Comparative Plastid Genome and Phylogenomic Analyses of Potamogeton Species.

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.

Infrageneric Plastid Genomes of Cotoneaster (Rosaceae): Implications for the Plastome Evolution and Origin of C. wilsonii on Ulleung Island.

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.

The complete plastid genome sequence of the enigmatic moss, Takakia lepidozioides (Takakiopsida, Bryophyta): evolutionary perspectives on the largest collection of genes in mosses and the intensive RNA editing.

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 trnPGGG. 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.

Nuclear-cytoplasmic balance: whole genome duplications induce elevated organellar genome copy number.

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.

Plastome-based phylogenomics elucidate relationships in rare Isoëtes species groups from the Neotropics.

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.

Phylogeny of Euclidieae (Brassicaceae) based on plastome and nuclear ribosomal DNA data.

Euclidieae, a morphologically diverse tribe in the family Brassicaceae (Cruciferae), consists of 29 genera and more than 150 species distributed mainly in Asia. Prior phylogenetic analyses on Euclidieae are inadequate. In this study, sequence data from the plastid genome and nuclear ribosomal DNA of 72 species in 27 genera of Euclidieae were used to infer the inter- and intra-generic relationships within. The well-resolved and strongly supported plastome phylogenies revealed that Euclidieae could be divided into five clades. Both Cymatocarpus and Neotorularia are polyphyletic in nuclear and plastome phylogenies. Besides, the conflicts of systematic positions of three species of Braya and two species of Solms-laubachia s.l. indicated that hybridization and or introgression might have happened during the evolutionary history of the tribe. Results from divergence-time analyses suggested an early Miocene origin of Euclidieae, and it probably originated from the Central Asia, Pamir Plateau and West Himalaya. In addition, multiple ndh genes loss and pseudogenization were detected in eight species based on comparative genomic study.

Comparative analysis of plastid genomes within the Campanulaceae and phylogenetic implications.

The conflicts exist between the phylogeny of Campanulaceae based on nuclear ITS sequence and plastid markers, particularly in the subdivision of Cyanantheae (Campanulaceae). Besides, various and complicated plastid genome structures can be found in species of the Campanulaceae. However, limited availability of genomic information largely hinders the studies of molecular evolution and phylogeny of Campanulaceae. We reported the complete plastid genomes of three Cyanantheae species, compared them to eight published Campanulaceae plastomes, and shed light on a deeper understanding of the applicability of plastomes. We found that there were obvious differences among gene order, GC content, gene compositions and IR junctions of LSC/IRa. Almost all protein-coding genes and amino acid sequences showed obvious codon preferences. We identified 14 genes with highly positively selected sites and branch-site model displayed 96 sites under potentially positive selection on the three lineages of phylogenetic tree. Phylogenetic analyses showed that Cyananthus was more closely related to Codonopsis compared with Cyclocodon and also clearly illustrated the relationship among the Cyanantheae species. We also found six coding regions having high nucleotide divergence value. Hotpot regions were considered to be useful molecular markers for resolving phylogenetic relationships and species authentication of Campanulaceae.

Comparative Plastid Genomics of Cryptomonas Species Reveals Fine-Scale Genomic Responses to Loss of Photosynthesis.

Loss of photosynthesis is a recurring theme in eukaryotic evolution. In organisms that have lost the ability to photosynthesize, nonphotosynthetic plastids are retained because they play essential roles in processes other than photosynthesis. The unicellular algal genus Cryptomonas contains both photosynthetic and nonphotosynthetic members, the latter having lost the ability to photosynthesize on at least three separate occasions. To elucidate the evolutionary processes underlying the loss of photosynthesis, we sequenced the plastid genomes of two nonphotosynthetic strains, Cryptomonas sp. CCAC1634B and SAG977-2f, as well as the genome of the phototroph Cryptomonas curvata CCAP979/52. These three genome sequences were compared with the previously sequenced plastid genome of the nonphotosynthetic species Cryptomonas paramecium CCAP977/2a as well as photosynthetic members of the Cryptomonadales, including C. curvata FBCC300012D. Intraspecies comparison between the two C. curvata strains showed that although their genome structures are stable, the substitution rates of their genes are relatively high. Although most photosynthesis-related genes, such as the psa and psb gene families, were found to have disappeared from the nonphotosynthetic strains, at least ten pseudogenes are retained in SAG977-2f. Although gene order is roughly shared among the plastid genomes of photosynthetic Cryptomonadales, genome rearrangements are seen more frequently in the smaller genomes of the nonphotosynthetic strains. Intriguingly, the light-independent protochlorophyllide reductase comprising chlB, L, and N is retained in nonphotosynthetic SAG977-2f and CCAC1634B. On the other hand, whereas CCAP977/2a retains ribulose-1,5-bisphosphate carboxylase/oxygenase-related genes, including rbcL, rbcS, and cbbX, the plastid genomes of the other two nonphotosynthetic strains have lost the ribulose-1,5-bisphosphate carboxylase/oxygenase protein-coding genes.

Conservation and innovation: Plastome evolution during rapid radiation of Rhodiola on the Qinghai-Tibetan Plateau.

The amount of plastome sequence data available has soared in the last decade, but the nature of plastome evolution during rapid radiations is largely unknown. Moreover, although there is increasing evidence showing that plastomes may have undergone adaptive evolution in order to allow adaptation to various environments, few studies have systematically investigated the role of the plastome in alpine adaptation. To address these questions, we sequenced and analyzed 12 representative species of Rhodiola, a genus which includes ca. 70 perennial herbs mainly growing in alpine habitats in the Qinghai-Tibet Plateau and the Hengduan Mountains. Rapid radiation in this genus was triggered by the uplift of the Qinghai-Tibet Plateau. We also included nine species of Crassulaceae as the outgroups. All plastomes were conserved with respect to size, structure, and gene content and order, with few variations: each contained 134 genes, including 85 protein-coding genes, 37 tRNAs, 8 rRNAs, and 4 potential pseudogenes. Four types of repeat sequence were detected. Slight contraction and expansion of the inverted repeats were also revealed. Both the genome-wide alignment and sequence polymorphism analyses showed that the inverted repeats and coding regions were more conserved than the single-copy regions and the non-coding regions. Positive selection analyses identified three genes containing sites of positive selection (rpl16, ndhA, ndhH), and one gene with a faster than average rate of evolution (psaA). The products of these genes may be involved in the adaptation of Rhodiola to alpine environments such as low CO2 concentration and high-intensity light.

Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development.

Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post-meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI-based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring-shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20-750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20-fold even within individual organelles, with average values between 2.6-fold and 6.7-fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70-130 copies in chloroplasts of about 7 µm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600-3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed-field electrophoresis, restriction of high-molecular-weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single-stranded and double-stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.

Chloroplast competition is controlled by lipid biosynthesis in evening primroses.

In most eukaryotes, organellar genomes are transmitted preferentially by the mother, but molecular mechanisms and evolutionary forces underlying this fundamental biological principle are far from understood. It is believed that biparental inheritance promotes competition between the cytoplasmic organelles and allows the spread of so-called selfish cytoplasmic elements. Those can be, for example, fast-replicating or aggressive chloroplasts (plastids) that are incompatible with the hybrid nuclear genome and therefore maladaptive. Here we show that the ability of plastids to compete against each other is a metabolic phenotype determined by extremely rapidly evolving genes in the plastid genome of the evening primrose Oenothera Repeats in the regulatory region of accD (the plastid-encoded subunit of the acetyl-CoA carboxylase, which catalyzes the first and rate-limiting step of lipid biosynthesis), as well as in ycf2 (a giant reading frame of still unknown function), are responsible for the differences in competitive behavior of plastid genotypes. Polymorphisms in these genes influence lipid synthesis and most likely profiles of the plastid envelope membrane. These in turn determine plastid division and/or turnover rates and hence competitiveness. This work uncovers cytoplasmic drive loci controlling the outcome of biparental chloroplast transmission. Here, they define the mode of chloroplast inheritance, as plastid competitiveness can result in uniparental inheritance (through elimination of the "weak" plastid) or biparental inheritance (when two similarly "strong" plastids are transmitted).

Cytonuclear interactions remain stable during allopolyploid evolution despite repeated whole-genome duplications in Brassica.

Several plastid macromolecular protein complexes are encoded by both nuclear and plastid genes. Therefore, cytonuclear interactions are held in place to prevent genomic conflicts that may lead to incompatibilities. Allopolyploidy resulting from hybridization and genome doubling of two divergent species can disrupt these fine-tuned interactions, as newly formed allopolyploid species confront biparental nuclear chromosomes with a uniparentally inherited plastid genome. To avoid any deleterious effects of unequal genome inheritance, preferential transcription of the plastid donor over the other donor has been hypothesized to occur in allopolyploids. We used Brassica as a model to study the effects of paleopolyploidy in diploid parental species, as well as the effects of recent and ancient allopolyploidy in Brassica napus, on genes implicated in plastid protein complexes. We first identified redundant nuclear copies involved in those complexes. Compared with cytosolic protein complexes and with genome-wide retention rates, genes involved in plastid protein complexes show a higher retention of genes in duplicated and triplicated copies. Those redundant copies are functional and are undergoing strong purifying selection. We then compared transcription patterns and sequences of those redundant gene copies between resynthesized allopolyploids and their diploid parents. The neopolyploids showed no biased subgenome expression or maternal homogenization via gene conversion, despite the presence of some non-synonymous substitutions between plastid genomes of parental progenitors. Instead, subgenome dominance was observed regardless of the maternal progenitor. Our results provide new insights on the evolution of plastid protein complexes that could be tested and generalized in other allopolyploid species.

Phylogeny, character evolution and spatiotemporal diversification of the species-rich and world-wide distributed tribe Rubieae (Rubiaceae).

The Rubiaceae tribe Rubieae has a world-wide distribution with up to 1,000 species. These collectively exhibit an enormous ecological and morphological diversity, making Rubieae an excellent group for macro- and microevolutionary studies. Previous molecular phylogenetic analyses used only a limited sampling within the tribe or missed lineages crucial for understanding character evolution in this group. Here, we analyze sequences from two plastid spacer regions as well as morphological and biogeographic data from an extensive and evenly distributed sampling to establish a sound phylogenetic framework. This framework serves as a basis for our investigation of the evolution of important morphological characters and the biogeographic history of the Rubieae. The tribe includes three major clades, the Kelloggiinae Clade (Kelloggia), the Rubiinae Clade (Didymaea, Rubia) and the most species-rich Galiinae Clade (Asperula, Callipeltis, Crucianella, Cruciata, Galium, Mericarpaea, Phuopsis, Sherardia, Valantia). Within the Galiinae Clade, the largest genera Galium and Asperula are para- and polyphyletic, respectively. Smaller clades, however, usually correspond to currently recognized taxa (small genera or sections within genera), which may be used as starting points for a refined classification in this clade. Life-form (perennial versus annual), flower shape (long versus short corolla tube) and fruit characters (dry versus fleshy, with or without uncinate hairs) are highly homoplasious and have changed multiple times independently. Inference on the evolution of leaf whorls, a characteristic feature of the tribe, is sensitive to model choice. Multi-parted leaf whorls appear to have originated from opposite leaves with two small interpetiolar stipules that are subsequently enlarged and increased in number. Early diversification of Rubieae probably started during the Miocene in western Eurasia. Disjunctions between the Old and the New World possibly are due to connections via a North Atlantic land bridge. Diversification of the Galiineae Clade started later in the Miocene, probably in the Mediterranean, from where lineages reached, often multiple times, Africa, eastern Asia and further on the Americas and Australia.

Using phylogenomics to reconstruct phylogenetic relationships within tribe Polygonateae (Asparagaceae), with a special focus on Polygonatum.

Polygonatum is a widespread temperate genus with approximately 75 species centered in the Eastern Himalaya and Indo-Burma biodiversity hotspots. A complete assessment of the remarkable diversity of Polygonatum in these areas requires an accurate circumscription of the genus, as well as a clear understanding of generic and infrageneric relationships, both of which have been problematic in the past. In this study, we reconstruct phylogenetic relationships within Polygonatum and test its monophyly using a phylogenomic approach. For that, we built a comprehensive dataset that includes complete or nearly-complete plastid genomes of 19 species of Polygonatum, one of Disporopsis, and four of Heteropolygonatum. Their plastid genomes do not present any major structural differences and range from 153,821 to 155,580 bp in length. Molecular phylogenetic analyses of the chloroplast coding regions indicate that Polygonatum and Heteropolygonatum are monophyletic, providing support for their recognition as distinct genera and corroborating recent adjustments of their circumscriptions. An expanded analysis with higher species sampling using the petA-psbJ plastid gene region combined with the nuclear ribosomal ITS provided support for the recognition of three distinct sections within Polygonatum. These same sections are further supported by chromosome data: Polygonatum sect. Sibirica (x = 12); Polygonatum sect. Polygonatum (x = 9-11); and, Polygonatum sect. Verticillata (x = 13-15). Populations of P. multiflorum from northwestern Himalaya are here shown to be best treated as a separate taxon, P. govanianum. Furthermore, P. verticillatum is shown to be polyphyletic, indicating that it represents a species-complex that includes multiple Asiatic species. Despite that, additional studies are still needed until the proper nomenclatural adjustments can be made.

Plastid genome analysis of three Nemaliophycidae red algal species suggests environmental adaptation for iron limited habitats.

The red algal subclass Nemaliophycidae includes both marine and freshwater taxa that contribute to more than half of the freshwater species in Rhodophyta. Given that these taxa inhabit diverse habitats, the Nemaliophycidae is a suitable model for studying environmental adaptation. For this purpose, we characterized plastid genomes of two freshwater species, Kumanoa americana (Batrachospermales) and Thorea hispida (Thoreales), and one marine species Palmaria palmata (Palmariales). Comparative genome analysis identified seven genes (ycf34, ycf35, ycf37, ycf46, ycf91, grx, and pbsA) that were different among marine and freshwater species. Among currently available red algal plastid genomes (127), four genes (pbsA, ycf34, ycf35, ycf37) were retained in most of the marine species. Among these, the pbsA gene, known for encoding heme oxygenase, had two additional copies (HMOX1 and HMOX2) that were newly discovered and were reported from previously red algal nuclear genomes. Each type of heme oxygenase had a different evolutionary history and special modifications (e.g., plastid targeting signal peptide). Based on this observation, we suggest that the plastid-encoded pbsA contributes to the iron controlling system in iron-deprived conditions. Thus, we highlight that this functional requirement may have prevented gene loss during the long evolutionary history of red algal plastid genomes.

The dynamic history of plastid genomes in the Campanulaceae sensu lato is unique among angiosperms.

Why have some plants lost the organizational stability in plastid genomes (plastomes) that evolved in their algal ancestors? During the endosymbiotic transformation of a cyanobacterium into the eukaryotic plastid, most cyanobacterial genes were transferred to the nucleus or otherwise lost from the plastome, and the resulting plastome architecture in land plants confers organizational stability, as evidenced by the conserved gene order among bryophytes and lycophytes, whereas ferns, gymnosperms, and angiosperms share a single, 30-kb inversion. Although some additional gene losses have occurred, gene additions to angiosperm plastomes were previously unknown. Plastomes in the Campanulaceae sensu lato have incorporated dozens of large ORFs (putative protein-coding genes). These insertions apparently caused many of the 125+ large inversions now known in this small eudicot clade. This phylogenetically restricted phenomenon is not biogeographically localized, which indicates that these ORFs came from the nucleus or (less likely) a cryptic endosymbiont.

Plastid marker gene excision in greenhouse-grown tobacco by agrobacterium-delivered Cre recombinase.

Uniform transformation of the thousands of plastid genome (ptDNA) copies in a cell is driven by selection for plastid markers. When each of the plastid genome copies is uniformly altered, the marker gene is no longer needed. Plastid markers have been efficiently excised by site-specific recombinases expressed from nuclear genes either by transforming tissue culture cells or introducing the genes by pollination. Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the bar (au) gene, confers a visual aurea leaf phenotype; thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The bar (au) marker-free plastids are transmitted through seed to the progeny. PCR and DNA gel blot (Southern) protocols to confirm transgene integration and plastid marker excision are also provided herein.

Bayesian inference of phylogeny, morphology and range evolution reveals a complex evolutionary history in St. John's wort (Hypericum).

The genus Hypericum L. ("St. John's wort", Hypericaceae) comprises nearly 500 species of shrubs, trees and herbs distributed mainly in temperate regions of the Northern Hemisphere, but also in high-altitude tropical and subtropical areas. Until now, molecular phylogenetic hypotheses on infra-generic relationships have been based solely on the nuclear marker ITS. Here, we used a full Bayesian approach to simultaneously reconstruct phylogenetic relationships, divergence times, and patterns of morphological and range evolution in Hypericum, using nuclear (ITS) and plastid DNA sequences (psbA-trnH, trnS-trnG, trnL-trnF) of 186 species representing 33 of the 36 described morphological sections. Consistent with other studies, we found that corrections of the branch length prior helped recover more realistic branch lengths in by-gene partitioned Bayesian analyses, but the effect was also seen within single genes if the overall mutation rate differed considerably among sites or regions. Our study confirms that Hypericum is not monophyletic with the genus Triadenum embedded within, and rejects the traditional infrageneric classification, with many sections being para- or polyphyletic. The small Western Palearctic sections Elodes and Adenotrias are the sister-group of a geographic dichotomy between a mainly New World clade and a large Old World clade. Bayesian reconstruction of morphological character states and range evolution show a complex pattern of morphological plasticity and inter-continental movement within the genus. The ancestors of Hypericum were probably tropical shrubs that migrated from Africa to the Palearctic in the Early Tertiary, concurrent with the expansion of tropical climates in northern latitudes. Global climate cooling from the Mid Tertiary onwards might have promoted adaptation to temperate conditions in some lineages, such as the development of the herbaceous habit or unspecialized corollas.

Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants.

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.

Biolistic co-transformation of the nuclear and plastid genomes.

Particle gun-mediated (so-called 'biolistic') transformation represents a universal genetic transformation technology that is widely applied in nearly all groups of organisms. The mechanism of how accelerated DNA-coated particles, after their entry into the cell, deliver the foreign DNA to the target compartment is not known. Here we have studied this process in plants by performing co-transformation experiments with vectors targeted to two different cellular compartments, the nucleus and the plastids (chloroplasts). We find that coating of particles with both plastid and nuclear transformation vectors can result in co-transformation of chloroplasts and the nucleus. In contrast, mixing of particles coated individually with the vectors does not produce co-transformed plants. Our data suggest that a single DNA-coated particle can transform more than one compartment of the plant cell, opening up the possibility to generate doubly transgenic plants in one step. Importantly, co-transformation can also be obtained in the absence of selection, thus providing a method to produce marker-free transgenic genomes. In addition, our findings raise the possibility of occasional inadvertent co-transformation of two genomes and, therefore, have important implications for the molecular characterization and regulation of transgenic plants.