TrnL-trnF sequence data imply paraphyly of Aquilaria and Gyrinops (Thymelaeaceae) and provide new perspectives for agarwood identification

The genera Aquilaria and Gyrinops (Thymelaeaceae, Malvales) are well known for the production of agarwood which is a highly wanted forest product of substantial economic value. The taxonomic status of Aquilaria and Gyrinops as separate genera is doubted as they are only distinguished by the number of stamens. We investigated their status by conducting phylogenetic analyses of DNA sequences from the plastid trnL-trnF spacer. Control of international trade of agarwood is currently hampered by the failure of traditional methods such as microscopy to identify samples to species level. We therefore evaluated the potential of molecular identification of agarwood by searching for species- and region-specific plastid DNA polymorphisms. DNA sequences were obtained from 31 Thymelaeaceae accessions encompassing 20 different species in six genera. Aquilaria and Gyrinops appear to be paraphyletic. Success in sequencing wood samples demonstrates that molecular markers provide new perspectives for agarwood identification.     

Molecular phylogenetics supports a clade of red algal parasites retaining native plastids: taxonomy and terminology revised

Parasitism is a life strategy that has repeatedly evolved within the Florideophyceae. Historically, the terms adelphoparasite and alloparasite have been used to distinguish parasites based on the relative phylogenetic relationship of host and parasite. However, analyses using molecular phylogenetics indicate that nearly all red algal parasites infect within their taxonomic family, and a range of relationships exist between host and parasite. To date, all investigated adelphoparasites have lost their plastid, and instead, incorporate a host‐derived plastid when packaging spores. In contrast, a highly reduced plastid lacking photosynthesis genes was sequenced from the alloparasite Choreocolax polysiphoniae. Here we present the complete Harveyella mirabilis plastid genome, which has also lost genes involved in photosynthesis, and a partial plastid genome from Leachiella pacifica. The H. mirabilis plastid shares more synteny with free‐living red algal plastids than that of C. polysiphoniae. Phylogenetic analysis demonstrates that C. polysiphoniae, H. mirabilis, and L. pacifica form a robustly supported clade of parasites, which retain their own plastid genomes, within the Rhodomelaceae. We therefore transfer all three genera from the exclusively parasitic family, Choreocolacaceae, to the Rhodomelaceae. Additionally, we recommend applying the terms archaeplastic parasites (formerly alloparasites), and neoplastic parasites (formerly adelphoparasites) to distinguish red algal parasites using a biological framework rather than taxonomic affiliation with their hosts.     

Complex Patterns of Plastid 16S rRNA Gene Evolution in Nonphotosynthetic Green Algae

This study provides a phylogenetic/comparative approach to deciphering the processes underlying the evolution of plastid rRNA genes in genomes under relaxed functional constraints. Nonphotosynthetic green algal taxa that belong to two distinct classes, Chlorophyceae (Polytoma) and Trebouxiophyceae (Prototheca), were investigated. Similar to the situation described previously for plastid 16S rRNA genes in nonphotosynthetic land plants, nucleotide substitution levels, extent of structural variations, and percentage AT values are increased in nonphotosynthetic green algae compared to their closest photosynthetic relatives. However, the mutational processes appear to be different in many respects. First, with the increase in AT content, more transversions are noted in Polytoma and holoparasite angiosperms, while more transitions characterize the evolution of the 16S rDNA sequences in Prototheca. Second, although structural variations do accumulate in both Polytoma and Prototheca (as well as holoparasitic plastid 16S rRNAs), insertions as large as 1.6 kb characterize the plastid 16S rRNA genes in the former, whereas significantly smaller indels (not exceeding 24 bp) seem to be more prevalent in the latter group. The differences in evolutionary rates and patterns within and between lineages might be due to mutations in replication/repair-related genes; slipped-strand mispairing is likely the mechanism responsible for the expansion of insertions in Polytoma plastid 16S rRNA genes. Received: 29 December 2000 / Accepted: 18 May 2001     

Plastid marker gene excision by the phiC31 phage site-specific recombinase

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.     

Plastid DNA sequence data help to clarify phylogenetic relationships and reticulate evolution in Lycoris (Amaryllidaceae)

The temperate East Asian genus Lycoris is a well known lineage of ornamental geophytes consisting of at least 20 species, some of which are thought to be of natural hybrid origin. Previous genetic studies have supported this hypothesis, but these have relied solely on the use of karyology and/or nuclear ribosomal ITS sequences. No plastid DNA data have been available to address interspecific relationships within Lycoris until now. In this study, 500 individuals from 29 populations representing 16 of the 20 published Lycoris spp. were sampled, and DNA sequences were generated for two plastid markers (trnS‐trnfM and trnC‐ycf6). From these data we inferred phylogenetic relationships among the sampled taxa at the species and population levels using concatenated phylogenetic methods. A well resolved and strongly supported phylogenetic reconstruction for Lycoris was obtained. Although the plastid DNA topology differs from that derived previously using ITS, both genomes produce trees that cluster Lycoris spp. into three clades. One of these, containing polyploid taxa such as L. albiflora, L. caldwellii, L. straminea and L. houdyshelii, shows strong evidence of reticulation, and we discuss the identity of potential parents of these allopolyploids. In contrast, we offer evidence that challenges the hypothesis that triploid individuals of L. radiata are the result of hybridization. Instead, they appear to be autotriploids that have arisen in more than one location. By comparing the phylogenetic results obtained using nuclear genomic data to those from the plastid genome, a much clearer picture of the role that hybridization and reticulation have played in the evolution of Lycoris is emerging. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 115–126.     

PHYLOGENETIC IMPLICATIONS OF DIFFERENCES IN NUMBER OF PLASTID PHOSPHOGLUCOSE ISOMERASE ISOZYMES IN NORTH AMERICAN COREOPSIS (ASTERACEAE: HELIANTHEAE: COREOPSIDINAE)

Enzyme electrophoresis was employed to ascertain the number of loci encoding plastid phosphoglucose isomerase (PGI) in species representing all sections of North American Coreopsis. Several species from each of the closely related genera Bidens, Coreocarpus, Cosmos, and Thelesperma were also examined. Species in nine of the 11 sections of North American Coreopsis have two isozymes for plastid PGI, and nearly all species examined in the four other genera also have two (one species has three) isozymes. Since most diploid vascular plants have one plastid PGI isozyme, a gene duplication probably occurred in an ancestor that is common to Coreopsis and the other four genera. That is, two isozymes represent the ancestral number for Coreopsis. The two sections (Electra and Anathysana) apparently lacking the duplication are closely related woody plants restricted largely to Mexico. One gene encoding plastid PGI ostensibly was silenced in a common ancestor of these two sections. This is concordant with other data suggesting a close relationship between the two sections, i.e., they appear to represent a monophyletic group. The electrophoretic data also indicate that 1) the enigmatic monotypic section Silphidium is more closely related to eastern North American sections and not derived from section Electra; and 2) section Anathysana is not ancestral to the three California sections Leptosyne, Pugiopappus, and Tuckermannia; rather, it represents a terminal element closely related to and possibly derived from section Electra.     

Functional loss of all ndh genes in an otherwise relatively unaltered plastid genome of the holoparasitic flowering plant Cuscuta reflexa

We have cloned and sequenced an area of about 9.0 kb of the plastid DNA (ptDNA) from the holoparasitic flowering plant Cuscuta reflexa to investigate the evolutionary response of plastid genes to a reduced selective pressure. The region contains genes for the 16S rRNA, a subunit of a plastid NAD(P)H dehydrogenase (ndhB), three transfer RNAs (trnA, trnI, trnV) as well as the gene coding for the ribosomal protein S7 (rps7). While the other genes are strongly conserved in C. reflexa, the ndhB gene is a pseudogene due to many frameshift mutations. In addition we used heterologous gene probes to identify the other ndh genes encoded by the plastid genome in higher plants. No hybridization signals could be obtained, suggesting that these genes are either lost or strongly altered in the ptDNA of C. reflexa. Together with evidence of deleted genes in the ptDNA of C. reflexa, the plastid genome can be grouped into four classes reflecting a different evolutionary rate in each case. The phylogenetic position of Cuscuta and the significance of ndh genes in the plastid genome of higher plants are discussed.     

Gene-rich plastid genomes of two parasitic red algal species,Laurencia australis and L. verruciformis (Rhodomelaceae,Ceramiales), and a taxonomic revision of Janczewskia

Parasitic red algae are an interesting system for investigating the genetic changes that occur in parasites. These parasites have evolved independently multiple times within the red algae. The functional loss of plastid genomes can be investigated in these multiple independent examples, and fine-scale patterns may be discerned. The only plastid genomes from red algal parasites known so far are highly reduced and missing almost all photosynthetic genes. Our study assembled and annotated plastid genomes from the parasites Janczewskia tasmanica and its two Laurencia host species (Laurencia elata and one unidentified Laurencia sp. A25) from Australia and Janczewskia verruciformis, its host species (Laurencia catarinensis), and the closest known free-living relative (Laurencia obtusa) from the Canary Islands (Spain). For the first time we show parasitic red algal plastid genomes that are similar in size and gene content to free-living host species without any gene loss or genome reduction. The only exception was two pseudogenes (moeB and ycf46) found in the plastid genome of both isolates of J. tasmanica, indicating potential for future loss of these genes. Further comparative analyses with the three highly reduced plastid genomes showed possible gene loss patterns, in which photosynthetic gene categories were lost followed by other gene categories. Phylogenetic analyses did not confirm monophyly of Janczewskia, and the genus was subsumed into Laurencia. Further investigations will determine if any convergent small-scale patterns of gene loss exist in parasitic red algae and how these are applicable to other parasitic systems.     

Plant phosphoglucose isomerase genes lack introns and are expressed in Escherichia coli

Nuclear genes that appear to encode both cytosolic and plastid isozymes of phosphoglucose isomerase (PGI), an essential glycolytic enzyme, have been isolated from three diploid species of the annual wild flower genus Clarkia (Onagraceae). The genes do not contain introns and are expressed to varying degrees in Escherichia coli when cloned in either Charon 35 phage or pUC plasmid vectors. The PGI proteins synthesized in E. coli form dimers, are catalytically active, and their electrophoretic mobilities are similar to those of appropriate Clarkia PGIs. The nucleotide sequence of a gene encoding a plastid isozyme of C. unguiculata is described.     

Simple and efficient plastid transformation system for the liverwort <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> L. suspension-culture cells

We have established a simple and efficient plastid transformation system for liverwort, Marchantia polymorpha L., suspension-culture cells, which are homogenous, chloroplast-rich and␣rapidly growing. Plasmid pCS31 was constructed to integrate an aadA expression cassette for spectinomycin-resistance into the trnI–trnA intergenic region of the liverwort plastid DNA by homologous recombination. Liverwort suspension-culture cells were bombarded with pCS31-coated gold projectiles and selected on a medium containing spectinomycin. Plastid transformants were reproducibly isolated from the obtained spectinomycin-resistant calli. Selection on a sucrose-free medium greatly improved the efficiency of selection of plastid transformants. Homoplasmic plastid transformant lines were established by␣successive subculturing for 14 weeks or longer on the spectinomycin-containing medium. The plastid transformation system of liverwort suspension-culture cells should facilitate the investigation of the fundamental genetic systems of plastid DNA, such as replication.     

Precise excision of plastid DNA by the large serine recombinase Bxb1

Marker genes are essential for the selection and identification of rarely occurring transformation events generated in biotechnology. This includes plastid transformation, which requires that multiple copies of the modified chloroplast genome be present to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here, we demonstrate the precise excision of attP‐ and attB‐flanked DNA from the plastid genome mediated by the large serine recombinase Bxb1. We transformed the tobacco plastid genome with the pTCH‐PB vector containing a stuffer fragment of DNA flanked by directly oriented nonhomologous attP and attB recombinase recognition sites. In the absence of the Bxb1 recombinase, the transformed plastid genomes were stable and heritable. Nuclear‐transformed transgenic tobacco plants expressing a plastid‐targeted Bxb1 recombinase were crossed with transplastomic pTCH‐PB plants, and the T₁ hybrids exhibited efficient excision of the target sequence. The Bxb1–att system should prove to be a useful tool for site‐specifically manipulating the plastid genome and generating marker‐free transplastomic plants.     

Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker

We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regenera- tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid-expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.     

Plastid transformants of tomato selected using mutations affecting ribosome structure

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.     

Comparative Analysis of the Complete Plastid Genome Sequence of the Red Alga Gracilaria tenuistipitata var. liui Provides Insights into the Evolution of Rhodoplasts and Their Relationship to Other Plastids

We sequenced to completion the circular plastid genome of the red alga Gracilaria tenuistipitata var. liui. This is the first plastid genome sequence from the subclass Florideophycidae (Rhodophyta). The genome is composed of 183,883 bp and contains 238 predicted genes, including a single copy of the ribosomal RNA operon. Comparisons with the plastid genome of Porphyra pupurea reveal strong conservation of gene content and order, but we found major genomic rearrangements and the presence of coding regions that are specific to Gracilaria. Phylogenetic analysis of a data set of 41 concatenated proteins from 23 plastid and two cyanobacterial genomes support red algal plastid monophyly and a specific evolutionary relationship between the Florideophycidae and the Bangiales. Gracilaria maintains a surprisingly ancient gene content in its plastid genome and, together with other Rhodophyta, contains the most complete repertoire of plastid genes known in photosynthetic eukaryotes.Supplementary material () is available for this article.[Reviewing Editor: Dr. W. Ford Doolittle]     

Selective feeding and foreign plastid retention in an Antarctic dinoflagellate

The peridinin‐containing plastid found in most photosynthetic dinoflagellates is thought to have been replaced in a few lineages by plastids of chlorophyte, diatom, or haptophyte origin. Other distinct lineages of phagotrophic dinoflagellates retain functional plastids obtained from algal prey for different durations and with varying source species specificity. 18S rRNA gene sequence analyses have placed a novel gymnodinoid dinoflagellate isolated from the Ross Sea (RSD) in the Kareniaceae, a family of dinoflagellates with permanent plastids of haptophyte origin. In contrast to other species in this family, the RSD contains kleptoplastids sequestered from its prey, Phaeocystis antarctica. Culture experiments were employed to determine whether the RSD fed selectively on P. antarctica when offered in combination with another polar haptophyte or cryptophyte species, and whether the RSD, isolated from its prey and starved, would take up plastids from P. antarctica or from other polar haptophyte or cryptophyte species. Evidence was obtained for selective feeding on P. antarctica, plastid uptake from P. antarctica, and increased RSD growth in the presence of P. antarctica. The presence of a peduncle‐like structure in the RSD suggests that kleptoplasts are obtained by myzocytosis. RSD cells incubated without P. antarctica were capable of survival for at least 29.5 months. This remarkable longevity of the RSD's kleptoplasts and its species specificity for prey and plastid source is consistent with its prolonged co‐evolution with P. antarctica. It may also reflect the presence of a plastid protein import mechanism and genes transferred to the dinokaryon from a lost permanent haptophyte plastid.     

GENETIC EVIDENCE FOR TRACE PATERNAL TRANSMISSION OF PLASTIDS IN LIRIODENDRON AND MAGNOLIA (MAGNOLIACEAE)

Transmission of plastids in Liriodendron and Magnolia was studied by examining inheritance of plastid DNA markers in interspecific crosses. In congeneric hybrids of Liriodendron and Magnolia, 2.9% and 11.1% of progeny, respectively, exhibited uniparental paternal transmission of plastids. This departure from strict uniparental maternal transmission of plastids indicates that mechanisms for the elimination of paternal plastids are not 100% efficient and that any proposed model for plastid transmission must account for a continuum of variation. Our results, and other results reviewed here, show a discrepancy with data obtained by cytological observations and point to the need of confirming the mode of plastid inheritance by genetic analysis. In addition, if paternal plastids are occasionally inherited, intensive sampling of plastid DNA in a putative hybrid population could identify both parents of hybrids and thereby aid in the phylogenetic reconstruction of hybrid zones.     

Phylogeny of Plastids Based on Cladistic Analysis of Gene Loss Inferred from Complete Plastid Genome Sequences

Based on the recent hypothesis on the origin of eukaryotic phototrophs, red algae, green plants, and glaucophytes constitute the primary photosynthetic eukaryotes (whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis), whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events (involving a phototrophic eukaryote and a host cell). Although phylogenetic analyses using multiple plastid genes from a wide range of eukaryotic lineages have been carried out, some of the major phylogenetic relationships of plastids remain ambiguous or conflict between different phylogenetic methods used for nucleotide or amino acid substitutions. Therefore, an alternative methodology to infer the plastid phylogeny is needed. Here, we carried out a cladistic analysis of the loss of plastid genes after primary endosymbiosis using complete plastid genome sequences from a wide range of eukaryotic phototrophs. Since it is extremely unlikely that plastid genes are regained during plastid evolution, we used the irreversible Camin-Sokal model for our cladistic analysis of the loss of plastid genes. The cladistic analysis of the 274 plastid protein-coding genes resolved the 20 operational taxonomic units representing a wide range of eukaryotic lineages (including three secondary plastid-containing groups) into two large monophyletic groups with high bootstrap values: one corresponded to the red lineage and the other consisted of a large clade composed of the green lineage (green plants and Euglena) and the basal glaucophyte plastid. Although the sister relationship between the green lineage and the Glaucophyta was not resolved in recent phylogenetic studies using amino acid substitutions from multiple plastid genes, it is consistent with the rbcL gene phylogeny and with a recent phylogenetic study using multiple nuclear genes. In addition, our analysis robustly resolved the conflicting/ambiguous phylogenetic positions of secondary plastids in previous phylogenetic studies: the Euglena plastid was sister to the chlorophycean (Chlamydomonas) lineage, and the secondary plastids from the diatom (Odontiella) and cryptophyte (Guillardia) were monophyletic within the red lineage.     

CYANOPHYTE AND CYANELLE DNA: A SEARCH FOR THE ORIGINS OF PLASTIDS1

Cyanophyte-like prokaryotes are widely presumed to be the progenitors of eukaryote plastids. A few rare protistan species bearing cyanophyte-like cyanelles may represent intermediate stages in the evolution of true organelles. Cyanophyte DNA disposition in the cell, so far as is known from electron microscopy, seems uniform within the group and distinctly different from the several known arrangements of DNA in plastids. Therefore a survey of representative cyanophytes and protistan cyanelles was undertaken to determine whether forms reminiscent of plastids could be found. DNA-specific fluorochromes were utilized, along with epifluorescent microscopy, to study the DNA arrangement in situ in whole cells. Only the endospore (baeocyte)-forming Cyanophyta contained more than one, centrally located DNA skein per cell, and then only for the period just preceding visible baeocyte formation. Such forms might, with modification, presage the “scattered nucleoid” DNA disposition found in plastids of several groups, including Rhodophytes, Cryptophytes, Chlorophytes and higher plants. The DNA arrangement in cyanelles of two protists, Cyanophora and Glaucocystis, appear different from each other and possibly related to, respectively, the cyanophytes Gloeobacter and Synechococcus. Cyanelles of the third protist, Glaucosphaera, like the cells of the unique prokaryote Prochloron, appear to have multiple sites of DNA, somewhat similar to those of the “scattered nucleoid” line of plastid evolution. No obvious precursor of the “ring nucleoid” or other types of plastid DNA conformation was found.