The complete plastome of Panax stipuleanatus: Comparative and phylogenetic analyses of the genus Panax (Araliaceae)

Panax stipuleanatus (Araliaceae) is an endangered and medicinally important plant endemic to China. However, phylogenetic relationships within the genus Panax have remained unclear. In this study, we sequenced the complete plastome of P. stipuleanatus and included previously reported Panax plastomes to better understand the relationships between species and plastome evolution within the genus Panax. The plastome of P. stipuleanatus is 156,069 base pairs (bp) in length, consisting of a pair of inverted repeats (IRs, each 25,887 bp) that divide the plastome into a large single copy region (LSC, 86,126 bp) and a small single copy region (SSC, 8169 bp). The plastome contains 114 unigenes (80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes). Comparative analyses indicated that the plastome gene content and order, as well as the expansion/contraction of the IR regions, are all highly conserved within Panax. No significant positive selection in the plastid protein-coding genes was observed across the eight Panax species, suggesting the Panax plastomes may have undergone a strong purifying selection. Our phylogenomic analyses resulted in a phylogeny with high resolution and supports for Panax. Nine proteincoding genes and 10 non-coding regions presented high sequence divergence, which could be useful for identifying different Panax species.     

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.     

Plastome phylogenomics and phylogenetic diversity of endangered and threatened grassland species (Poaceae) in a North American tallgrass prairie

Native grasslands are one of the most endangered ecosystems in North America. In this study, we examined the ecological and evolutionary roles of endangered and threatened (e/t) grasses by establishing robust evolutionary relationships with other nonthreatened native and introduced grass species of the community. We hypothesized that the phylogenomic distribution of e/t species of grasses in Illinois would be phylogenetically clustered because closely related species would be vulnerable to the same threats and have similar requirements for survival. This study presents the first time a phylogeny based on complete plastome DNA of Poaceae was analyzed by phylogenetic diversity analysis. To avoid the disturbance of e/t populations, DNA was extracted from herbarium specimens. Next‐generation sequencing (NGS) techniques were used to sequence DNA of plastid genomes (plastomes). The resulting phylogenomic tree was analyzed by phylogenetic diversity metrics. The extracted DNA successfully produced complete plastomes demonstrating that herbarium material is a practical source of DNA for genomic studies. The phylogenomic tree was strongly supported and defined Dichanthelium as a separate clade from Panicum. The phylogenetic metrics revealed phylogenetic clustering of e/t species, confirming our hypothesis.     

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.     

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 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.     

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.     

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.     

Molecular markers for phylogenetic applications derived from comparative plastome analysis of Prunus species

Interspecific and intergeneric relationships of Prunus s.l. are still unclear due to low levels of genetic variation among species, and resulting partially unresolved phylogenetic inferences. Here we sequenced and compared six complete plastomes from two subgenera of Prunus in order to choose molecular markers to increase the amount of genetic variation suitable for inference of Prunus phylogeny. The plastomes range between 157 817 and 158 995 bp in length, and we found different levels of inverted repeat (IR) contraction among the three sampled subgenera of Prunus s.l. Most regions in Prunus plastomes considered individually provide low phylogenetic resolution at the subgenus or species level compared to a tree constructed using all 78 coding regions combined. We compared levels of variation among 206 coding regions and noncoding (intergenic and intron) plastid regions and inferred phylogenies from each region considered individually. We then chose using two regions together for future studies of relationships in Prunus, ycf1 and trnT-L, that display high to moderate levels of variation among coding and intergenic regions, respectively, and that individually permit inference of resolved species-level trees in Prunus with moderate to strong branch support. Considered together, these two regions allow inference of the same topology of Prunus inferred using all coding plastid regions combined, with comparable levels of tree support to the full plastome set. These two loci should therefore be useful as a plastid phylogenetic marker set for further inference of relationships within Prunus s.l.     

CHLOROPLAST DNA POLYMORPHISM AND PHYLOGENY IN THE B GENOME OF GLYCINE SUBGENUS GLYCINE (LEGUMINOSAE)

The B genome of Glycine subgenus Glycine comprises three diploid species whose monophyly is supported by morphological, crossing, and chloroplast DNA (cpDNA) data. Previous cpDNA studies indicated low levels of divergence among these taxa and failed to resolve cladistic relationships among them. More intensive studies of cpDNA variation were initiated, using additional restriction endonucleases and accessions. Results from cladistic analyses of over 50 restriction site characters indicate that there is considerable cpDNA polymorphism within this group of species, with a minimum of 27 plastome types occurring among the 74 accessions sampled. Levels of homoplasy observed in this group are relatively high (15%) for closely related congeneric species. There is only limited congruence between plastome type and taxonomic classification based on morphological characters. Explanations for this lack of concordance include: 1) the early state of taxonomic understanding in this group, 2) lack of resolution in the cpDNA tree caused by homoplasy and the small number of synapomorphic characters, 3) introgression among these interfertile, often sympatric taxa, and 4) maintenance of ancestral cpDNA polymorphisms resulting in shared plastomes among species.     

Plastome genomics in South American maize landraces: chloroplast lineages parallel the geographical structuring of nuclear gene pools

Background and AimsThe number of plastome sequences has increased exponentially during the last decade. However, there is still little knowledge of the levels and distribution of intraspecific variation. The aims of this study were to estimate plastome diversity within Zea mays and analyse the distribution of haplotypes in connection with the landrace groups previously delimited for South American maize based on nuclear markers.MethodsWe obtained the complete plastomes of 30 South American maize landraces and three teosintes by means of next-generation sequencing (NGS) and used them in combination with data from public repositories. After quality filtering, the curated data were employed to search for single-nucleotide polymorphisms, indels and chloroplast simple sequence repeats. Exact permutational contingency tests were performed to assess associations between plastome and nuclear variation. Network and Bayesian phylogenetic analyses were used to infer evolutionary relationships among haplotypes.Key ResultsOur analyses identified a total of 124 polymorphic plastome loci, with the intergenic regions psbE-rps18, petN-rpoB, trnL_UAG-ndhF and rpoC2-atpI exhibiting the highest marker densities. Although restricted in number, these markers allowed the discrimination of 27 haplotypes in a total of 51 Zea mays individuals. Andean and lowland South American landraces differed significantly in haplotype distribution. However, overall differentiation patterns were not informative with respect to subspecies diversification, as evidenced by the scattered distribution of maize and teosinte plastomes in both the network and Bayesian phylogenetic reconstructions.ConclusionsKnowledge of intraspecific plastome variation provides the framework for a more comprehensive understanding of evolutionary processes at low taxonomic levels and may become increasingly important for future plant barcoding efforts. Whole-plastome sequencing provided useful variability to contribute to maize phylogeographic studies. The structuring of haplotype diversity in the maize landraces examined here clearly reflects the distinction between the Andean and South American lowland gene pools previously inferred based on nuclear markers.     

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.     

质体系统发育基因组解析旋花科系统发育关系

旋花科是一个世界广布的类群,具有丰富的形态特征和重要的经济价值。然而,目前该科主要分支或族间的系统发育关系问题一直未解决。为解析旋花科内系统发育关系,该研究代表性选取旋花科内8个族40个物种,基于质体全基因组数据,使用最大似然法和贝叶斯推论进行系统发育分析。结果表明：(1)旋花科质体基因组均为四分体结构,质体基因组大小为113 273～164 112 bp,蛋白质编码基因数目为66～79个。(2)基于五种DNA矩阵(即WCG、CDS、LSC、IR、SSC)的系统发育分析结果显示,WCG矩阵和CDS矩阵的拓扑结构基本一致,仅少数分支的支持率略有差异;LSC矩阵和WCG矩阵的拓扑结构差异在于菟丝子族、马蹄金族和盐帚花族的系统位置;AU检验和SH检验结果显示,WCG矩阵和SSC矩阵与IR矩阵的拓扑结构有显著冲突。(3)所有系统发育分析结果均显示,菟丝子属和马蹄金族都包括在旋花亚科内,应处理为族等级。(4)基于WCG矩阵和CDS矩阵较好地解决了旋花科8个族之间的系统发育关系,即心被藤族和丁公藤族聚为一支,最先从旋花亚科分化出来,随后是菟丝子族,剩下的5个族分成2个分支。(5)系统发育基因组分析...     

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.     

Prospects on the evolutionary mitogenomics of plants: A case study on the olive family (Oleaceae)

The mitogenome is rarely used to reconstruct the evolutionary history of plants, contrary to nuclear and plastid markers. Here, we evaluate the usefulness of mitochondrial DNA for molecular evolutionary studies in Oleaceae, in which cases of cytoplasmic male sterility (CMS) and of potentially contrasted organelle inheritance are known. We compare the diversity and the evolution of mitochondrial and chloroplast genomes by focusing on the olive complex and related genera. Using high‐throughput techniques, we reconstructed complete mitogenomes (ca. 0.7 Mb) and plastomes (ca. 156 kb) for six olive accessions and one Chionanthus. A highly variable organization of mitogenomes was observed at the species level. In olive, two specific chimeric genes were identified in the mitogenome of lineage E3 and may be involved in CMS. Plastid‐derived regions (mtpt) were observed in all reconstructed mitogenomes. Through phylogenetic reconstruction, we demonstrate that multiple integrations of mtpt regions have occurred in Oleaceae, but mtpt regions shared by all members of the olive complex derive from a common ancestor. We then assembled 52 conserved mitochondrial gene regions and complete plastomes of ten additional accessions belonging to tribes Oleeae, Fontanesieae and Forsythieae. Phylogenetic congruence between topologies based on mitochondrial regions and plastomes suggests a strong disequilibrium linkage between both organellar genomes. Finally, while phylogenetic reconstruction based on plastomes fails to resolve the evolutionary history of maternal olive lineages in the Mediterranean area, their phylogenetic relationships were successfully resolved with complete mitogenomes. Overall, our study demonstrates the great potential of using mitochondrial DNA in plant phylogeographic and metagenomic studies.     

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.     

Plastome characteristics of Cannabaceae

Cannabaceae is an economically important family that includes ten genera and ca.117 accepted species. To explore the structure and size variation of their plastomes,we sequenced ten plastomes representing all ten genera of Cannabaceae.Each plastome possessed the typical angiosperm quadripartite structure and contained a total of 128 genes.The Inverted Repeat (IR) regions in five plastomes had experienced small expansions (330-983 bp) into the Large Single-Copy (LSC) region.The plastome of Chaetachme aristata has experienced a 942-bp IR contraction and lost rpl22 and rps19 in its IRs.The substitution rates of rps19 and rpl22 decreased after they shifted from the LSC to IR.A 270-bp inversion was detected in the Parasponia rugosa plastome,which might have been mediated by 18-bp inverted repeats.Repeat sequences,simple sequence repeats,and nucleotide substitution rates varied among these plastomes. Molecular markers with more than 13% variable sites and 5% parsimony-informative sites were identified,which may be useful for further phylogenetic analysis and species identification.Our results show strong support for a sister relationship between Gironniera and Lozanell (BS=100).Celtis,Cannabis-Humulus,Chaetachme-Pteroceltis,and Trema-Parasponia formed a strongly supported clade,and their relationships were well resolved with strong support (BS=100).The availability of these ten plastomes provides valuable genetic information for accurately identifying species,clarifying taxonomy and reconstructing the intergeneric phylogeny of Cannabaceae.     

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.