Plastid genome sequencing,comparative genomics,and phylogenomics: Current status and prospects

Abstract More than 190 plastid genomes have been completely sequenced during the past two decades due to advances in DNA sequencing technologies. Based on this unprecedented abundance of data, extensive genomic changes have been revealed in the plastid genomes. Inversion is the most common mechanism that leads to gene order changes. Several inversion events have been recognized as informative phylogenetic markers, such as a 30‐kb inversion found in all living vascular plants minus lycopsids and two short inversions putatively shared by all ferns. Gene loss is a common event throughout plastid genome evolution. Many genes were independently lost or transferred to the nuclear genome in multiple plant lineages. The trnR‐CCG gene was lost in some clades of lycophytes, ferns, and seed plants, and all the ndh genes were absent in parasitic plants, gnetophytes, Pinaceae, and the Taiwan moth orchid. Certain parasitic plants have, in particular, lost plastid genes related to photosynthesis because of the relaxation of functional constraint. The dramatic growth of plastid genome sequences has also promoted the use of whole plastid sequences and genomic features to solve phylogenetic problems. Chloroplast phylogenomics has provided additional evidence for deep‐level phylogenetic relationships as well as increased phylogenetic resolutions at low taxonomic levels. However, chloroplast phylogenomics is still in its infant stage and rigorous analysis methodology has yet to be developed.     

Plastid genome sequences of legumes reveal parallel inversions and multiple losses of rps16 in papilionoids

To date, publicly available plastid genomes of legumes have for the most part been limited to the subfamily Papilionoideae. Here we report 13 new plastid genomes of legumes spanning all three subfamilies. The genomes representing Caesalpinioideae and Mimosoideae are highly conserved in gene content and gene order, similar to the ancestral angiosperm genome organization. Genomes within the Papilionoideae, however, have reduced sizes due to deletions in nine intergenic spacers primarily in the large single copy region. Our study also indicates that rps16 has been independently lost at least five times in legumes, with additional gene and intron losses scattered among the papilionoids. Additionally, genera from two distinct lineages within the papilionoids, Lupinus and Robinia, have a parallel inversion of 36 and 39 kb, respectively. This parallel inversion is novel as it appears to be caused by a 29 bp repeat within two trnS genes. This repeat is present in all available legume plastid genomes indicating that there is the potential for this inversion to be present in more species. This case of a homoplasious inversion is also evidence that some inversion events may not be reliable phylogenetic markers.     

Distribution and phylogenetic significance of the 71-kb inversion in the plastid genome in Funariidae (Bryophyta)

BACKGROUND AND AIMS: The recent assembly of the complete sequence of the plastid genome of the model taxon Physcomitrella patens (Funariaceae, Bryophyta) revealed that a 71-kb fragment, encompassing much of the large single copy region, is inverted. This inversion of 57% of the genome is the largest rearrangement detected in the plastid genomes of plants to date. Although initially considered diagnostic of Physcomitrella patens, the inversion was recently shown to characterize the plastid genome of two species from related genera within Funariaceae, but was lacking in another member of Funariidae. The phylogenetic significance of the inversion has remained ambiguous. METHODS: Exemplars of all families included in Funariidae were surveyed. DNA sequences spanning the inversion break ends were amplified, using primers that anneal to genes on either side of the putative end points of the inversion. Primer combinations were designed to yield a product for either the inverted or the non-inverted architecture. KEY RESULTS: The survey reveals that exemplars of eight genera of Funariaceae, the sole species of Disceliaceae and three generic representatives of Encalyptales all share the 71-kb inversion in the large single copy of the plastid genome. By contrast, the plastid genome of Gigaspermaceae (Funariales) is characterized by a gene order congruent with that described for other mosses, liverworts and hornworts, and hence it does not possess this inversion. CONCLUSIONS: The phylogenetic distribution of the inversion in the gene order supports a hypothesis only weakly supported by inferences from sequence data whereby Funariales are paraphyletic, with Funariaceae and Disceliaceae sharing a common ancestor with Encalyptales, and Gigaspermaceae sister to this combined clade. To reflect these relationships, Gigaspermaceae are excluded from Funariales and accommodated in their own order, Gigaspermales order nov., within Funariideae.     

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales)

Premise

Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome.

Methods

We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω).

Results

Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK.

Conclusions

We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.     

The complete chloroplast genome of the chlorarachniophyte Bigelowiella natans: evidence for independent origins of chlorarachniophyte and euglenid secondary endosymbionts

Chlorarachniophytes are amoeboflagellate cercozoans that acquired a plastid by secondary endosymbiosis. Chlorarachniophytes are the last major group of algae for which there is no completely sequenced plastid genome. Here we describe the 69.2-kbp chloroplast genome of the model chlorarachniophyte Bigelowiella natans. The genome is highly reduced in size compared with plastids of other photosynthetic algae and is closer in size to genomes of several nonphotosynthetic plastids. Unlike nonphotosynthetic plastids, however, the B. natans chloroplast genome has not sustained a massive loss of genes, and it retains nearly all of the functional photosynthesis-related genes represented in the genomes of other green algae. Instead, the genome is highly compacted and gene dense. The genes are organized with a strong strand bias, and several unusual rearrangements and inversions also characterize the genome; notably, an inversion in the small-subunit rRNA gene, a translocation of 3 genes in the major ribosomal protein operon, and the fragmentation of the cluster encoding the large photosystem proteins PsaA and PsaB. The chloroplast endosymbiont is known to be a green alga, but its evolutionary origin and relationship to other primary and secondary green plastids has been much debated. A recent hypothesis proposes that the endosymbionts of chlorarachniophytes and euglenids share a common origin (the Cabozoa hypothesis). We inferred phylogenies using individual and concatenated gene sequences for all genes in the genome. Concatenated gene phylogenies show a relationship between the B. natans plastid and the ulvophyte-trebouxiophyte-chlorophyte clade of green algae to the exclusion of Euglena. The B. natans plastid is thus not closely related to that of Euglena, which suggests that plastids originated independently in these 2 groups and the Cabozoa hypothesis is false.     

SecA is plastid-encoded in a red alga: implications for the evolution of plastid genomes and the thylakoid protein import apparatus

Summary Partial sequence analysis of the plastid DNA (ptDNA) from a red alga, Antithamnion sp., revealed the presence of a homologue to the Escherichia coli SecA gene as well as two open reading frames (ORF 510, ORF 179). In addition a sec Y homologue has been detected on the plastid genome by heterologous hybridization. None of these genes has been found in completely sequenced chlorophytic plastid genomes. SecA and secY gene copies were also detected in the ptDNA of a chromophytic alga, indicating that secAY may be ubiquitous in rhodophytes and chromophytes. The significance of these findings for the evolution of plastid genomes and the thylakoid protein import mechanism is discussed.     

Covarion structure in plastid genome evolution: a new statistical test

Covarion models of molecular evolution allow the rate of evolution of a site to vary through time. There are few simple and effective tests for covarion evolution, and consequently, little is known about the presence of covarion processes in molecular evolution. We describe two new tests for covarion evolution and demonstrate with simulations that they perform well under a wide range of conditions. A survey of covarion evolution in sequenced plastid genomes found evidence of covarion drift in at least 26 out of 57 genes. Covarion evolution is most evident in first and second codon positions of the plastid genes, and there is no evidence of covarion evolution in third codon positions. Therefore, the significant covarion tests are likely due to changes in the selective constraints of amino acids. The frequency of covarion evolution within the plastid genome suggests that covarion processes of evolution were important in generating the observed patterns of sequence variation among plastid genomes.     

Sequencing of whole plastid genomes and nuclear ribosomal DNA of Diospyros species (Ebenaceae) endemic to New Caledonia: many species,little divergence

Background and Aims Some plant groups, especially on islands, have been shaped by strong ancestral bottlenecks and rapid, recent radiation of phenotypic characters. Single molecular markers are often not informative enough for phylogenetic reconstruction in such plant groups. Whole plastid genomes and nuclear ribosomal DNA (nrDNA) are viewed by many researchers as sources of information for phylogenetic reconstruction of groups in which expected levels of divergence in standard markers are low. Here we evaluate the usefulness of these data types to resolve phylogenetic relationships among closely related Diospyros species.Methods Twenty-two closely related Diospyros species from New Caledonia were investigated using whole plastid genomes and nrDNA data from low-coverage next-generation sequencing (NGS). Phylogenetic trees were inferred using maximum parsimony, maximum likelihood and Bayesian inference on separate plastid and nrDNA and combined matrices.Key Results The plastid and nrDNA sequences were, singly and together, unable to provide well supported phylogenetic relationships among the closely related New Caledonian Diospyros species. In the nrDNA, a 6-fold greater percentage of parsimony-informative characters compared with plastid DNA was found, but the total number of informative sites was greater for the much larger plastid DNA genomes. Combining the plastid and nuclear data improved resolution. Plastid results showed a trend towards geographical clustering of accessions rather than following taxonomic species.Conclusions In plant groups in which multiple plastid markers are not sufficiently informative, an investigation at the level of the entire plastid genome may also not be sufficient for detailed phylogenetic reconstruction. Sequencing of complete plastid genomes and nrDNA repeats seems to clarify some relationships among the New Caledonian Diospyros species, but the higher percentage of parsimony-informative characters in nrDNA compared with plastid DNA did not help to resolve the phylogenetic tree because the total number of variable sites was much lower than in the entire plastid genome. The geographical clustering of the individuals against a background of overall low sequence divergence could indicate transfer of plastid genomes due to hybridization and introgression following secondary contact.     

石松类和蕨类植物质体基因组结构演化研究进展

近年来, 随着测序技术的发展, 石松类和蕨类植物的核基因组、质体基因组以及线粒体基因组研究发展迅速, 质体基因组研究工作更是呈爆发式增长。截至2019年3月1日, GenBank公布的石松类和蕨类植物的175个质体基因组中, 约3/4为最近两年新增。研究内容从早期对个别质体基因组结构和序列特征的简单报道, 逐渐发展到综合性的比较基因组学和系统发育基因组学研究。目前已发表的质体基因组覆盖了石松类和蕨类植物的所有目和大部分科, 这两大类群的质体基因组结构变异和系统发育的基本框架已逐渐清晰。这些研究为我们理解维管植物的早期演化提供了重要参考。本文对石松类和蕨类植物的质体基因组结构特征进行了系统梳理, 发现其结构变异主要包括大片段倒位、IR区边界变动、基因或内含子丢失等, 其中一些结构变异可作为较高分类阶元的共衍征。RNA编辑和长片段非编码序列插入普遍存在于石松类和蕨类植物的质体基因组中, 但其起源、演化机制和功能等仍不清楚。我们对质体基因组的应用、系统发育研究中质体和核基因组的优劣性, 以及系统发育基因组学的前景进行了评述。     

Identification of functional lox sites in the plastid genome

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.     

Structural organization and evolution of the plastid genome of Vaucheria sessilis (Xanthophyceae)

The plastid DNAs of 18 Vaucheria sessilis strains from various habitats in western Europe were digested with the restriction endonucleases Eco RI, Sal I, Bam HI and Pvu II. Their restriction patterns showed variable fragment divergencies. Two main groups of plastid genomes were recognized, which were substantiated by morphological features. The differences among the restriction patterns could be attributed to the loss or appearance of restriction sites and to minor size variations caused by deletions/insertions. The Sal I and Bam HI restriction sites which together discriminate six different plastid genomes were mapped on the circular molecule of 124 kilobase paris (kbp). The plastid genomes of several Vaucheria sessilis strains were shown to exist in two inversion isomers caused by intramolecular recombination within the inverted repeat segments.     

Genomic architectural variation of plant mitochondria—A review of multichromosomal structuring

Since the endosymbiont origin from α-Proteobacteria, mitochondrial genomes have undergone extremely divergent evolutionary trajectories among eukaryotic lineages. Compared with the relatively compact and conserved animal mitochondrial genomes, plant mitochondrial genomes have many unique features, especially their large and complex genomic arrangements. The sizes of fully sequenced plant mitochondrial genomes span over a 100-fold range from 66 kb in Viscum scurruloideum to 11 000 kb in Silene conica. In addition to the typical circular structure, some species of plants also possess linear, and even multichromosomal, architectures. In contrast with the thousands of fully sequenced animal mitochondrial genomes and plant plastid genomes, only around 200 fully sequenced land plant mitochondrial genomes have been published, with many being only draft assemblies. In this review, we summarize some of the known novel characteristics found in plant mitochondrial genomes, with special emphasis on multichromosomal structures described in recent publications. Finally, we discuss the future prospects for studying the inheritance patterns of multichromosomal plant mitochondria and examining architectural variation at different levels of taxonomic organization—including at the population level.     

Dictyochophyceae Plastid Genomes Reveal Unusual Variability in Their Organization

Dictyochophyceae (silicoflagellates) are unicellular freshwater and marine algae (Heterokontophyta, stramenopiles). Despite their abundance in global oceans and potential ecological significance, discovered in recent years, neither nuclear nor organellar genomes of representatives of this group were sequenced until now. Here, we present the first complete plastid genome sequences of Dictyochophyceae, obtained from four species: Dictyocha speculum, Rhizochromulina marina, Florenciella parvula and Pseudopedinella elastica. Despite their comparable size and genetic content, these four plastid genomes exhibit variability in their organization: plastid genomes of F. parvula and P. elastica possess conventional quadripartite structure with a pair of inverted repeats, R. marina instead possesses two direct repeats with the same orientation and D. speculum possesses no repeats at all. We also observed a number of unusual traits in the plastid genome of D. speculum, including expansion of the intergenic regions, presence of an intron in the otherwise non‐intron‐bearing psaA gene, and an additional copy of the large subunit of RuBisCO gene (rbcL), the last of which has never been observed in any plastid genome. We conclude that despite noticeable gene content similarities between the plastid genomes of Dictyochophyceae and their relatives (pelagophytes, diatoms), the number of distinctive features observed in this lineage strongly suggests that additional taxa require further investigation.     

Evolution of Red Algal Plastid Genomes: Ancient Architectures,Introns, Horizontal Gene Transfer,and Taxonomic Utility of Plastid Markers

Red algae have the most gene-rich plastid genomes known, but despite their evolutionary importance these genomes remain poorly sampled. Here we characterize three complete and one partial plastid genome from a diverse range of florideophytes. By unifying annotations across all available red algal plastid genomes we show they all share a highly compact and slowly-evolving architecture and uniquely rich gene complements. Both chromosome structure and gene content have changed very little during red algal diversification, and suggest that plastid-to nucleus gene transfers have been rare. Despite their ancient character, however, the red algal plastids also contain several unprecedented features, including a group II intron in a tRNA-Met gene that encodes the first example of red algal plastid intron maturase – a feature uniquely shared among florideophytes. We also identify a rare case of a horizontally-acquired proteobacterial operon, and propose this operon may have been recruited for plastid function and potentially replaced a nucleus-encoded plastid-targeted paralogue. Plastid genome phylogenies yield a fully resolved tree and suggest that plastid DNA is a useful tool for resolving red algal relationships. Lastly, we estimate the evolutionary rates among more than 200 plastid genes, and assess their usefulness for species and subspecies taxonomy by comparison to well-established barcoding markers such as cox1 and rbcL. Overall, these data demonstrates that red algal plastid genomes are easily obtainable using high-throughput sequencing of total genomic DNA, interesting from evolutionary perspectives, and promising in resolving red algal relationships at evolutionarily-deep and species/subspecies levels.     

HSP90, tubulin and actin are retained in the tertiary endosymbiont genome of Kryptoperidinium foliaceum

The dinoflagellate Kryptoperidinium foliaceum has replaced its ancestral peridinin-containing plastid with a fucoxanthin-containing diatom plastid via tertiary endosymbiosis. The diatom endosymbiont of K. foliaceum is much less reduced than well-studied endosymbiotic intermediates, such as cryptophytes and chlorarachniophytes, where relict nuclear genomes are retained in secondary endosymbionts. The K. foliaceum endosymbiont retains a prominent nucleus, multiple four-membrane plastids, and mitochondria, all within a relatively large volume of cytoplasm that is separated from the host cytoplasm by a single membrane. Here we report the first protein-coding gene sequences from the K. foliaceum endosymbiont and host nuclear genomes. We have characterised genes for nucleus-encoded cytosolic proteins, actin (from endosymbiont), alpha-tubulin (from both), beta-tubulin (from host), and HSP90 (from both), in addition to homologues from pennate diatoms Nitzschia thermalis and Phaeodactylum tricornutum. Phylogenetic reconstruction shows that the actin is diatom-derived, the beta-tubulin dinoflagellate-derived, while both diatom- and dinoflagellate-derived alpha-tubulin and HSP90 genes were found. The base composition biases of these genes co-varied with their phylogenetic position, suggesting that the genes still reside in their respective genomes. The presence of these genes implies they are still functional and more generally indicates that the endosymbiont is less genetically reduced than those of cryptophytes or chlorarachniophytes, raising the interesting question of whether any genes have transferred between the two nuclear genomes.     

Nucleomorph genomes: structure, function, origin and evolution

The cryptomonads and chlorarachniophytes are two unicellular algal lineages with complex cellular structures and fascinating evolutionary histories. Both groups acquired their photosynthetic abilities through the assimilation of eukaryotic endosymbionts. As a result, they possess two distinct cytosolic compartments and four genomes--two nuclear genomes, an endosymbiont-derived plastid genome and a mitochondrial genome derived from the host cell. Like mitochondrial and plastid genomes, the genome of the endosymbiont nucleus, or 'nucleomorph', of cryptomonad and chlorarachniophyte cells has been greatly reduced through the combined effects of gene loss and intracellular gene transfer. This article focuses on the structure, function, origin and evolution of cryptomonad and chlorarachniophyte nucleomorph genomes in light of recent comparisons of genome sequence data from both groups. It is now possible to speculate on the reasons that nucleomorphs persist in cryptomonads and chlorarachniophytes but have been lost in all other algae with plastids of secondary endosymbiotic origin.     

The mitochondrial genome of Arabidopsis is composed of both native and immigrant information

Plants contain large mitochondrial genomes, which are several times as complex as those in animals, fungi or algae. However, genome size is not correlated with information content. The mitochondrial genome (mtDNA) of Arabidopsis specifies only 58 genes in 367 kb, whereas the 184 kb mtDNA in the liverwort Marchantia polymorpha codes for 66 genes, and the 58 kb genome in the green alga Prototheca wickerhamii encodes 63 genes. In Arabidopsis’ mtDNA, genes for subunits of complex II, for several ribosomal proteins and for 16 tRNAs are missing, some of which have been transferred recently to the nuclear genome. Numerous integrated fragments originate from alien genomes, including 16 sequence stretches of plastid origin, 41 fragments of nuclear (retro)transposons and two fragments of fungal viruses. These immigrant sequences suggest that the large size of plant mitochondrial genomes is caused by secondary expansion as a result of integration and propagation, and is thus a derived trait established during the evolution of land plants.     

Comparative plastid genomics of Pinus species: Insights into sequence variations and phylogenetic relationships

Pinus L. is the largest genus of conifers and provides a classical model for studying species divergence and phylogenetic evolution by gymnosperms. However, our poor understanding of sequence divergence in the whole plastid genomes of Pinus species severely hinders studies of their evolution and phylogeny. Thus, we analyzed the sequences of 97 Pinus plastid genomes, including four newly sequenced genomes and 93 previously published plastomes, to explore the evolution and phylogenetic relationships in the genus Pinus. The complete chloroplast genomes of Pinus species ranged in size from 109 640 bp (P. cembra L.) to 121 976 bp (P. glabra Walter), and these genomes comprised circular DNA molecules in a similar manner to those of most gymnosperms. We identified 9108 repeats where most of the repeats comprised the dispersed type with 3983 (44%), followed by tandem repeats with 2999 (33%), and then palindromic repeats with 2126 (23%). Sixteen divergence hotspot regions were identified in Pinus plastid genomes, which could be useful molecular markers for future population genetics studies. Phylogenetic analysis showed that Pinus species could be divided into two diverged clades comprising the subgenera Strobus (single needle section) and Pinus (double needles section). Molecular dating suggested that the genus Pinus originated approximately 130.38 Mya during the late Cretaceous. The two subgenera subsequently split 85.86 Mya, which was largely consistent with the other molecular results based on partial DNA markers. These findings provide important insights into the sequence variations and phylogenetic evolution of Pinus plastid genomes.     

Ins and outs of plastid genome evolution

Recent findings have established cracks in the straight-laced image of the plastid genome as a molecule whose sole function is photosynthesis and whose gene content is highly conserved. Genes for numerous non-photosynthetic functions have been identified. Algal plastid genomes contain many genes with no homologs in angiosperms, and the recent transfer of genes from the plastid to the nuclear genome has been described. Wholesale abandonment of genes encoding photosynthetic and gene-expression functions has occurred in the plastid genomes of a non-green plant and alga. The origins of plastid DNA, its use in phylogenetic studies, and the origins of plastid introns are also reviewed.