The chicken or the egg? Plastome evolution and an independent loss of the inverted repeat in papilionoid legumes

The plastid genome (plastome), while surprisingly constant in gene order and content across most photosynthetic angiosperms, exhibits variability in several unrelated lineages. During the diversification history of the legume family Fabaceae, plastomes have undergone many rearrangements, including inversions, expansion, contraction and loss of the typical inverted repeat (IR), gene loss and repeat accumulation in both shared and independent events. While legume plastomes have been the subject of study for some time, most work has focused on agricultural species in the IR-lacking clade (IRLC) and the plant model Medicago truncatula. The subfamily Papilionoideae, which contains virtually all of the agricultural legume species, also comprises most of the plastome variation detected thus far in the family. In this study three non-papilioniods were included among 34 newly sequenced legume plastomes, along with 33 publicly available sequences, to assess plastome structural evolution in the subfamily. In an effort to examine plastome variation across the subfamily, approximately 20% of the sampling represents the IRLC with the remainder selected to represent the early-branching papilionoid clades. A number of IR-related and repeat-mediated changes were identified and examined in a phylogenetic context. Recombination between direct repeats associated with ycf2 resulted in intraindividual plastome heteroplasmy. Although loss of the IR has not been reported in legumes outside of the IRLC, one genistoid taxon was found to completely lack the typical plastome IR. The role of the IR and non-IR repeats in the progression of plastome change is discussed.     

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.     

Total duplication of the small single copy region in the angiosperm plastome: Rearrangement and inverted repeat instability in Asarum

Premise of the Study

As more plastomes are assembled, it is evident that rearrangements, losses, intergenic spacer expansion and contraction, and syntenic breaks within otherwise functioning plastids are more common than was thought previously, and such changes have developed independently in disparate lineages. However, to date, the magnoliids remain characterized by their highly conserved plastid genomes (plastomes).

Methods

Illumina HiSeq and MiSeq platforms were used to sequence the plastomes of Saruma henryi and those of representative species from each of the six taxonomic sections of Asarum. Sequenced plastomes were compared in a phylogenetic context provided by maximum likelihood and parsimony inferences made using an additional 18 publicly available plastomes from early‐diverging angiosperm lineages.

Key Results

In contrast to previously published magnoliid plastomes and the newly sequenced Saruma henryi plastome published here, Asarum plastomes have undergone extensive disruption and contain extremely lengthy AT‐repeat regions. The entirety of the small single copy region (SSC) of A. canadense and A. sieboldii var. sieboldii has been incorporated into the inverted repeat regions (IR), and the SSC of A. delavayi is only 14 bp long. All sampled Asarum plastomes share an inversion of a large portion of the large single copy region (LSC) such that trnE‐UUC is adjacent to the LSC‐IR boundary.

Conclusions

Plastome divergence in Asarum appears to be consistent with trends seen in highly rearranged plastomes of the monocots and eudicots. We propose that plastome instability in Asarum is due to repetitive motifs that serve as recombinatory substrates and reduce genome stability.     

Mutational meltdown or controlled chain reaction: The dynamics of rapid plastome evolution in the hyperdiversity of Poaceae

The study of genomic structural evolution associated with accelerated evolutionary rates that result in avoidance of meltdown and increase biodiversity is becoming ever more possible as the number of available plastomes increases. To more comprehensively analyze rate heterogeneity among monocots and within Poaceae, we sequenced plastomes from four Poaceae species, combined them with publicly available data from ~200 plastomes, and conducted comparative analyses to quantify the pattern of rate heterogeneity between different lineages, functional groups, and periods of evolutionary time. We compared structural differences across the Poaceae to quantify how changes in plastome size correspond to different genomic subunits and the evolution of IR–SC junction boundaries. The substitution rates among ancestral Poaceae were inferred to be exceptionally rapid compared to other monocots but slowed after divergence into extant lineages, which could not be sufficiently explained by positive selection. As such, rapid rates in the ancestral lineage leading to Poaceae might be more closely linked to large-scale structural changes like the loss of ycf1 and ycf2. The total increase in plastome size across Poaceae was positively correlated with the total length of intergenic spacers, tandem repeats, and dispersed repeats as well as large single copy, and inverted repeats (IRs). The continuous evolution of IR–SC junction boundaries was asynchronous with sizes of total genome and subunits across Poaceae. Future work is needed to better understand what factors in ancestral Poaceae evolved to harness such rapid rates of plastome evolution, avoid a mutational meltdown, and escape the stagnation of strong purifying selection as well as if these factors could be utilized to synthetically control rates.     

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.     

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.     

The first complete plastome sequence of the basal asterid family Styracaceae (Ericales) reveals a large inversion

Plastome sequences are rich sources of information for resolving difficult phylogenetic relationships and provide genomic data for conservation studies. Here, the complete plastome sequence of Alniphyllum eberhardtii Guillaumin is reported, representing the first plastome of the basal asterid family Styracaceae (Ericales). The plastome is 155,384 bp in length and contains 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes, totaling 113 unique genes with 19 genes in the inverted repeat region. Unusual features of the plastome include the presence a large 20-kb inversion in the Large Single-Copy region, the pseudogenization of the accD gene, and the loss of the second intron from clpP. The 20-kb inversion includes 14 genes and has not been previously reported in other Ericales plastomes. Thirty-nine plastid simple sequence repeats (SSRs) that may provide genetic resources for the conservation of this economically import timber plant are characterized. Phylogenetic results inferred from ML and MP analyses of 66 plastid genes and 26 taxa reveal that the Styracaceae are sister to a clade including Actinidiaceae and Ericaceae and suggest that complete plastomes are likely to be very helpful in resolving the basal relationships among Ericales families, which have resisted resolution in smaller phylogenetic data sets.     

Genetic markers in Andean Puya species (Bromeliaceae) with implications on plastome evolution and phylogeny

The Andean plant endemic Puya is a striking example of recent and rapid diversification from central Chile to the northern Andes, tracking mountain uplift. This study generated 12 complete plastomes representing nine Puya species and compared them to five published plastomes for their features, genomic evolution, and phylogeny. The total size of the Puya plastomes ranged from 159,542 to 159,839 bp with 37.3%–37.4% GC content. The Puya plastomes were highly conserved in organization and structure with a typical quadripartite genome structure. Each of the 17 consensus plastomes harbored 133 genes, including 87 protein‐coding genes, 38 tRNA (transfer RNA) genes, and eight rRNA (ribosomal RNA) genes; we found 69–78 tandem repeats, 45–60 SSRs (simple sequence repeats), and 8–22 repeat structures among 13 species. Four protein‐coding genes were identified under positive site‐specific selection in Puya. The complete plastomes and hypervariable regions collectively provided pronounced species discrimination in Puya and a practical tool for future phylogenetic studies. The reconstructed phylogeny and estimated divergence time for the lineage suggest that the diversification of Puya is related to Andean orogeny and Pleistocene climatic oscillations. This study provides plastome resources for species delimitation and novel phylogenetic and biogeographic studies.     

Strong plastid degradation is consistent within section Chondrophyllae,the most speciose lineage of Gentiana

Recovering phylogenetic relationships in lineages experiencing intense diversification has always been a persistent challenge in evolutionary studies, including in Gentiana section Chondrophyllae sensu lato (s.l.). Indeed, this subcosmopolitan taxon encompasses more than 180 mostly annual species distributed around the world. We sequenced and assembled 22 new plastomes representing 21 species in section Chondrophyllae s.l. In addition to previously released plastome data, our study includes all main lineages within the section. We reconstructed their phylogenetic relationships based on protein‐coding genes and recombinant DNA (rDNA) cistron sequences, and then investigated plastome structural evolution as well as divergence time. Despite an admittedly humble species cover overall, we recovered a well‐supported phylogenetic tree based on plastome data, and found significant discordance between phylogenetic relationships and taxonomic treatments. Our results show that G. capitata and G. leucomelaena diverged early within the section, which is then further divided into two clades. The divergence time estimation showed that section Chondrophyllae s.l. evolved in the second half of the Oligocene. We found that section Chondrophyllae s.l. had the smallest average plastome size (128 KB) in tribe Gentianeae (Gentianaceae), with frequent gene and sequence losses such as the ndh complex and its flanking regions. In addition, we detected both expansion and contraction of the inverted repeat (IR) regions. Our study suggests that plastome degradation parallels the diversification of this group, and illustrates the strong discordance between phylogenetic relationships and taxonomic treatments, which now need to be carefully revised.     

Complete Plastid Genome Sequence of the Basal Asterid Ardisia polysticta Miq. and Comparative Analyses of Asterid Plastid Genomes

Ardisia is a basal asterid genus well known for its medicinal values and has the potential for development of novel phytopharmaceuticals. In this genus of nearly 500 species, many ornamental species are commonly grown worldwide and some have become invasive species that caused ecological problems. As there is no completed plastid genome (plastome) sequence in related taxa, we sequenced and characterized the plastome of Ardisia polysticta to find plastid markers of potential utility for phylogenetic analyses at low taxonomic levels. The complete A. polysticta plastome is 156,506 bp in length and has gene content and organization typical of most asterids and other angiosperms. We identified seven intergenic regions as potentially informative markers with resolution for interspecific relationships. Additionally, we characterized the diversity of asterid plastomes with respect to GC content, plastome organization, gene content, and repetitive sequences through comparative analyses. The results demonstrated that the genome organizations near the boundaries between inverted repeats (IRs) and single-copy regions (SCs) are polymorphic. The boundary organization found in Ardisia appears to be the most common type among asterids, while six other types are also found in various asterid lineages. In general, the repetitive sequences in genic regions tend to be more conserved, whereas those in noncoding regions are usually lineage-specific. Finally, we inferred the whole-plastome phylogeny with the available asterid sequences. With the improvement in taxon sampling of asterid orders and families, our result highlights the uncertainty of the position of Gentianales within euasterids I.     

Balanced Gene Losses,Duplications and Intensive Rearrangements Led to an Unusual Regularly Sized Genome in Arbutus unedo Chloroplasts

Completely sequenced plastomes provide a valuable source of information about the duplication, loss, and transfer events of chloroplast genes and phylogenetic data for resolving relationships among major groups of plants. Moreover, they can also be useful for exploiting chloroplast genetic engineering technology. Ericales account for approximately six per cent of eudicot diversity with 11,545 species from which only three complete plastome sequences are currently available. With the aim of increasing the number of ericalean complete plastome sequences, and to open new perspectives in understanding Mediterranean plant adaptations, a genomic study on the basis of the complete chloroplast genome sequencing of Arbutus unedo and an updated phylogenomic analysis of Asteridae was implemented. The chloroplast genome of A. unedo shows extensive rearrangements but a medium size (150,897 nt) in comparison to most of angiosperms. A number of remarkable distinct features characterize the plastome of A. unedo: five-fold dismissing of the SSC region in relation to most angiosperms; complete loss or pseudogenization of a number of essential genes; duplication of the ndhH-D operon and its location within the two IRs; presence of large tandem repeats located near highly re-arranged regions and pseudogenes. All these features outline the primary evolutionary split between Ericaceae and other ericalean families. The newly sequenced plastome of A. unedo with the available asterid sequences allowed the resolution of some uncertainties in previous phylogenies of Asteridae.     

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.      

Plastomes of nine hornbeams and phylogenetic implications

Poor phylogenetic resolution and inconsistency of gene trees are major complications when attempting to construct trees of life for various groups of organisms. In this study, we addressed these issues in analyses of the genus Carpinus (hornbeams) of the Betulaceae. We assembled and annotated the chloroplast (cp) genomes (plastomes) of nine hornbeams representing main clades previously distinguished in this genus. All nine plastomes are highly conserved, with four regions, and about 158–160 kb long, including 121–123 genes. Phylogenetic analyses of whole plastome sequences, noncoding sequences, and the well‐aligned coding genes resulted in high resolution of the sampled species in contrast to the failure based on a few cpDNA markers. Phylogenetic relationships in a few clades based only on the coding genes are slightly inconsistent with those based on the noncoding and total plastome datasets. Moreover, these plastome trees are highly incongruent with those based on bi‐parentally inherited internal transcribed spacer (ITS) sequence variations. Such high inconsistencies suggest widespread occurrence of incomplete lineage sorting and hybrid introgression during diversification of these hornbeams.     

Testing and using complete plastomes and ribosomal DNA sequences as the next generation DNA barcodes in Panax (Araliaceae)

Complete plastid genome (plastome) sequences and nuclear ribosomal DNA (nrDNA) regions have been proposed as candidates for the next generation of DNA barcodes for plant species discrimination. However, the efficacy of this approach still lacks comprehensive evaluation. We carried out a case study in the economically important but phylogenetically and taxonomically difficult genus Panax (Araliaceae). We generated a large data set of plastomes and nrDNA sequences from multiple accessions per species. Our data improved the phylogenetic resolution and levels of species discrimination in Panax, compared to any previous studies using standard DNA barcodes. This provides new insights into the speciation, lineage diversification and biogeography of the genus. However, both plastome and nrDNA failed to completely resolve the phylogenetic relationships in the Panax bipinnatifidus species complex, and only half of the species within it were recovered as monophyletic units. The results suggest that complete plastome and ribosomal DNA sequences can substantially increase species discriminatory power in plants, but they are not powerful enough to fully resolve phylogenetic relationships and discriminate all species, particularly in evolutionarily young and complex plant groups. To gain further resolving power for closely related species, the addition of substantial numbers of nuclear markers is likely to be required.     

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.     

Plastid genome of <Emphasis Type="Italic">Seseli montanum</Emphasis>: Complete sequence and comparison with plastomes of other members of the Apiaceae family

This work reports the complete plastid (pt) DNA sequence of Seseli montanum L. of the Apiaceae family, determined using next-generation sequencing technology. The complete genome sequence has been deposited in GenBank with accession No. KM035851. The S. montanum plastome is 147,823 bp in length. The plastid genome has a typical structure for angiosperms and contains a large single-copy region (LSC) of 92,620 bp and a small single-copy region (SSC) of 17,481 bp separated by a pair of 18,861 bp inverted repeats (IRa and IRb). The composition, gene order, and AT-content in the S. montanum plastome are similar to that of a typical flowering plant pt DNA. One hundred fourteen unique genes have been identified, including 30 tRNA genes, four rRNA genes, and 80 protein genes. Of 18 intron-containing genes found, 16 genes have one intron, and two genes (ycf3, clpP) have two introns. Comparative analysis of Apiaceae plastomes reveals in the S. montanum plastome a LSC/IRb junction shift, so that the part of the ycf2 (4980 bp) gene is located in the LSC, but the other part of ycf2 (1301 bp) is within the inverted repeat. Thus, structural rearrangements in the plastid genome of S. montanum result in an enlargement of the LSC region by means of capture of a large part of ycf2, in contrast to eight Apiaceae plastomes where the complete ycf2 gene sequence is located in the inverted repeat.     

The Complete Chloroplast Genome of <Emphasis Type="Italic">Coix lacryma-jobi</Emphasis> and a Comparative Molecular Evolutionary Analysis of Plastomes in Cereals

Graminoid molecular evolution was investigated by chloroplast genome (plastome) scale analyses. A complete plastome from Coix lacryma-jobi (Poaceae) and a draft plastome from Joinvillea plicata (Joinvilleaceae) were sequenced and analyzed. The draft plastome included conserved protein-coding loci routinely analyzed in previous studies plus one additional locus of demonstrated phylogenetic utility. The methodological approach was to directly sequence overlapping amplicons from known plastome regions. Over 100 pairs of amplification and sequencing primers were designed and positioned to flank overlapping 1,200-base pair fragments around the entire plastome. Newly determined sequences were analyzed with published plastomes from representatives of Panicoideae, Ehrhartoideae, and Pooideae. Considerable variation was found for studies within the family and even within Andropogoneae. Readily interpreted mutation patterns were observed, such as small inversions in hairpin-loop regions and indels, which were common in intergenic spacers. Maximum or near-maximum bootstrap support was observed in all analyses resolving relationships between subfamilies. However, the addition of characters from noncoding regions increased the number of parsimony-informative characters and lengthened short internal branches (Andropogoneae), better defining intergeneric relationships. Thus, characters in complete plastomes can be used over a wide scope of phylogenetic studies.     

Chloroplast Genome of Native Silene latifolia subsp. alba from Fennoscandia Shows High Level of Differences from Invasive White Campion

Silene latifolia is an herbaceous plant with great invasive potential. Spread along trade routes from Europe to almost all continents, white campion became particularly widespread in North America. We sequenced the chloroplast genome of S. latifolia subsp. alba from a native range in southeast Fennoscandia. The chloroplast genome of native S. latifolia subsp. alba forms a 151,747-bp circle, has two inverted repeat regions (25,993 bp each), large single copy (82,708 bp), and small single copy (17,106 bp) regions. It contains 77 protein-coding genes, 30 tRNA genes, and four rRNA genes. SSRs and long DNA repeats were identified. Comparison of a newly sequenced plastome of S. latifolia subsp. alba with plastomes of invasive specimens of species from North America and Japan revealed a high level of single nucleotide polymorphisms (SNPs) among them. A total of 214 SNPs were found, among which 110 were identified in intergenic spacers, 74 in exons, and 30 in introns. Intraspecific shifts in inverted repeat boundaries were identified. Our research suggests that high polymorphic regions may be potential molecular markers for population studies and that high intraspecific genetic polymorphism may contribute to a species’ invasive success.