The evolution of chloroplast genome structure in ferns

The plastid genome (plastome) is a rich source of phylogenetic and other comparative data in plants. Most land plants possess a plastome of similar structure. However, in a major group of plants, the ferns, a unique plastome structure has evolved. The gene order in ferns has been explained by a series of genomic inversions relative to the plastome organization of seed plants. Here, we examine for the first time the structure of the plastome across fern phylogeny. We used a PCR-based strategy to map and partially sequence plastomes. We found that a pair of partially overlapping inversions in the region of the inverted repeat occurred in the common ancestor of most ferns. However, the ancestral (seed plant) structure is still found in early diverging branches leading to the osmundoid and filmy fern lineages. We found that a second pair of overlapping inversions occurred on a branch leading to the core leptosporangiates. We also found that the unique placement of the gene matK in ferns (lacking a flanking intron) is not a result of a large-scale inversion, as previously thought. This is because the intron loss maps to an earlier point on the phylogeny than the nearby inversion. We speculate on why inversions may occur in pairs and what this may mean for the dynamics of plastome evolution.     

Evolution of the rpoB-psbZ region in fern plastid genomes: notable structural rearrangements and highly variable intergenic spacers

Background  

The rpoB-psbZ (BZ) region of some fern plastid genomes (plastomes) has been noted to go through considerable genomic changes. Unraveling its evolutionary dynamics across all fern lineages will lead to clarify the fundamental process shaping fern plastome structure and organization.     

The evolution of the plastid chromosome in land plants: gene content, gene order, gene function

This review bridges functional and evolutionary aspects of plastid chromosome architecture in land plants and their putative ancestors. We provide an overview on the structure and composition of the plastid genome of land plants as well as the functions of its genes in an explicit phylogenetic and evolutionary context. We will discuss the architecture of land plant plastid chromosomes, including gene content and synteny across land plants. Moreover, we will explore the functions and roles of plastid encoded genes in metabolism and their evolutionary importance regarding gene retention and conservation. We suggest that the slow mode at which the plastome typically evolves is likely to be influenced by a combination of different molecular mechanisms. These include the organization of plastid genes in operons, the usually uniparental mode of plastid inheritance, the activity of highly effective repair mechanisms as well as the rarity of plastid fusion. Nevertheless, structurally rearranged plastomes can be found in several unrelated lineages (e.g. ferns, Pinaceae, multiple angiosperm families). Rearrangements and gene losses seem to correlate with an unusual mode of plastid transmission, abundance of repeats, or a heterotrophic lifestyle (parasites or myco-heterotrophs). While only a few functional gene gains and more frequent gene losses have been inferred for land plants, the plastid Ndh complex is one example of multiple independent gene losses and will be discussed in detail. Patterns of ndh-gene loss and functional analyses indicate that these losses are usually found in plant groups with a certain degree of heterotrophy, might rendering plastid encoded Ndh1 subunits dispensable.     

Passiflora plastome sequencing reveals widespread genomic rearrangements

Although past studies have included Passiflora among angiosperm lineages with highly rearranged plastid genomes (plastomes), knowledge about plastome organization in the genus is limited. So far only one draft and one complete plastome have been published. Expanded sampling of Passiflora plastomes is needed to understand the extent of the genomic rearrangement in the genus, which is also unusual in having biparental plastid inheritance and plastome‐genome incompatibility. We sequenced 15 Passiflora plastomes using either Illumina paired‐end or shotgun cloning and Sanger sequencing approaches. Assembled plastomes were annotated using Dual Organellar GenoMe Annotator (DOGMA) and tRNAscan‐SE. The Populus trichocarpa plastome was used as a reference to estimate genomic rearrangements in Passiflora by performing whole genome alignment in progressiveMauve. The phylogenetic distribution of rearrangements was plotted on the maximum likelihood tree generated from 64 plastid encoded protein genes. Inverted repeat (IR) expansion/contraction and loss of the two largest hypothetical open reading frames, ycf1 and ycf2, account for most plastome size variation, which ranges from 139 262 base pairs (bp) in P. biflora to 161 494 bp in P. pittieri. Passiflora plastomes have experienced numerous inversions, gene and intron losses along with multiple independent IR expansions and contractions resulting in a distinct organization in each of the three subgenera examined. Each Passiflora subgenus has a unique plastome structure in terms of gene content, order and size. The phylogenetic distribution of rearrangements shows that Passiflora has experienced widespread genomic changes, suggesting that such events may not be reliable phylogenetic markers.     

Comparative analysis of inverted repeats of polypod fern (Polypodiales) plastomes reveals two hypervariable regions

Background

Ferns are large and underexplored group of vascular plants (~ 11 thousands species). The genomic data available by now include low coverage nuclear genomes sequences and partial sequences of mitochondrial genomes for six species and several plastid genomes.

Results

We characterized plastid genomes of three species of Dryopteris, which is one of the largest fern genera, using sequencing of chloroplast DNA enriched samples and performed comparative analysis with available plastomes of Polypodiales, the most species-rich group of ferns. We also sequenced the plastome of Adianthum hispidulum (Pteridaceae). Unexpectedly, we found high variability in the IR region, including duplication of rrn16 in D. blanfordii, complete loss of trnI-GAU in D. filix-mas, its pseudogenization due to the loss of an exon in D. blanfordii. Analysis of previously reported plastomes of Polypodiales demonstrated that Woodwardia unigemmata and Lepisorus clathratus have unusual insertions in the IR region. The sequence of these inserted regions has high similarity to several LSC fragments of ferns outside of Polypodiales and to spacer between tRNA-CGA and tRNA-TTT genes of mitochondrial genome of Asplenium nidus. We suggest that this reflects the ancient DNA transfer from mitochondrial to plastid genome occurred in a common ancestor of ferns. We determined the marked conservation of gene content and relative evolution rate of genes and intergenic spacers in the IRs of Polypodiales. Faster evolution of the four intergenic regions had been demonstrated (trnA- orf42, rrn16-rps12, rps7-psbA and ycf2-trnN).

Conclusions

IRs of Polypodiales plastomes are dynamic, driven by such events as gene loss, duplication and putative lateral transfer from mitochondria.

     

Exploring the plastid genome disparity of liverworts

Sequencing the plastid genomes of land plants provides crucial improvements to our understanding of the plastome evolution of land plants. Although the number of available complete plastid genome sequences has rapidly increased in the recent years, only a few sequences have been yet released for the three bryophyte lineages, namely hornworts, liverworts, and mosses. Here, we explore the disparity of the plastome structure of liverworts by increasing the number of sequenced liverwort plastomes from five to 18. The expanded sampling included representatives of all major lineages of liverworts including the genus Haplomitrium. The disparity of the liverwort genomes was compared with other 2386 land plant plastomes with emphasis on genome size and GC‐content. We found evidence for structural conservatism of the plastid genomes in liverworts and a trend towards reduced plastome sequence length in liverworts and derived mosses compared to other land plants, including hornworts and basal lineages of mosses. Furthermore, Aneura and Haplomitrium were distinct from other liverworts by an increased GC content, with the one found in Haplomitrium only second to the lycophyte Selaginella. The results suggest the hypothesis that liverworts and other land plants inherited and conserved the plastome structure of their most recent algal ancestors.     

Complete sequencing and comparative analyses of the pepper (Capsicum annuum L.) plastome revealed high frequency of tandem repeats and large insertion/deletions on pepper plastome

Plants in the family Solanaceae are used as model systems in comparative and evolutionary genomics. The complete chloroplast genomes of seven solanaceous species have been sequenced, including tobacco, potato and tomato, but not peppers. We analyzed the complete chloroplast genome sequence of the hot pepper, Capsicum annuum. The pepper chloroplast genome was 156,781 bp in length, including a pair of inverted repeats (IR) of 25,783 bp. The content and the order of 133 genes in the pepper chloroplast genome were identical to those of other solanaceous plastomes. To characterize pepper plastome sequence, we performed comparative analysis using complete plastome sequences of pepper and seven solanaceous plastomes. Frequency and contents of large indels and tandem repeat sequences and distribution pattern of genome-wide sequence variations were investigated. In addition, a phylogenetic analysis using concatenated alignments of coding sequences was performed to determine evolutionary position of pepper in Solanaceae. Our results revealed two distinct features of pepper plastome compared to other solanaceous plastomes. Firstly, large indels, including insertions on accD and rpl20 gene sequences, were predominantly detected in the pepper plastome compared to other solanaceous plastomes. Secondly, tandem repeat sequences were particularly frequent in the pepper plastome. Taken together, our study represents unique features of evolution of pepper plastome among solanaceous plastomes.     

The complete nucleotide sequences of the five genetically distinct plastid genomes of Oenothera, subsection Oenothera: I. sequence evaluation and plastome evolution

The flowering plant genus Oenothera is uniquely suited for studying molecular mechanisms of speciation. It assembles an intriguing combination of genetic features, including permanent translocation heterozygosity, biparental transmission of plastids, and a general interfertility of well-defined species. This allows an exchange of plastids and nuclei between species often resulting in plastome–genome incompatibility. For evaluation of its molecular determinants we present the complete nucleotide sequences of the five basic, genetically distinguishable plastid chromosomes of subsection Oenothera (=Euoenothera) of the genus, which are associated in distinct combinations with six basic genomes. Sizes of the chromosomes range from 163 365 bp (plastome IV) to 165 728 bp (plastome I), display between 96.3% and 98.6% sequence similarity and encode a total of 113 unique genes. Plastome diversification is caused by an abundance of nucleotide substitutions, small insertions, deletions and repetitions. The five plastomes deviate from the general ancestral design of plastid chromosomes of vascular plants by a subsection-specific 56 kb inversion within the large single-copy segment. This inversion disrupted operon structures and predates the divergence of the subsection presumably 1 My ago. Phylogenetic relationships suggest plastomes I–III in one clade, while plastome IV appears to be closest to the common ancestor.     

First insights into fern matK phylogeny

MatK, the only maturase gene in the land plant plastid genome, is a very popular phylogenetic marker that has been extensively applied in reconstructing angiosperm phylogeny. However, the use of matK in fern phylogeny is largely unknown, due to difficulties with amplification: ferns have lost the flanking trnK exons, typically the region used for designing stable priming sites. We developed primers that are either universal or lineage-specific that successfully amplify matK across all fern families. To evaluate whether matK is as powerful a phylogenetic marker in ferns as in angiosperms, we compared its sequence characteristics and phylogenetic performance to those of rbcL and atpA. Among these three genes, matK has the highest variability and substitution evenness, yet shows the least homoplasy. Most importantly, applying matK in fern phylogenetics better resolved relationships among families, especially within eupolypods I and II. Here we demonstrate the power of matK for fern phylogenetic reconstruction, as well as provide primers and extensive sequence data that will greatly facilitate future evolutionary studies of ferns.     

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。     

Plastid genome evolution in mycoheterotrophic Ericaceae

Unlike parasitic plants, which are linked to their hosts directly through haustoria, mycoheterotrophic (MHT) plants derive all or part of their water and nutrients from autothrophs via fungal mycorrhizal intermediaries. Ericaceae, the heather family, are a large and diverse group of plants known to form elaborate symbiotic relationships with mycorrhizal fungi. Using PHYA sequence data, we first investigated relationships among mycoheterotrophic Ericaceae and their close autotrophic relatives. Phylogenetic results suggest a minimum of two independent origins of MHT within this family. Additionally, a comparative investigation of plastid genomes (plastomes) grounded within this phylogenetic framework was conducted using a slot-blot Southern hybridization approach. This survey encompassed numerous lineages of Ericaceae with different life histories and trophic levels, including multiple representatives from mixotrophic Pyroleae and fully heterotrophic Monotropeae and Pterosporeae. Fifty-four probes derived from all categories of protein coding genes typically found within the plastomes of flowering plants were used. Our results indicate that the holo-mycoheterotrophic Ericaceae exhibit extensive loss of genes relating to photosynthetic function and expression of the plastome but retain genes with possible functions outside photosynthesis. Mixotrophic taxa tend to retain most genes relating to photosynthetic functions but are varied regarding the plastid ndh gene content. This investigation extends previous inferences that the loss of the NDH complex occurs prior to becoming holo-heterotrophic and it shows that the pattern of gene losses among mycoheterotrophic Ericaceae is similar to that of haustorial parasites. Additionally, we identify the most desirable candidate species for entire plastome sequencing.     

Lineage‐specific plastid degradation in subtribe Gentianinae (Gentianaceae)

The structure and sequence of plastid genomes is highly conserved across most land plants, except for a minority of lineages that show gene loss and genome degradation. Understanding the early stages of plastome degradation may provide crucial insights into the repeatability and predictability of genomic evolutionary trends. We investigated these trends in subtribe Gentianinae of the Gentianaceae, which encompasses ca. 450 species distributed around the world, particularly in alpine and subalpine environments. We sequenced, assembled, and annotated the plastomes of 41 species, representing all six genera in subtribe Gentianinae and all main sections of the species‐rich genus Gentiana L. We reconstructed the phylogeny, estimated divergence times, investigated the phylogenetic distribution of putative gene losses, and related these to substitution rate shifts and species’ habitats. We obtained a strongly supported topology consistent with earlier studies, with all six genera in Gentianinae recovered as monophyletic and all main sections of Gentiana having full support. While closely related species have very similar plastomes in terms of size and structure, independent gene losses, particularly of the ndh complex, have occurred in multiple clades across the phylogeny. Gene loss was usually associated with a shift in the boundaries of the small single‐copy and inverted repeat regions. Substitution rates were variable between clades, with evidence for both elevated and decelerated rate shifts. Independent lineage‐specific loss of ndh genes occurred at a wide range of times, from Eocene to Pliocene. Our study illustrates that diverse degradation patterns shape the evolution of the plastid in this species‐rich plant group.     

The first complete plastid genome of Burmannia disticha L. from the mycoheterotrophic monocot family Burmanniaceae

Burmanniaceae is one major group within the monocot order Dioscoreales that has not had its plastome sequenced. Members of Burmanniaceae are mostly achlorophyllous, although the genus Burmannia also includes autotrophs. Here, we report sequencing and analysis of the first Burmanniaceae plastid genome from Burmannia disticha L.. This plastome is 157,480 bp and was assembled as a circular sequence with the typical quadripartite structure of plant plastid genomes. This plastome has a regular number of potentially functional genes with a total of 111, including 78 protein coding genes, 4 ribosomal RNA (rRNA) genes, and 29 tRNA genes. The ratio of the total length of genic:intergenic DNA is 1.58:1, and the mean length of intergenic regions is 398 bp, the longest being 1918 bp. The overall GC content of the B. disticha plastome is 34.90%, and the IR regions in B. disticha are more GC rich (39.50%) than the LSC (32.30%) and SSC (28.80%) regions. Phylogenetic analysis of protein-coding sequences from plastomes of related species in the order Dioscoreales support a clade comprising Burmanniaceae and Dioscoreaceae. This phylogenetic placement is congruent with previous findings based on nuclear and mitochondrial evidence.     

The plastid genome of the mycoheterotrophic Corallorhiza striata (Orchidaceae) is in the relatively early stages of degradation

? Premise of the study: Plastid genomes of nonphotosynthetic, mycoheterotrophic plants represent apt systems in which to study effects of relaxed evolutionary constraints. The few mycoheterotrophic angiosperm plastomes sequenced to date display drastic patterns of degradation/reduction relative to those of photosynthetic relatives. The goal of this study was to focus on a mycoheterotrophic orchid hypothesized to be in the "early" stages of plastome degradation, to provide perspective on this process. ? Methods: Short-read sequencing was used to generate a complete plastome sequence for Corallorhiza striata var. vreelandii, a mycoheterotrophic orchid, to investigate the extent of plastome degradation. Patterns of nonsynonymous/synonymous mutations were also assessed, and comparisons were made between Corallorhiza and other heterotrophic plant lineages. ? Key results: Corallorhiza yielded a plastome of 137505 bp, with several photosynthesis-related genes either lost or pseudogenized. Members of all major photosynthesis complexes, except ATP-synthase genes, were affected. "Housekeeping" genes were intact, despite the loss of a single tRNA. Intact photosynthesis genes (excluding atp genes) together displayed elevated nonsynonymous changes, while housekeeping genes did not. ? Conclusions: The Corallorhiza plastome is not drastically reduced in overall size (～6% reduction relative to that of photosynthetic Oncidium), but displays a pattern congruent with a loss of photosynthetic function. Comparing Corallorhiza with other heterotrophs allows some emergent evolutionary patterns to be inferred, but these remain as hypotheses to be tested, especially at lower taxonomic levels, and in lineages illustrating transitions from autotrophy to heterotrophy. The independent, unique processes of plastome modification among mycoheterotrophic lineages illustrate the urgency of their conservation.     

Mechanisms of Functional and Physical Genome Reduction in Photosynthetic and Nonphotosynthetic Parasitic Plants of the Broomrape Family

Nonphotosynthetic plants possess strongly reconfigured plastomes attributable to convergent losses of photosynthesis and housekeeping genes, making them excellent systems for studying genome evolution under relaxed selective pressures. We report the complete plastomes of 10 photosynthetic and nonphotosynthetic parasites plus their nonparasitic sister from the broomrape family (Orobanchaceae). By reconstructing the history of gene losses and genome reconfigurations, we find that the establishment of obligate parasitism triggers the relaxation of selective constraints. Partly because of independent losses of one inverted repeat region, Orobanchaceae plastomes vary 3.5-fold in size, with 45 kb in American squawroot (Conopholis americana) representing the smallest plastome reported from land plants. Of the 42 to 74 retained unique genes, only 16 protein genes, 15 tRNAs, and four rRNAs are commonly found. Several holoparasites retain ATP synthase genes with intact open reading frames, suggesting a prolonged function in these plants. The loss of photosynthesis alters the chromosomal architecture in that recombinogenic factors accumulate, fostering large-scale chromosomal rearrangements as functional reduction proceeds. The retention of DNA fragments is strongly influenced by both their proximity to genes under selection and the co-occurrence with those in operons, indicating complex constraints beyond gene function that determine the evolutionary survival time of plastid regions in nonphotosynthetic plants.     

Plastid genome structure and phylogenomics of Nymphaeales: conserved gene order and new insights into relationships

The plastid genomes of early-diverging angiosperms were among the first land plant plastomes investigated. Despite their importance to understanding angiosperm evolution, no investigation has so far compared gene content or gene synteny of these plastid genomes with a focus on the Nymphaeales. Here, we report an evaluation and comparison of gene content, gene synteny and inverted repeat length for a set of 15 plastid genomes of early-diverging angiosperms. Seven plastid genomes of the Nymphaeales were newly sequenced for this investigation. We compare gene order and inverted repeat (IR) length across all genomes, review the gene annotations of previously published genomes, generate a multi-gene alignment of 77 plastid-encoded genes and reconstruct the phylogenetic relationships of the taxa under study. Our results show that gene content and synteny are highly conserved across early-diverging angiosperms: All species analyzed display complete gene synteny when accounting for expansions and contractions of the IRs. This conservation was initially obscured by ambiguous and potentially incorrect gene annotations in previously published genomes. We also report the presence of intact open reading frames across all taxa analyzed. The multi-gene phylogeny displays maximum support for the families Cabombaceae and Hydatellaceae, but no support for a clade of all Nymphaeaceae. It further indicates that the genus Victoria is embedded within Nymphaea. Plastid genomes of Trithuria were found to deviate by numerous substitutions and length changes in the IRs. Phylogenetic analyses further indicate that a previously published plastome named Nymphaea mexicana falls into a clade of N. odorata and should be re-evaluated.     

Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication

The spacer between the 16S and 23S rRNA genes of the chloroplast DNA has been implicated as an origin of replication in several species of plants. In the evening primrose, Oenothera, this site was found to vary greatly in size, with plastid genomes (plastomes) being readily distinguished. To determine whether plastome "strength" in transmission could be correlated with variation at oriB, the 16S rRNA-trnI spacer was sequenced from five plastomes. The size variation was found to be due to differential amplification (and deletion) of combinations of sequences belonging to seven families of direct repeats. From these comparisons, one short series of direct repeats and one region capable of forming a hairpin structure were identified as candidates for the factor that could be responsible for the differences between strong and weak plastome types. Ample sequence variation allowed phylogenetic inferences to be made about the relationships among the plastomes. Phylogenetic trees also could be constructed for most of the families of direct repeats. The amplifications and deletions of repeats that account for the size variation at oriB are proposed to have occurred through extensive replication slippage at this site.      

Do nonasterid holoparasitic flowering plants have plastid genomes?

Past work involving the plastid genome (plastome) of holoparasitic plants has been confined to Scrophulariaceae (or Orobanchaceae) which have truncated plastomes owing to loss of photosynthetic and other genes. Nonasterid holoparasites from Balanophoraceae (Corynaea), Hydnoraceae (Hydnora) and Cytinaceae (Cytinus) were tested for the presence of plastid genes and a plastome. Using PCR, plastid 16S rDNA was successfully amplified and sequenced from the above three holoparasites. The sequence of Cytinus showed 121 single base substitutions relative to Nicotiana (8% of the molecule) whereas higher sequence divergence was observed in Hydnora and Corynaea (287 and 513 changes, respectively). Secondary structural models for these 16S rRNAs show that most changes are compensatory, thus suggesting they are functional. Probes constructed for 16S rDNA and for four plastid-encoded ribosomal protein genes (rps2, rps4, rps7 and rpl16) were used in Southern blots of digested genomic DNA from the three holoparasites. Positive hybridizations were obtained using each of the five probes only for Cytinus. For SmaI digests, all plastid gene probes hybridized to a common fragment ca. 20 kb in length in this species. Taken together, these data provide preliminary evidence suggestive of the retention of highly diverged and truncated plastid genome in Cytinus. The greater sequence divergence for 16S rDNA and the negative hybridization results for Hydnora and Corynaea suggests two possibilities: the loss of typically conserved elements of their plastomes or the complete absence of a plastome.