poorly understood. Here, we reconstructed Apiales phylogeny based on 72 plastid genes from 280 species plastid genomes representing six of seven families of this order. Highly supported phylogenetic relationships were detected, which revealed that each family of Apiales is monophyletic and confirmed that Pennanticeae is a member of Apiales. Genera Centella and Dickinsia are members of Apiaceae, and the genus Hydrocotyle previously classified into Apiaceae is confirmed to belong to Araliaceae. Besides, coalescent phylogenetic analysis and gene trees cluster revealed ten genes that can be used for distinguishing species among families of Apiales. Molecular dating suggested that the Apiales originated during the mid-Cretaceous (109.51 Ma), with the families' radiation occurring in the Late Cretaceous. Apiaceae species exhibit higher differentiation compared to other families. Ancestral trait reconstruction suggested that fruit morphological evolution may be related to shifts in plant types (herbaceous or woody), which in turn is related to the distribution areas and species numbers. Codon bias and positive selection analyses suggest that negative selection and mutation pressure may play important roles in environmental adaptation of Apiales members. Our results improve the phylogenetic framework of Apiales and provide insights into the origin, divergence, and adaptive evolution of this order and its members., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: The study presents results of research on the evolution of plastid genomes in Stipa L. which is a large genus of the Poaceae family, comprising species diverse in terms of geographic distribution, growing under highly variated habitat conditions. Complete plastome sequences of 43 taxa from Stipeae and Ampelodesmae tribes were analyzed for the variability of the coding regions against the background of phylogenetic relationships within the genus Stipa. The research hypothesis put forward in our research was that some of coding regions are affected by a selection pressure differentiated between individual phylogenetic lines of Stipa, potentially reducing the phylogenetic informativeness of these CDS. The study aimed to answer the question, which genes evolve in Stipa most rapidly and what kind of changes in the properties of encoded amino acids this entails. Another goal of this research was to find out whether individual genes are affected by positive selection and finally, whether selective pressure is uniform within the genus or does it vary between particular evolutionary lines within the genus., Results: Results of our study proved the presence of selective pressure in 11 genes: ccsA, matK, ndhC, ndhF, ndhK, rbcL, rpoA rpoC1, rpoC2, rps8 and rps11. For the first time the effect of positive selection on the rps8, rps11, and ndhK genes was documented in grasses. The varied pace of evolution, different intensity and effects of selective pressure have been demonstrated between particular phylogenetic lines of the genus tested., Conclusions: Positive selection in plastid genome in Stipa mostly affects photosynthetic genes. The potential strongest adaptive pressure was observed in the rbcL gene, especially in the oldest evolutionary group comprising Central Asian high-mountain species: S. basiplumosa, S. klimesii, S. penicillata and S. purpurea, where adaptive pressure probably affected the amino acids directly related to the efficiency of CO 2 assimilation., (© 2022. The Author(s).)

Background: Mycoheterotrophy is a unique survival strategy adapted to dense forests and has attracted biologists' attention for centuries. However, its evolutionary origin and related plastome degradation are poorly understood. The tribe Neottieae contains various nutrition types, i.e., autotrophy, mixotrophy, and mycoheterotrophy. Here, we present a comprehensive phylogenetic analysis of the tribe based on plastome and nuclear ITS data. We inferred the evolutionary shift of nutrition types, constructed the patterns of plastome degradation, and estimated divergence times and ancestral ranges. We also used an integration of molecular dating and ecological niche modeling methods to investigate the disjunction between the Loess Plateau and Changbai Mountains in Diplandrorchis, a mycoheterotrophic genus endemic to China that was included in a molecular phylogenetic study for the first time., Results: Diplandrorchis was imbedded within Neottia and formed a clade with four mycoheterotrophic species. Autotrophy is the ancestral state in Neottieae, mixotrophy independently originated at least five times, and three shifts from mixotrophy to mycoheterotrophy independently occurred. The five mixotrophic lineages possess all plastid genes or lost partial/all ndh genes, whereas each of the three mycoheterotroph lineages has a highly reduced plastome: one lost part of its ndh genes and a few photosynthesis-related genes, and the other two lost almost all ndh, photosynthesis-related, rpo, and atp genes. These three mycoheterotrophic lineages originated at about 26.40 Ma, 25.84 Ma, and 9.22 Ma, respectively. Diplandrorchis had presumably a wide range in the Pliocene and migrated southward in the Pleistocene., Conclusions: The Pleistocene climatic fluctuations and the resultant migration resulted in the Loess Plateau-Changbai Mountains disjunction of Diplandrorchis. In the evolution of mycoheterotrophic lineages, the loss of plastid-encoded genes and plastome degradation are staged and irreversible, constraining mycoheterotrophs to inhabit understories with low light levels. Accordingly, the rise of local forests might have promoted the origin of conditions in which mycoheterotrophy is advantageous., (© 2022. The Author(s).)

Background: Orobanchaceae is the only flowering plant family with species from free-living nonparasite, hemi-parasite to holoparasite, making it an ideal system for studying the evolution of parasitism. However, both plastid and mitochondrial genome have been sequenced in only few parasitic species in Orobanchaceae. Therefore, further comparative study is wanted to investigate the impact of holoparasitism on organelle genomes evolution between close relatives. Here, we sequenced organelle genomes and transcriptome of holoparasitic Christisonia kwangtungensis and compared it with its closely related groups to analyze similarities and differences in adaption strategies to the holoparasitic lifestyle., Results: The plastid genome of C. kwangtungensis has undergone extensive pseudogenization and gene loss, but its reduction pattern is different from that of Aeginetia indica, the close relative of C. kwangtungensis. Similarly, the gene expression detected in the photosynthetic pathway of these two genera is different. In Orobanchaceae, holoparasites in Buchnereae have more plastid gene loss than Rhinantheae, which reflects their longer history of holoparasitism. Distinct from severe degradation of the plastome, protein-coding genes in the mitochondrial genome of C. kwangtungensis are relatively conserved. Interestingly, besides intracellularly transferred genes which are still retained in its plastid genome, we also found several horizontally transferred genes of plastid origin from diverse donors other than their current hosts in the mitochondrial genome, which probably indicate historical hosts., Conclusion: Even though C. kwangtungensis and A. indica are closely related and share severe degradation of plastome, they adapt organelle genomes to the parasitic lifestyle in different ways. The difference between their gene loss and gene expression shows they ultimately lost photosynthetic genes but through different pathways. Our study exemplifies how parasites part company after achieving holoparasitism., (© 2022. The Author(s).)

Angiosperm genome evolution was marked by many clade-specific whole-genome duplication events. The Microlepidieae is one of the monophyletic clades in the mustard family (Brassicaceae) formed after an ancient allotetraploidization. Postpolyploid cladogenesis has resulted in the extant c. 17 genera and 60 species endemic to Australia and New Zealand (10 species). As postpolyploid genome diploidization is a trial-and-error process under natural selection, it may proceed with different intensity and be associated with speciation events. In Microlepidieae, different extents of homoeologous recombination between the two parental subgenomes generated clades marked by slow ("cold") versus fast ("hot") genome diploidization. To gain a deeper understanding of postpolyploid genome evolution in Microlepidieae, we analyzed phylogenetic relationships in this tribe using complete chloroplast sequences, entire 35S rDNA units, and abundant repetitive sequences. The four recovered intra-tribal clades mirror the varied diploidization of Microlepidieae genomes, suggesting that the intrinsic genomic features underlying the extent of diploidization are shared among genera and species within one clade. Nevertheless, even congeneric species may exert considerable morphological disparity (e.g. in fruit shape), whereas some species within different clades experience extensive morphological convergence despite the different pace of their genome diploidization. We showed that faster genome diploidization is positively associated with mean morphological disparity and evolution of chloroplast genes (plastid-nuclear genome coevolution). Higher speciation rates in perennials than in annual species were observed. Altogether, our results confirm the potential of Microlepidieae as a promising subject for the analysis of postpolyploid genome diploidization in Brassicaceae., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Cistanche is an important genus of Orobanchaceae, with critical medicinal, economic, and desertification control values. However, the phylogenetic relationships of Cistanche genus remained obscure. To date, no effective molecular markers have been reported to discriminate effectively the Cistanche closely related species reported here. In this study, we obtained and characterized the plastomes of four Cistanche species from China, to clarify the phylogenetic relationship within the genus, and to develop molecular markers for species discrimination.  RESULTS: Four Cistanche species (Cistanche deserticola, Cistanche salsa, Cistanche tubulosa and Cistanche sinensis), were deep-sequenced with Illumina. Their plastomes were assembled using SPAdes and annotated using CPGAVAS2. The plastic genomes were analyzed in detail, finding that all showed the conserved quadripartite structure (LSC-IR-SSC-IR) and with full sizes ranging from 75 to 111 Kbp. We observed a significant contraction of small single copy region (SSC, ranging from 0.4-29 Kbp) and expansion of inverted repeat region (IR, ranging from 6-30 Kbp), with C. deserticola and C. salsa showing the smallest SSCs with only one gene (rpl32). Compared with other Orobanchaceae species, Cistanche species showed extremely high rates of gene loss and pseudogenization, as reported for other parasitic Orobanchaceae species. Furthermore, analysis of sequence divergence on protein-coding genes showed the three genes (rpl22, clpP and ycf2) had undergone positive selection in the Cistanche species under study. In addition, by comparison of all available Cistanche plastomes we found 25 highly divergent intergenic spacer (IGS) regions that were used to predict two DNA barcode markers (Cis-mk01 and Cis-mk02 based on IGS region trnR-ACG-trnN-GUU) and eleven specific DNA barcode markers using Ecoprimer software. Experimental validation showed 100% species discrimination success rate with both type of markers., Conclusion: Our findings have shown that Cistanche species are an ideal model to investigate the structure variation, gene loss and pseudogenization during the process of plastome evolution in parasitic species, providing new insights into the evolutionary relationships among the Cistanche species. In addition, the developed DNA barcodes markers allow the proper species identification, ensuring the effective and safe use of Cistanche species as medicinal products., (© 2022. The Author(s).)

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cotoneaster is a taxonomically and ornamentally important genus in the family Rosaceae; however, phylogenetic relationships among its species are complicated owing to insufficient morphological diagnostic characteristics and hybridization associated with polyploidy and apomixis. In this study, we sequenced the complete plastomes of seven Cotoneaster species ( C. dielsianus , C. hebephyllus , C. integerrimus , C. mongolicus , C. multiflorus , C. submultiflorus , and C. tenuipes ) and included the available complete plastomes in a phylogenetic analysis to determine the origin of C. wilsonii , which is endemic to Ulleung Island, Korea. Furthermore, based on 15 representative lineages within the genus, we carried out the first comparative analysis of Cotoneaster plastid genomes to gain an insight into their molecular evolution. The plastomes were highly conserved, with sizes ranging from 159,595 bp ( C. tenuipes ) to 160,016 bp ( C. hebephyllus ), and had a GC content of 36.6%. The frequency of codon usage showed similar patterns among the 15 Cotoneaster species, and 24 of the 35 protein-coding genes were predicted to undergo RNA editing. Eight of the 76 common protein-coding genes, including ccsA , matK , ndhD , ndhF , ndhK , petA , rbcL , and rpl16 , were positively selected, implying their potential roles in adaptation and speciation. Of the 35 protein-coding genes, 24 genes (15 photosynthesis-related, seven self-replications, and three others) were found to harbor RNA editing sites. Furthermore, several mutation hotspots were identified, including trnG -UCC/ trnR -UCU/ atpA and trnT -UGU/ trnL -UAA. Maximum likelihood analysis based on 57 representative plastomes of Cotoneaster and two Heteromeles plastomes as outgroups revealed two major lineages within the genus, which roughly correspond to two subgenera, Chaenopetalum and Cotoneaster . The Ulleung Island endemic, C. wilsonii , shared its most recent common ancestor with two species, C. schantungensis and C. zabelii , suggesting its potential origin from geographically close members of the subgenus Cotoneaster , section Integerrimi .

Prunus subgenus Cerasus (cherry) is an economically important group that distributed in temperate regions of the northern hemisphere. However, shared interspecific morphological traits and variability across taxa of Cerasus are among the impediments to taxonomic efforts to correctly delimit taxa. This is further complicated by a lack of genetic information on these taxa, with no focused genomic or phylogenetic studies being done on Cerasus. In this study, we conducted comparative analysis on the complete plastid genomes (plastomes) of 20 Cerasus species to gain a greater understanding of the attributes of the plastome of these taxa while helping resolve their phylogenetic placement in Prunus sensu lato and interspecific relationships within the subgenus. Our results displayed that (1) the plastomes of the 20 Cerasus species studied exhibited a typical quadripartite structure with conversed genome arrangement, structure, and moderate divergence. (2) The average size of complete plastomes for the Cerasus taxa studied was 157,861 bp, ranging from 157,458 to 158,024 bp. A total of 134 genes were annotated, including 86 protein-coding genes, 40 tRNAs, and 8 rRNAs across all species. In simple sequence repeat analysis, we found Cerasus had a comparable number of dispersed and tandem repeats to those identified in other angiosperm taxa, with only P. pseudocerasus found to contain trinucleotide repeats. Nucleotide diversity analysis revealed that the trnG-GCC gene and rpl32-trnL region had the highest Pi value showing potential as phylogenetic markers. (3) Two phylogenetic trees of the plastomes verified the monophyletic relationship of Cerasus and provided a more resolved species-level phylogeny. Our study provides detailed plastome information for exploring the phylogeny of subg. Cerasus taxa. We identified various types of repeats and nucleotide diversity hotspots, which can be a reference for species identification and reconstruction of phylogenetic relationships., Competing Interests: The authors have declared that no competing interests exist.

Background: Plastid genomes (plastomes) present great potential in resolving multiscale phylogenetic relationship but few studies have focused on the influence of genetic characteristics of plastid genes, such as genetic variation and phylogenetic discordance, in resolving the phylogeny within a lineage. Here we examine plastome characteristics of Cycas L., the most diverse genus among extant cycads, and investigate the deep phylogenetic relationships within Cycas by sampling 47 plastomes representing all major clades from six sections., Results: All Cycas plastomes shared consistent gene content and structure with only one gene loss detected in Philippine species C. wadei. Three novel plastome regions (psbA-matK, trnN-ndhF, chlL-trnN) were identified as containing the highest nucleotide variability. Molecular evolutionary analysis showed most of the plastid protein-coding genes have been under purifying selection except ndhB. Phylogenomic analyses that alternatively included concatenated and coalescent methods, both identified four clades but with conflicting topologies at shallow nodes. Specifically, we found three species-rich Cycas sections, namely Stangerioides, Indosinenses and Cycas, were not or only weakly supported as monophyly based on plastomic phylogeny. Tree space analyses based on different tree-inference methods both revealed three gene clusters, of which the cluster with moderate genetic properties showed the best congruence with the favored phylogeny., Conclusions: Our exploration in plastomic data for Cycas supports the idea that plastid protein-coding genes may exhibit discordance in phylogenetic signals. The incongruence between molecular phylogeny and morphological classification reported here may largely be attributed to the uniparental attribute of plastid, which cannot offer sufficient information to resolve the phylogeny. Contrasting to a previous consensus that genes with longer sequences and a higher proportion of variances are superior for phylogeny reconstruction, our result implies that the most effective phylogenetic signals could come from loci that own moderate variation, GC content, sequence length, and underwent modest selection., (© 2022. The Author(s).)

Northern Hemisphere forests changed drastically in the early Eocene with the diversification of the oak family (Fagaceae). Cooling climates over the next 20 million years fostered the spread of temperate biomes that became increasingly dominated by oaks and their chestnut relatives. Here we use phylogenomic analyses of nuclear and plastid genomes to investigate the timing and pattern of major macroevolutionary events and ancient genome-wide signatures of hybridization across Fagaceae. Innovation related to seed dispersal is implicated in triggering waves of continental radiations beginning with the rapid diversification of major lineages and resulting in unparalleled transformation of forest dynamics within 15 million years following the K-Pg extinction. We detect introgression at multiple time scales, including ancient events predating the origination of genus-level diversity. As oak lineages moved into newly available temperate habitats in the early Miocene, secondary contact between previously isolated species occurred. This resulted in adaptive introgression, which may have further amplified the diversification of white oaks across Eurasia., (© 2022. The Author(s).)

The species tree paradigm that dominates current molecular systematic practice infers species trees from collections of sequences under assumptions of the multispecies coalescent (MSC), that is, that there is free recombination between the sequences and no (or very low) recombination within them. These coalescent genes (c-genes) are thus defined in an historical rather than molecular sense and can in theory be as large as an entire genome or as small as a single nucleotide. A debate about how to define c-genes centers on the contention that nuclear gene sequences used in many coalescent analyses undergo too much recombination, such that their introns comprise multiple c-genes, violating a key assumption of the MSC. Recently a similar argument has been made for the genes of plastid (e.g., chloroplast) and mitochondrial genomes, which for the last 30 or more years have been considered to represent a single c-gene for the purposes of phylogeny reconstruction because they are nonrecombining in an historical sense. Consequently, it has been suggested that these genomes should be analyzed using coalescent methods that treat their genes-over 70 protein-coding genes in the case of most plastid genomes (plastomes)-as independent estimates of species phylogeny, in contrast to the usual practice of concatenation, which is appropriate for generating gene trees. However, although recombination certainly occurs in the plastome, as has been recognized since the 1970's, it is unlikely to be phylogenetically relevant. This is because such historically effective recombination can only occur when plastomes with incongruent histories are brought together in the same plastid. However, plastids sort rapidly into different cell lineages and rarely fuse. Thus, because of plastid biology, the plastome is a more canonical c-gene than is the average multi-intron mammalian nuclear gene. The plastome should thus continue to be treated as a single estimate of the underlying species phylogeny, as should the mitochondrial genome. The implications of this long-held insight of molecular systematics for studies in the phylogenomic era are explored. [c-gene; coalescent gene; concatalescence; organelle genome; plastome; recombination; species tree.]., (© The Author(s) 2021. Published by Oxford University Press, on behalf of Society of Systematic Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pinus is one of the most economical and ecological important conifers, model specie for studying sequence divergence and molecular phylogeney of gymnosperms. The less availability of information for genome resources enable researchers to conduct evolutionary studies of Pinus species. To improve understanding, we firstly reported, previously released chloroplast genome of 72 Pinus species, the sequence variations, phylogenetic relationships and genome divergence among Pinus species. The results displayed 7 divergent hotspot regions (trnD-GUC, trnY-GUA, trnH-GUG, ycf1, trnL-CAA, trnK-UUU and trnV-GAC) in studied Pinus species, which holds potential to utilized as molecular genetic markers for future phylogenetic studies in Pinnus species. In addition, 3 types of repeats (tandem, palindromic and dispersed) were also studied in Pinus species under investigation. The outcome showed P. nelsonii had the highest, 76 numbers of repeats, while P. sabiniana had the lowest, 13 13 numbers of repeats. It was also observed, constructed phylogenetic tree displayed division into two significant diverged clades: single needle (soft pine) and double-needle (hard pine). Theoutcome of present investigation, based on the whole chloroplast genomes provided novel insights into the molecular based phylogeny of the genus Pinus which holds potential for its utilization in future studies focusing genetic diversity in Pinnus species., Competing Interests: The authors have declared that no competing interests exist.

Transformation of the chloroplast genome offers key advantages over traditional methods for generating transgenic plants, but this approach is limited to a few plant species. Nakazato et al. have developed a novel technique that will help to extend the technology to other plant species that are recalcitrant to current tissue culture-based chloroplast transformation protocols., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Key Message: Complete chloroplast genome sequence of a moss, Takakia lepidozioides (Takakiopsida) is reported. The largest collection of genes in mosses and the intensive RNA editing were discussed from evolutionary perspectives. We assembled the entire plastid genome sequence of Takakia lepidozioides (Takakiopsida), emerging from the first phylogenetic split among extant mosses. The genome sequences were assembled into a circular molecule 149,016 bp in length, with a quadripartite structure comprising a large and a small single-copy region separated by inverted repeats. It contained 88 genes coding for proteins, 32 for tRNA, four for rRNA, two open reading frames, and at least one pseudogene (tufA). This is the largest number of genes of all sequenced plastid genomes in mosses and Takakia is the only moss that retains the seven coding genes ccsA, cysA, cysT, petN rpoA, rps16 and trnP GGG . Parsimonious interpretation of gene loss suggests that the last common ancestor of bryophytes had all seven genes and that mosses lost at least three of them during their diversification. Analyses of the plastid transcriptome identified the extraordinary frequency of RNA editing with more than 1100 sites. We indicated a close correlation between the monoplastidy of vegetative tissue and the intensive RNA editing sites in the plastid genome in land plant lineages. Here, we proposed a hypothesis that the small population size of plastids in each vegetative cell of some early diverging land plants, including Takakia, might cause the frequent fixation of mutations in plastid genome through the intracellular genetic drift and that deleterious mutations might be continuously compensated by RNA editing during or following transcription., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The plant genome is partitioned across three distinct subcellular compartments: the nucleus, mitochondria, and plastids. Successful coordination of gene expression among these organellar genomes and the nuclear genome is critical for plant function and fitness. Whole genome duplication (WGD) events in the nucleus have played a major role in the diversification of land plants and are expected to perturb the relative copy number (stoichiometry) of nuclear, mitochondrial, and plastid genomes. Thus, elucidating the mechanisms whereby plant cells respond to the cytonuclear stoichiometric imbalance that follows WGDs represents an important yet underexplored question in understanding the evolutionary consequences of genome doubling. We used droplet digital PCR to investigate the relationship between nuclear and organellar genome copy numbers in allopolyploids and their diploid progenitors in both wheat and Arabidopsis. Polyploids exhibit elevated organellar genome copy numbers per cell, largely preserving the cytonuclear stoichiometry observed in diploids despite the change in nuclear genome copy number. To investigate the timescale over which cytonuclear stoichiometry may respond to WGD, we also estimated the organellar genome copy number in Arabidopsis synthetic autopolyploids and in a haploid-induced diploid line. We observed corresponding changes in organellar genome copy number in these laboratory-generated lines, indicating that at least some of the cellular response to cytonuclear stoichiometric imbalance is immediate following WGD. We conclude that increases in organellar genome copy numbers represent a common response to polyploidization, suggesting that maintenance of cytonuclear stoichiometry is an important component in establishing polyploid lineages., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC., Results: We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences., Conclusions: Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes., (© 2021. The Author(s).)

We present the first comparative plastome study of Pleurothallidinae with analyses of structural and molecular characteristics and identification of the ten most-variable regions to be incorporated in future phylogenetic studies. We sequenced complete plastomes of eight species in the subtribe and compared phylogenetic results of these to parallel analyses of their nuclear ribosomal DNA operon (26S, 18S, and 5.8S plus associated spacers) and partial mitochondrial genome sequences (29-38 genes and partial introns). These plastomes have the typical quadripartite structure for which gene content is similar to those of other orchids, with variation only in the composition of the ndh genes. The independent loss of ndh genes had an impact on which genes border the inverted repeats and thus the size of the small single-copy region, leading to variation in overall plastome length. Analyses of 68 coding sequences indicated the same pattern of codon usage as in other orchids, and 13 protein-coding genes under positive selection were detected. Also, we identified 62 polymorphic microsatellite loci and ten highly variable regions, for which we designed primers. Phylogenomic analyses showed that the top ten mutational hotspots represent well the phylogenetic relationships found with whole plastome sequences. However, strongly supported incongruence was observed among plastid, nuclear ribosomal DNA operon, and mitochondrial DNA trees, indicating possible occurrence of incomplete lineage sorting and/or introgressive hybridization. Despite the incongruence, the mtDNA tree retrieved some clades found in other analyses. These results, together with performance in recent studies, support a future role for mitochondrial markers in Pleurothallidinae phylogenetics., Competing Interests: The authors have declared that no competing interests exist.

Background: In most flowering plants, the plastid genome exhibits a quadripartite genome structure, comprising a large and a small single copy as well as two inverted repeat regions. Thousands of plastid genomes have been sequenced and submitted to public sequence repositories in recent years. The quality of sequence annotations in many of these submissions is known to be problematic, especially regarding annotations that specify the length and location of the inverted repeats: such annotations are either missing or portray the length or location of the repeats incorrectly. However, many biological investigations employ publicly available plastid genomes at face value and implicitly assume the correctness of their sequence annotations., Results: We introduce airpg, a Python package that automatically assesses the frequency of incomplete or incorrect annotations of the inverted repeats among publicly available plastid genomes. Specifically, the tool automatically retrieves plastid genomes from NCBI Nucleotide under variable search parameters, surveys them for length and location specifications of inverted repeats, and confirms any inverted repeat annotations through self-comparisons of the genome sequences. The package also includes functionality for automatic identification and removal of duplicate genome records and accounts for taxa that genuinely lack inverted repeats. A survey of the presence of inverted repeat annotations among all plastid genomes of flowering plants submitted to NCBI Nucleotide until the end of 2020 using airpg, followed by a statistical analysis of potential associations with record metadata, highlights that release year and publication status of the genome records have a significant effect on the frequency of complete and equal-length inverted repeat annotations., Conclusion: The number of plastid genomes on NCBI Nucleotide has increased dramatically in recent years, and many more genomes will likely be submitted over the next decade. airpg enables researchers to automatically access and evaluate the inverted repeats of these plastid genomes as well as their sequence annotations and, thus, contributes to increasing the reliability of publicly available plastid genomes. The software is freely available via the Python package index at http://pypi.python.org/pypi/airpg ., (© 2021. The Author(s).)

With some 7300 species of small nonvascular spore-producing plants, liverworts represent one of the major lineages of land plants. Although multi-locus molecular phylogenetic studies have elucidated relationships of liverworts at different taxonomic categories, the backbone phylogeny of liverworts is still to be fully resolved, especially for the placement of Ptilidiales and the relationships within Jungermanniales and Marchantiales. Here, we provided phylogenomic inferences of liverworts based on 42 newly sequenced and 24 published liverwort plastid genomes representing all but two orders of liverworts, and characterized the evolution of the plastome in liverworts. The structure of the plastid genome is overall conserved across the phylogeny of liverworts, with only two structural variants detected from simple thalloids, besides 18 out of 43 liverwort genera showing intron variations in their plastomes. Complex thalloid liverworts maintain the most plastid genes, and seem to undergo fewer gene deletions and pseudogenization events than other liverworts. Plastid phylogenetic inferences yielded mostly robustly supported relationships, and consistently resolved Ptilidiales as the sister to Porellales. The relative ratio of silent substitutions across the three genetic compartments (i.e., 1:15:10, for mitochondrial:plastid:nuclear) suggests that liverwort plastid genes have the potential to evolve faster than their nuclear counterparts, unlike in any other major land plant lineages where the mutation rate of nuclear genes overwhelm those of their plastid and mitochondrial counterparts., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The genus Isoëtes is globally distributed. Within the Neotropics, Isoëtes occurs in various habitats and ecosystems, making it an interesting case study to address phylogenetic and biogeographic questions. We sequenced and assembled plastomes and ribosomal DNA (rDNA) sequences to reconstruct phylogenetic relationships in Isoëtes from tropical regions in the Neotropics. The ploidy level of nine taxa was established to address the potential source of phylogenetic incongruence in the genus. Node ages were estimated using MCMCTree. The ancestral range estimates were conducted in BioGeoBEARS. Plastome-based phylogenies were congruent throughout distinct matrices and partition schemes, exhibiting high support for almost all nodes. Whereas, we found incongruences between the rDNA and plastome datasets. Chromosome counts identified three diploids, five tetraploids and one likely hexaploid among Neotropical species. Plastome-based node age estimates showed that the radiation of the crown Isoëtes group occurred at 20 Ma, with the diversification of the tropical American (TAA) clade taking place in the Pleistocene at 1.7 Ma. Ancestral range estimates showed that the ancestor of the TAA clade may have evolved first in the dry diagonal area in South America before reaching more humid regions. In addition, the colonization of the Brazilian semiarid region occurred three times, while the occupation of the Cerrado and Amazon regions occurred twice and once, respectively. Our study showed a large unobserved diversity within the genus in warm-dry regions in the Neotropics. Plastomes provided sufficient genomic information to establish a robust phylogenetic framework to answer evolutionary questions in Isoëtes from the Neotropics., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Epimedium L., the largest herbaceous genus of Berberidaceae, is one of the most taxonomically difficult representatives. The classification and phylogenetic relationships within Epimedium are controversial and unresolved., Objectives: For the first time, we systematically studied the phylogeny and evolution of Epimedium based on plastid genome (plastome) data for better understanding this enigmatic genus., Methods: We explored the molecular phylogeny, assessed the infrageneric classification, estimated the divergence times, and inferred the ancestral states for flower traits of Epimedium based on 45 plastomes from 32 species., Results: The Epimedium plastome length ranged from 156,635 bp to 159,956 bp. Four types of plastome organization with different inverted repeat boundary changes were identified. Phylogenetic analysis revealed a strong support for the sister relationship of sect. Macroceras and sect. Diphyllon but did not provide a distinct route for petal evolution in sect. Diphyllon . Disharmony between phylogenetic relationships and traditional classification of sect. Diphyllon was observed. Results from divergence time analysis showed that Epimedium diverged in the early Pleistocene (~2.11 Ma, 95% HPD = 1.88-2.35 Ma). Ancestral character state reconstructions indicated transitions from long spur (large-flowered group) to other petal types (small-flowered group) in Epimedium ., Conclusion: These findings provide new insights into the relationships among Epimedium species and pave the way for better elucidation of the classification and evolution of this genus., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

Background and Aims: Horizontal gene transfer (HGT) is an important evolutionary mechanism because it transfers genetic material that may code for traits or functions between species or genomes. It is frequent in mitochondrial and nuclear genomes but has not been demonstrated between plastid genomes of different green land plant species., Methods: We Sanger-sequenced the nuclear internal transcribed spacers (ITS1 and 2) and the plastid rpl16 G2 intron (rpl16). In five individuals with foreign rpl16 we also sequenced atpB-rbcL and trnLUAA-trnFGAA., Key Results: We discovered 14 individuals of a moss species with typical nuclear ITSs but foreign plastid rpl16 from a species of a distant lineage. None of the individuals with three plastid markers sequenced contained all foreign markers, demonstrating the transfer of plastid fragments rather than the entire plastid genome, i.e. entire plastids were not transferred. The two lineages diverged 165-185 Myr BP. The extended time interval since lineage divergence suggests that the foreign rpl16 is more likely explained by HGT than by hybridization or incomplete lineage sorting., Conclusions: We provide the first conclusive evidence of interspecific plastid-to-plastid HGT among land plants. Two aspects are critical: it occurred at several localities during the massive colonization of recently disturbed open habitats that were created by large-scale liming as a freshwater biodiversity conservation measure; and it involved mosses whose unique life cycle includes spores that first develop a filamentous protonema phase. We hypothesize that gene transfer is facilitated when protonema filaments of different species intermix intimately when colonizing disturbed early succession habitats., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Key Message: The plastomes of E. edulis and E. oleracea revealed several molecular markers useful for genetic studies in natural populations and indicate specific evolutionary features determined by vicariant speciation. Arecaceae is a large and diverse family occurring in tropical and subtropical ecosystems worldwide. E. oleracea is a hyperdominant species of the Amazon forest, while E. edulis is a keystone species of the Atlantic forest. It has reported that E. edulis arose from vicariant speciation after the emergence of the belt barrier of dry environment (Cerrado and Caatinga biomes) between Amazon and Atlantic forests, isolating the E. edulis in the Atlantic forest. We sequenced the complete plastomes of E. edulis and E. oleracea and compared them concerning plastome structure, SSRs, tandem repeats, SNPs, indels, hotspots of nucleotide polymorphism, codon Ka/Ks ratios and RNA editing sites aiming to investigate evolutionary traits possibly affected by distinct environments. Our analyses revealed 303 SNPs, 91 indels, and 82 polymorphic SSRs among both species. Curiously, the narrow correlation among localization of repetitive sequences and indels strongly suggests that replication slippage is involved in plastid DNA mutations in Euterpe. Moreover, most non-synonymous substitutions represent amino acid variants in E. edulis that evolved specifically or in a convergent manner across the palm phylogeny. Amino acid variants observed in several plastid proteins in E. edulis were also identified as positive signatures across palm phylogeny. The higher incidence of specific amino acid changes in plastid genes of E. edulis in comparison with E. oleracea probably configures adaptive genetic variations determined by vicariant speciation. Our data indicate that the environment generates a selective pressure on the plastome making it more adapted to specific conditions.

Ligusticum L., one of the largest members in Apiaceae, encompasses medicinally important plants, the taxonomic statuses of which have been proved to be difficult to resolve. In the current study, the complete chloroplast genomes of seven crucial plants of the best-known herbs in Ligusticum were presented. The seven genomes ranged from 148,275 to 148,564 bp in length with a highly conserved gene content, gene order and genomic arrangement. A shared dramatic decrease in genome size resulted from a lineage-specific inverted repeat (IR) contraction, which could potentially be a promising diagnostic character for taxonomic investigation of Ligusticum, was discovered, without affecting the synonymous rate. Although a higher variability was uncovered in hotspot divergence regions that were unevenly distributed across the chloroplast genome, a concatenated strategy for rapid species identification was proposed because separate fragments inadequately provided variation for fine resolution. Phylogenetic inference using plastid genome-scale data produced a concordant topology receiving a robust support value, which revealed that L. chuanxiong had a closer relationship with L. jeholense than L. sinense, and L. sinense cv. Fuxiong had a closer relationship to L. sinense than L. chuanxiong, for the first time. Our results not only furnish concrete evidence for clarifying Ligusticum taxonomy but also provide a solid foundation for further pharmaphylogenetic investigation.

Phylogenomic data from a rapidly increasing number of studies provide new evidence for resolving relationships in recently radiated clades, but they also pose new challenges for inferring evolutionary histories. Most existing methods for reconstructing phylogenetic hypotheses rely solely on algorithms that only consider incomplete lineage sorting (ILS) as a cause of intra- or intergenomic discordance. Here, we utilize a variety of methods, including those to infer phylogenetic networks, to account for both ILS and introgression as a cause for nuclear and cytoplasmic-nuclear discordance using phylogenomic data from the recently radiated flowering plant genus Polemonium (Polemoniaceae), an ecologically diverse genus in Western North America with known and suspected gene flow between species. We find evidence for widespread discordance among nuclear loci that can be explained by both ILS and reticulate evolution in the evolutionary history of Polemonium. Furthermore, the histories of organellar genomes show strong discordance with the inferred species tree from the nuclear genome. Discordance between the nuclear and plastid genome is not completely explained by ILS, and only one case of discordance is explained by detected introgression events. Our results suggest that multiple processes have been involved in the evolutionary history of Polemonium and that the plastid genome does not accurately reflect species relationships. We discuss several potential causes for this cytoplasmic-nuclear discordance, which emerging evidence suggests is more widespread across the Tree of Life than previously thought. [Cyto-nuclear discordance, genomic discordance, phylogenetic networks, plastid capture, Polemoniaceae, Polemonium, reticulations.]., (© The Author(s) 2020. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plant mitochondria and plastids display an array of inheritance patterns and varying levels of heteroplasmy, where individuals harbor more than 1 version of a mitochondrial or plastid genome. Organelle inheritance in plants has the potential to be quite complex and can vary with plant growth, development, and reproduction. Few studies have sought to investigate these complicated patterns of within-individual variation and inheritance using experimental crosses in plants. We carried out crosses in carrot, Daucus carota L. (Apiaceae), which has previously been shown to exhibit organellar heteroplasmy. We used mitochondrial and plastid markers to begin to disentangle the patterns of organellar inheritance and the fate of heteroplasmic variation, with special focus on cases where the mother displayed heteroplasmy. We also investigated heteroplasmy across the plant, assaying leaf samples at different development stages and ages. Mitochondrial and plastid paternal leakage was rare and offspring received remarkably similar heteroplasmic mixtures to their heteroplasmic mothers, indicating that heteroplasmy is maintained over the course of maternal inheritance. When offspring did differ from their mother, they were likely to exhibit a loss of the genetic variation that was present in their mother. Finally, we found that mitochondrial variation did not vary significantly over plant development, indicating that substantial vegetative sorting did not occur. Our study is one of the first to quantitatively investigate inheritance patterns and heteroplasmy in plants using controlled crosses, and we look forward to future studies making use of whole genome information to study the complex evolutionary dynamics of plant organellar genomes., (© The American Genetic Association 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plastids and mitochondria are endosymbiotic organelles that store genetic information. The genomes of these organelles generally exhibit contrasting patterns regarding genome architecture and genetic content. However, they have similar genetic features in Selaginellaceae, and little is known about what causes parallel evolution. Here, we document the multipartite plastid genomes (plastomes) and the highly divergent mitochondrial genomes (mitogenomes) from spikemoss obtained by combining short- and long-reads. The 188-kb multipartite plastome has three ribosomal operon copies in the master genomic conformation, creating the alternative subgenomic conformation composed of 110- and 78-kb subgenomes. The long-read data indicated that the two different genomic conformations were present in almost equal proportions in the plastomes of Selaginella nipponica. The mitogenome of S. nipponica was assembled into 27 contigs with a total size of 110 kb. All contigs contained directly arranged repeats at both ends, which introduced multiple conformations. Our results showed that plastomes and mitogenomes share high tRNA losses, GC-biased nucleotides, elevated substitution rates and complicated organization. The exploration of nuclear-encoded organelle DNA replication, recombination and repair proteins indicated that, several single-targeted proteins, particularly plastid-targeted recombinase A1, have been lost in Selaginellaceae; conversely, the dual-targeted proteins remain intact. According to the reported function of recombinase A1, we propose that the plastomes of spikemoss often fail to pair homologous sequences during recombination, and the dual-targeted proteins play a key role in the convergent genetic features of plastomes and mitogenomes. Our results provide a distinctive evolutionary pattern of the organelle genomes in Selaginellaceae and evidence of their convergent evolution., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)