Plastome Structure Research Articles

Does IR-loss promote plastome structural variation and sequence evolution?

Plastids are one of the main distinguishing characteristics of the plant cell. The plastid genome (plastome) of most autotrophic seed plants possesses a highly conserved quadripartite structure containing a large single-copy (LSC) and a small single-copy (SSC) region separated by two copies of the inverted repeat (termed as IRA and IRB). The IRs have been inferred to stabilize the plastid genome via homologous recombination-induced repair mechanisms. IR loss has been documented in seven autotrophic flowering plant lineages and two autotrophic gymnosperm lineages, and the plastomes of these species (with a few exceptions) are rearranged to a great extent. However, some plastomes containing normal IRs also show high structural variation. Therefore, the role of IRs in maintaining plastome stability is still controversial. In this study, we first integrated and compared genome structure and sequence evolution of representative plastomes of all nine reported IR-lacking lineages and those of their closest relative(s) with canonical inverted repeats (CRCIRs for short) to explore the role of the IR in maintaining plastome structural stability and sequence evolution. We found the plastomes of most IR-lacking lineages have experienced significant structural rearrangement, gene loss and duplication, accumulation of novel small repeats, and acceleration of synonymous substitution compared with those of their CRCIRs. However, the IR-lacking plastomes show similar structural variation and sequence evolution rate, and even less rearrangement distance, dispersed repeat number, tandem repeat number, indels frequency and GC3 content than those of IR-present plastomes with variation in Geraniaceae. We argue that IR loss is not a driver of these changes but is instead itself a consequence of other processes that more broadly shape both structural and sequence-level plastome evolution.

Plastome structure, phylogenomic analyses and molecular dating of Arecaceae.

Arecaceae is a species-rich clade of Arecales, while also being regarded as a morphologically diverse angiosperm family with numerous species having significant economic, medicinal, and ornamental value. Although in-depth studies focused on the chloroplast structure of Arecaceae, as well as inferring phylogenetic relationships using gene fragments, have been reported in recent years, a comprehensive analysis of the chloroplast structure of Arecaceae is still needed. Here we perform a comprehensive analysis of the structural features of the chloroplast genome of Arecaceae, compare the variability of gene sequences, infer phylogenetic relationships, estimate species divergence times, and reconstruct ancestral morphological traits. In this study, 74 chloroplast genomes of Arecaceae were obtained, covering five subfamilies. The results show that all chloroplast genomes possess a typical tetrad structure ranging in size between 153,806-160,122 bp, with a total of 130-137 genes, including 76-82 protein-coding genes, 29-32 tRNA genes, and 4 rRNA genes. Additionally, the total GC content was between 36.9-37.7%. Analysis of the SC/IR boundary indicated that the IR region underwent expansion or contraction. Phylogenetic relationships indicate that all five subfamilies in Arecaceae are monophyletic and that Ceroxyloideae and Arecoideae are sister groups (BS/PP = 100/1). The results of molecular dating indicate that the age of the crown group of Arecaceae is likely to be 96.60 [84.90-107.60] Ma, while the age of the stem group is 102.40 [93.44-111.17] Ma. Reconstruction of ancestral traits indicate that the ancestral characteristics of the family include monoecious plants, one seed, six stamens, and a smooth pericarp.

New plastome structural rearrangements discovered in core Tillandsioideae (Bromeliaceae) support recently adopted taxonomy.

Full plastome sequences for land plants have become readily accessible thanks to the development of Next Generation Sequencing (NGS) techniques and powerful bioinformatic tools. Despite this vast amount of genomic data, some lineages remain understudied. Full plastome sequences from the highly diverse (>1,500 spp.) subfamily Tillandsioideae (Bromeliaceae, Poales) have been published for only three (i.e., Guzmania, Tillandsia, and Vriesea) out of 22 currently recognized genera. Here, we focus on core Tillandsioideae, a clade within subfamily Tillandsioideae, and explore the contribution of individual plastid markers and data categories to inform deep divergences of a plastome phylogeny. We generated 37 high quality plastome assemblies and performed a comparative analysis in terms of plastome structure, size, gene content and order, GC content, as well as number and type of repeat motifs. Using the obtained phylogenetic context, we reconstructed the evolution of these plastome attributes and assessed if significant shifts on the evolutionary traits’ rates have occurred in the evolution of the core Tillandsioideae. Our results agree with previously published phylogenetic hypotheses based on plastid data, providing stronger statistical support for some recalcitrant nodes. However, phylogenetic discordance with previously published nuclear marker-based hypotheses was found. Several plastid markers that have been consistently used to address phylogenetic relationships within Tillandsioideae were highly informative for the retrieved plastome phylogeny and further loci are here identified as promising additional markers for future studies. New lineage-specific plastome rearrangements were found to support recently adopted taxonomic groups, including large inversions, as well as expansions and contractions of the inverted repeats. Evolutionary trait rate shifts associated with changes in size and GC content of the plastome regions were found across the phylogeny of core Tillandsioideae.

Complete Plastome of Three Korean Asarum (Aristolochiaceae): Confirmation Tripartite Structure within Korean Asarum and Comparative Analyses.

The genus Asarum (Aristolochiaceae) is a well-known resource of medicinal and ornamental plants. However, the taxonomy of Korean Asarum is ambiguous due to their considerable morphological variations. Previously, a unique plastome structure has been reported from this genus. Therefore, we investigated the structural change in the plastomes within three Korean Asarum species and inferred their phylogenetic relationships. The plastome sizes of Asarum species assembled here range from 190,168 to 193,356 bp, which are longer than a typical plastome size (160 kb). This is due to the incorporation and duplication of the small single copy into the inverted repeat, which resulted in a unique tripartite structure. We first verified this unique structure using the Illumina Miseq and Oxford Nanopore MinION platforms. We also investigated the phylogeny of 26 Aristolochiaceae species based on 79 plastid protein-coding genes, which supports the monophyly of Korean Asarum species. Although the 79 plastid protein-coding gene data set showed some limitations in supporting the previous classification, it exhibits its effectiveness in delineating some sections and species. Thus, it can serve as an effective tool for resolving species-level phylogeny in Aristolochiaceae. Last, we evaluated variable sites and simple sequence repeats in the plastome as potential molecular markers for species delimitation.

The genome of a thorny species: comparative genomic analysis among South and North American Cactaceae.

The first South American cactus nuclear genome assembly associated with comparative genomic analyses provides insights into nuclear and plastid genomic features, such as size, transposable elements, and metabolic processes related to cactus development. Here, we assembled the partial genome, plastome, and transcriptome of Cereus fernambucensis(Cereeae, Cactaceae), a representative species of the South American core Cactoideae. We accessed other genomes and transcriptomes available for cactus species to compare the heterozygosity level, genome size, transposable elements, orthologous genes, and plastome structure. These estimates were obtained from the literature or using the same pipeline adopted for C. fermabucensis. In addition to the C. fernambucensis plastome, we also performed de novo plastome assembly of Pachycereus pringlei, Stenocereus thurberi, and Pereskia humboldtii based on the sequences available in public databases. We estimated a genome size of ~ 1.58Gb for C. fernambucensis, the largest genome among the compared species. The genome heterozygosity was 0.88% in C. fernambucensis but ranged from 0.36 (Carnegiea gigantea) to 17.4% (Lophocereus schottii) in the other taxa. The genome lengths of the studied cacti are constituted by a high amount of transposable elements, ranging from ~ 57 to ~ 67%. Putative satellite DNAs are present in all species, excepting C. gigantea. The plastome of C. fernambucensis was ~ 104kb, showing events of translocation, inversion, and gene loss. We observed a low number of shared unique orthologs, which may suggest gene duplication events and the simultaneous expression of paralogous genes. We recovered 37 genes that have undergone positive selection along the Cereus branch that are associated with different metabolic processes, such as improving photosynthesis during drought stress and nutrient absorption, which may be related to the adaptation to xeric areas of the Neotropics.

Plastid phylogenomics improves resolution of phylogenetic relationship in the Cheirostylis and Goodyera clades of Goodyerinae (Orchidoideae, Orchidaceae)

Goodyerinae are one of phylogenetically unresolved groups of Orchidaceae. The lack of resolution achieved through the analyses of previous molecular sequences from one or a few markers has long confounded phylogenetic estimation and generic delimitation. Here, we present large-scale phylogenomic data to compare the plastome structure of the two main clades (Goodyera and Cheirostylis) in this subtribe and further adopt two strategies, combining plastid coding sequences and the whole plastome, to investigate phylogenetic relationships. A total of 46 species in 16 genera were sampled, including 39 species in 15 genera sequenced in this study. The plastomes of heterotrophic species are not drastically reduced in overall size, but display a pattern congruent with a loss of photosynthetic function. The plastomes of autotrophic species ranged from 147 to 165 kb and encoded from 132 to 137 genes. Three unusual structural features were detected: a 1.0-kb inversion in the large single-copy region of Goodyera schlechtendaliana; the loss and/or pseudogenization of ndh genes only in two species, Cheirostylis chinensis and C. montana; and the expansion of inverted repeat regions and contraction of small single-copy region in Hetaeria oblongifolia. Phylogenomic analyses provided improved resolution for phylogenetic relationships. All genera were recovered as monophyletic, except for Goodyera and Hetaeria, which were each recovered as non-monophyletic. Nomenclatural changes are needed until the broader sampling and biparental inherited markers. This study provides a phylogenetic framework of Goodyerinae and insight into plastome evolution of Orchidaceae.

Plastome evolution and organisation in the Hoya group (Apocynaceae)

The genus Hoya is highly diverse and many of its species are popular ornamental plants. However, the relationships between Hoya and related genera (the Hoya group) are not fully resolved. In this study, we report 20 newly sequenced plastomes of species in the Hoya group. The complete plastomes vary in length from 175,405 to 178,525 bp while the LSCs vary from 90,248 to 92,364 bp and the complete SSCs vary from 2,285 to 2,304 bp, making the SSC in the Hoya group one of the shortest known in the angiosperms. The plastome structure in the Hoya group is characterised by a massive increase in the size of the inverted repeats as compared to the outgroups. In all ingroup species, the IR/SSC boundary moved from ycf1 to ndhF while this was not observed in outgroup taxa, making it a synapomorphy for the Hoya group. We have also assembled the mitogenome of Hoya lithophytica, which, at 718,734 bp, is the longest reported in the family. The phylogenetic analysis using exons from 42 taxa in the Hoya group and three outgoups confirms that the earliest divergent genus in the Hoya group is Papuahoya, followed by Dischidia. The relationship between Dischidia and the clade which includes all Hoya and Oreosparte taxa, is not fully supported. Oreosparte is nested in Hoya making it paraphyletic unless Clemensiella is recognised as a separate genus.

Plastid NDH Pseudogenization and Gene Loss in a Recently Derived Lineage from the Largest Hemiparasitic Plant Genus Pedicularis (Orobanchaceae).

The plastid genome (plastome) is highly conserved in both gene order and content and has a lower mutation rate than the nuclear genome. However, the plastome is more variable in heterotrophic plants. To date, most such studies have investigated just a few species or only holoheterotrophic groups, and few have examined plastome evolution in recently derived lineages at an early stage of transition from autotrophy to heterotrophy. In this study, we investigated the evolutionary dynamics of plastomes in the monophyletic and recently derived Pedicularis sect. Cyathophora (Orobanchaceae). We obtained 22 new plastomes, 13 from the six recognized species of section Cyathophora, six from hemiparasitic relatives and three from autotrophic relatives. Comparative analyses of gene content, plastome structure and selection pressure showed dramatic differences among species in section Cyathophora and in Pedicularis as a whole. In comparison with autotrophic relatives and other Pedicularis spp., we found that the inverted repeat (IR) region in section Cyathophora had expansions to the small single-copy region, with a large expansion event and two independent contraction events. Moreover, NA(D)H dehydrogenase, accD and ccsA have lost function multiple times, with the function of accD being replaced by nuclear copies of an accD-like gene in Pedicularis spp. The ccsA and ndhG genes may have evolved under selection in association with IR expansion/contraction events. This study is the first to report high plastome variation in a recently derived lineage of hemiparasitic plants and therefore provides evidence for plastome evolution in the transition from autotrophy to heterotrophy.

Comparative Plastomes and Phylogenetic Analysis of Cleistogenes and Closely Related Genera (Poaceae).

Cleistogenes (Orininae, Cynodonteae, Chloridoideae, Poaceae) is an ecologically important genus. The phylogenetic placement of Cleistogenes and phylogenetic relationships among Cleistogenes taxa remain controversial for a long time. To resolve the intra- and inter-generic relationships of Cleistogenes, the plastomes of 12 Cleistogenes taxa (including 8 species and 4 varieties), one Orinus species, 15 Triodia species, two Tripogon species, and two Aeluropus species were included in the present study. All the taxa showed a similar pattern in plastome structure, gene order, gene content, and IR boundaries. The number of simple sequence repeats ranged from 145 (O. kokonorica) to 161 (T. plurinervata and T. schinzii). Moreover, 1,687 repeats were identified in these taxa, including 1,012 forward, 650 palindromic, 24 reverse, and one complement. Codon usage analysis revealed that these plastomes contained 16,633 (T. stipoides) to 16,678 (T. tomentosa) codons. Sequence divergence analysis among Cleistogenes and closely related genera identified five non-coding regions (trnS-UGA-psbZ, rpl32-trnL-UAG, trnQ-UUG-psbK, trnD-GUC-psbM, trnT-GGU-trnE-UUC). Phylogenetic analysis of complete plastomes indicated that Cleistogenes is sister to a clade composed of Orinus and Triodia, whereas it did not support the sister relationship between the recently proposed subtribe Orininae (Cleistogenes and Orinus) and Triodia. The subtribe Orininae was not supported by our complete plastome data. The split between Cleistogenes and Orinus-Triodia clade go back to 14.01 Ma. Besides, our findings suggested that C. squarrosa and C. songorica are the successive early diverging groups in the phylogenetic analysis. The other 10 taxa are divided into two groups: a monophyletic group composed of Cleistogenes sp. nov. and C. caespitosa var. ramosa is sister to other eight Cleistogenes taxa. Cleistogenes was estimated to have experienced rapid divergence within a short period, which could be a major obstacle in resolving phylogenetic relationships within Cleistogenes. Collectively, our results provided valuable insights into the phylogenetic study of grass species.

Complete plastome sequence of Macaranga tanarius (L.) Muell. Arg. (Euphorbiaceae): a fast-growing timber species

Macaranga tanarius (L.) Muell. Arg. is a tree species within Euphorbiaceae, which can be used for building timber and its extract can be used to treat diabetes. In this report, we describe the complete plastome sequence of Macaranga tanarius. The complete plastome of Macaranga tanarius (L.) Muell. Arg. is of 165,362 bp in length, and it is with typical plastome structure and gene content of angiosperm plastome, including two reverse repeat regions (IRs) of 27,503 bp, large single copy region (LSC) of 91,443 bp, and small single copy (SSC) region of 18,913 bp. The plastome contains 131 genes, including 85 protein coding genes, 38 tRNA genes, eight rRNA genes (i.e., 5S rRNA, 4.5S rRNA, 16S rRNA, and 23S rRNA). The total G/C content of Macaranga tanarius (L.) Muell. Arg.plastome is 35.6%. The complete plastome sequence is conducive to the development and utilization of Euphorbiaceae resources and the phylogenetic study.

Plastid genome evolution in Amazonian açaí palm (Euterpe oleracea Mart.) and Atlantic forest açaí palm (Euterpe edulis Mart.).

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.

The Complete Plastid Genome of Artocarpus camansi: A High Degree of Conservation of the Plastome Structure in the Family Moraceae

Understanding the plastid genome is extremely important for the interpretation of the genetic mechanisms associated with essential physiological and metabolic functions, the identification of possible marker regions for phylogenetic or phylogeographic analyses, and the elucidation of the modes through which natural selection operates in different regions of this genome. In the present study, we assembled the plastid genome of Artocarpus camansi, compared its repetitive structures with Artocarpus heterophyllus, and searched for evidence of synteny within the family Moraceae. We also constructed a phylogeny based on 56 chloroplast genes to assess the relationships among three families of the order Rosales, that is, the Moraceae, Rhamnaceae, and Cannabaceae. The plastid genome of A. camansi has 160,096 bp, and presents the typical circular quadripartite structure of the Angiosperms, comprising a large single copy (LSC) of 88,745 bp and a small single copy (SSC) of 19,883 bp, separated by a pair of inverted repeat (IR) regions each with a length of 25,734 bp. The total GC content was 36.0%, which is very similar to Artocarpus heterophyllus (36.1%) and other moraceous species. A total of 23,068 codons and 80 SSRs were identified in the A. camansi plastid genome, with the majority of the SSRs being mononucleotide (70.0%). A total of 50 repeat structures were observed in the A. camansi plastid genome, in contrast with 61 repeats in A. heterophyllus. A purifying selection signal was found in 70 of the 79 protein-coding genes, indicating that they have all been highly conserved throughout the evolutionary history of the genus. The comparative analysis of the structural characteristics of the chloroplast among different moraceous species found a high degree of similarity in the sequences, which indicates a highly conserved evolutionary model in these plastid genomes. The phylogenetic analysis also recovered a high degree of similarity between the chloroplast genes of A. camansi and A. heterophyllus, and reconfirmed the hypothesis of the intense conservation of the plastome in the family Moraceae.

Plastome comparison and evolution within the tribes of Plantaginaceae: Insights from an Asian gypsyweed.

In spite of availability of several plastomes representing different tribes of Plantaginaceae, sparse attempts have been made to understand the plastome structure, evolution, and phylogenomics. In the present study, we have made an effort to understand the gene content and plastome evolution in the family Plantaginaceae using the newly generated plastome sequence of Veronica ovata subsp. kiusiana, a taxon native to SE Asia. In the first-ever attempt, plastomes of seven out of 10 tribes of Plantaginaceae have been compared to understand the evolution across the tribes of Plantaginaceae. The size of the plastome of V. ovata subsp. kiusiana is 152,249 bp, showing a typical quadripartite structure containing LSC, SSC, and two IRs with the sizes of 83,187, 17,704, and 25,679 respectively. The plastome comparison revealed the unique deletions in ycf2 and ndhF genes of members of different tribes, and also revealed high nucleotide variable hotspots. The study also revealed six highly variable genes and intergenic spacer viz. rps16, rps15-ycf1, ccsA-ndhD, ndhC-trnV, petN-psbM, and ycf1-trnN as potential DNA barcodes for the genus Veronica. The phylogenomic study revealed the sister relationship between V. ovata subsp. kiusiana and V. persica and also suggested the tentative placement of seven tribes in the family Plantaginaceae.

Comparative plastomic analysis and insights into the phylogeny of Salvia (Lamiaceae)

Salvia is the largest genus of Lamiaceae, with almost 1000 species, and has been divided into 11 subgenera. Salvia subg. Glutinaria, native to East Asia, is particularly important because of its potential medicinal value. However, the interspecific relationships of this subgenus have not been resolved and the plastomes of Salvia have rarely been studied. In the current study, we compared plastid genome structure and organization of 19 species of Salvia (14 newly sequenced and 5 previously published). Our comparative analysis showed that all Salvia plastomes examined have a quadripartite structure typical of most angiosperms and contain an identical set of 114 unique genes (80 protein-coding genes, 4 rRNA genes, and 30 tRNA genes). The plastome structure of all Salvia species is highly conserved like other Lamiaceae plastomes. Gene content, gene order, and GC content were highly similar in these plastomes. The inverted repeats/single copy region (IR/SC) boundaries of Salvia are highly conserved, and IR contraction only occurred in two species (Salvia mekongensis and S. rosmarinus). In Salvia, sequence divergence was higher in non-coding regions than in coding regions. We found that using large single copy (LSC) and small single copy regions (SSC) with exclusion of the rapidly evolving sites produced the highest resolution in phylogenetic analysis of Salvia, suggesting that using suitable informative sites to build trees is more conducive in phylogenetic research. This study assembled a powerful matrix data set for studying the phylogeny of Salvia, resolving the interspecific relationship of Salvia subg. Glutinaria. The newly sequenced plastid genomes will also enrich the plastome database of Salvia, providing the scientific basis for the development and utilization of germplasm resources of this large and important genus.

Chloroplast mRNAs are 3' polyuridylylated in the Green Alga Pithophora roettleri (Cladophorales).

Species within the green algal order Cladophorales have an unconventional plastome structure where individual coding regions or small numbers of genes occur as linear single-stranded DNAs folded into hairpin structures. Another group of photosynthetic organisms with an equivalently reduced chloroplast genome are the peridinin dinoflagellates of the Alveolata eukaryotic lineage whose plastomes are mini-circles carrying one or a few genes required for photosynthesis. One unusual aspect of the Alveolata is the polyuridylylation of mRNA 3' ends among peridinin dinoflagellates and the chromerid algae. This study was conducted to understand if an unconventional highly reduced plastome structure co-occurs with unconventional RNA processing. To address this, the 5' and 3' mRNA termini of the known chloroplast genes of Pithophora roettleri (order Cladophorales) were analyzed for evidence of post-transcriptional processing. Circular Reverse Transcriptase PCR (cRT-PCR) followed by deep sequencing of the amplicons was used to analyze 5' and 3' mRNA termini. Evidence of several processing events were collected, most notably the 3' termini of six of the eight genes were polyuridylylated, which has not been reported for any lineage outside of the Alveolata. Other processing events include poly(A) and heteropolymeric 3' additions, 5' primary transcript start sites, as well as the presence of circularized RNAs. Five other species representing other green algal lineages were also tested and poly(U) additions appear to be limited to the order Cladophorales. These results demonstrate that chloroplast mRNA polyuridylylation is not the sole provenance of photosynthetic alveolates and may have convergently evolved in two distinct photosynthetic lineages.

Characteristics and Mutational Hotspots of Plastomes in Debregeasia (Urticaceae).

Debregeasia is an economically important genus of the nettle family (Urticaceae). Previous systematic studies based on morphology, or using up to four plastome regions, have not satisfactorily resolved relationships within the genus. Here, we report 25 new plastomes for Urticaceae, including 12 plastomes from five Debregeasia species and 13 plastomes from other genera. Together with the one published plastome for Debregeasia, we analyzed plastome structure and character, identified mutation hotspots and loci under selection, and constructed phylogenies. The plastomes of Debregeasia were found to be very conservative, with a size from 155,743 bp to 156,065 bp, and no structural variation. Eleven mutation hotspots were identified, including three (rpoB-trnC-GCA, trnT-GGU-psbD and ycf1) that are highly variable both within Debregeasia and among genera; these show high potential value for future DNA barcoding, population genetics and phylogenetic reconstruction. Selection pressure analysis revealed nine genes (clpP, ndhF, petB, psbA, psbK, rbcL, rpl23, ycf2, and ycf1) that may experience positive selection. Phylogenomic analyses results suggest that Debregeasia was monophyletic, and closest to Boehmeria among genera examined. Within Debregeasia, D. longifolia was sister to D. saeneb, whereas D. elliptica, D. orientalis with D. squamata formed the other subclade. This study enriches organelle genome resources for Urticaceae, and highlights the utility of plastome data for detecting mutation hotspots for evolutionary and systematic analysis.

The Loss of the Inverted Repeat in the Putranjivoid Clade of Malpighiales.

The typical plastid genome (plastome) of photosynthetic angiosperms comprises a pair of Inverted Repeat regions (IRs), which separate a Large Single Copy region (LSC) from a Small Single Copy region (SSC). The independent losses of IRs have been documented in only a few distinct plant lineages. The majority of these taxa show uncommonly high levels of plastome structural variations, while a few have otherwise conserved plastomes. For a better understanding of the function of IRs in stabilizing plastome structure, more taxa that have lost IRs need to be investigated. We analyzed the plastomes of eight species from two genera of the putranjivoid clade of Malpighiales using Illumina paired-end sequencing, the de novo assembly strategy GetOrganelle, as well as a combination of two annotation methods. We found that all eight plastomes of the putranjivoid clade have lost their IRB, representing the fifth case of IR loss within autotrophic angiosperms. Coinciding with the loss of the IR, plastomes of the putranjivoid clade have experienced significant structural variations including gene and intron losses, multiple large inversions, as well as the translocation and duplication of plastome segments. However, Balanopaceae, one of the close relatives of the putranjivoid clade, exhibit a relatively conserved plastome organization with canonical IRs. Our results corroborate earlier reports that the IR loss and additional structural reorganizations are closely linked, hinting at a shared mechanism that underpins structural disturbances.

Insights Into Chloroplast Genome Evolution Across Opuntioideae (Cactaceae) Reveals Robust Yet Sometimes Conflicting Phylogenetic Topologies.

Chloroplast genomes (plastomes) are frequently treated as highly conserved among land plants. However, many lineages of vascular plants have experienced extensive structural rearrangements, including inversions and modifications to the size and content of genes. Cacti are one of these lineages, containing the smallest plastome known for an obligately photosynthetic angiosperm, including the loss of one copy of the inverted repeat (∼25 kb) and the ndh gene suite, but only a few cacti from the subfamily Cactoideae have been sufficiently characterized. Here, we investigated the variation of plastome sequences across the second-major lineage of the Cactaceae, the subfamily Opuntioideae, to address (1) how variable is the content and arrangement of chloroplast genome sequences across the subfamily, and (2) how phylogenetically informative are the plastome sequences for resolving major relationships among the clades of Opuntioideae. Our de novo assembly of the Opuntia quimilo plastome recovered an organelle of 150,347 bp in length with both copies of the inverted repeat and the presence of all the ndh gene suite. An expansion of the large single copy unit and a reduction of the small single copy unit was observed, including translocations and inversion of genes, as well as the putative pseudogenization of some loci. Comparative analyses among all clades within Opuntioideae suggested that plastome structure and content vary across taxa of this subfamily, with putative independent losses of the ndh gene suite and pseudogenization of genes across disparate lineages, further demonstrating the dynamic nature of plastomes in Cactaceae. Our plastome dataset was robust in resolving three tribes with high support within Opuntioideae: Cylindropuntieae, Tephrocacteae and Opuntieae. However, conflicting topologies were recovered among major clades when exploring different assemblies of markers. A plastome-wide survey for highly informative phylogenetic markers revealed previously unused regions for future use in Sanger-based studies, presenting a valuable dataset with primers designed for continued evolutionary studies across Cactaceae. These results bring new insights into the evolution of plastomes in cacti, suggesting that further analyses should be carried out to address how ecological drivers, physiological constraints and morphological traits of cacti may be related with the common rearrangements in plastomes that have been reported across the family.

Plastome Structural Conservation and Evolution in the Clusioid Clade of Malpighiales

The clusioid clade of Malpighiales is comprised of five families: Bonnetiaceae, Calophyllaceae, Clusiaceae, Hypericaceae and Podostemaceae. Recent studies have found the plastome structure of Garcinia mangostana L. from Clusiaceae was conserved, while plastomes of five riverweed species from Podostemaceae showed significant structural variations. The diversification pattern of plastome structure of the clusioid clade worth a thorough investigation. Here we determined five complete plastomes representing four families of the clusioid clade. Our results found that the plastomes of the early diverged three families (Clusiaceae, Bonnetiaceae and Calophyllaceae) in the clusioid clade are relatively conserved, while the plastomes of the other two families show significant variations. The Inverted Repeat (IR) regions of Tristicha trifaria and Marathrum foeniculaceum (Podostemaceae) are greatly reduced following the loss of the ycf1 and ycf2 genes. An inversion over 50 kb spanning from trnK-UUU to rbcL in the LSC region is shared by Cratoxylum cochinchinense (Hypericaceae), T. trifaria and Ma. foeniculaceum (Podostemaceae). The large inversed colinear block in Hypericaceae and Podostemaceae contains all the genes in the 50-kb inversed colinear block in a clade of Papilionoideae, with two extra genes (trnK-UUU and matK) at one end. Another endpoint of both inversions in the two clusioids families and Papilionoideae is located between rbcL and accD. This study greatly helped to clarify the plastome evolution in the clusioid clade.

Unprecedented Intraindividual Structural Heteroplasmy in Eleocharis (Cyperaceae, Poales) Plastomes.

Plastid genomes (plastomes) of land plants have a conserved quadripartite structure in a gene-dense unit genome consisting of a large inverted repeat that separates two single copy regions. Recently, alternative plastome structures were suggested in Geraniaceae and in some conifers and Medicago the coexistence of inversion isomers has been noted. In this study, plastome sequences of two Cyperaceae, Eleocharis dulcis (water chestnut) and Eleocharis cellulosa (gulf coast spikerush), were completed. Unlike the conserved plastomes in basal groups of Poales, these Eleocharis plastomes have remarkably divergent features, including large plastome sizes, high rates of sequence rearrangements, low GC content and gene density, gene duplications and losses, and increased repetitive DNA sequences. A novel finding among these features was the unprecedented level of heteroplasmy with the presence of multiple plastome structural types within a single individual. Illumina paired-end assemblies combined with PacBio single-molecule real-time sequencing, long-range polymerase chain reaction, and Sanger sequencing data identified at least four different plastome structural types in both Eleocharis species. PacBio long read data suggested that one of the four E. dulcis plastome types predominates.