The plastomes of Lepismium cruciforme (Vell.) Miq and Schlumbergera truncata (Haw.) Moran reveal tribe-specific rearrangements and the first loss of the trnT-GGU gene in Cactaceae.

BACKGROUND: Recent studies have revealed atypical features in the plastomes of the family Cactaceae, the largest lineage of succulent species adapted to arid and semi-arid regions. Most plastomes sequenced to date are from short-globose and cylindrical cacti, while little is known about plastomes of epiphytic cacti. Published cactus plastomes reveal reduction and complete loss of IRs, loss of genes, pseudogenization, and even degeneration of tRNA structures. Aiming to contribute with new insights into the plastid evolution of Cactaceae, particularly within the tribe Rhipsalideae, we de novo assembled and analyzed the plastomes of Lepismium cruciforme and Schlumbergera truncata, two South American epiphytic cacti. METHODS AND RESULTS: Our data reveal many gene losses in both plastomes and the first loss of functionality of the trnT-GGU gene in Cactaceae. The trnT-GGU is a pseudogene in L. cruciforme plastome and appears to be degenerating in the tribe Rhipsalideae. Although the plastome structure is conserved among the species of the tribe Rhipsalideae, with tribe-specific rearrangements, we mapped around 200 simple sequence repeats and identified nine nucleotide polymorphism hotspots, useful to improve the phylogenetic resolutions of the Rhipsalideae. Furthermore, our analysis indicated high gene divergence and rapid evolution of RNA editing sites in plastid protein-coding genes in Cactaceae. CONCLUSIONS: Our findings show that some characteristics of the Rhipsalideae tribe are conserved, such as plastome structure with IRs containing only the ycf2 and two tRNA genes, structural degeneration of the trnT-GGU gene and ndh complex, and lastly, pseudogenization of rpl33 and rpl23 genes, both plastid translation-related genes.

Plastome evolution in Santalales involves relaxed selection prior to loss of ndh genes and major boundary shifts of the inverted repeat.

BACKGROUND AND AIMS: Biological aspects of haustorial parasitism have significant effects on the configuration of the plastid genome. Approximately half the diversity of haustorial parasites belongs to the order Santalales, where a clearer picture of plastome evolution in relation to parasitism is starting to emerge. However, in previous studies of plastome evolution there is still a notable under-representation of members from non-parasitic and deep-branching hemiparasitic lineages, limiting evolutionary inference around the time of transition to a parasitic lifestyle. To expand taxon sampling relevant to this transition we therefore targeted three families of non-parasites (Erythropalaceae, Strombosiaceae, and Coulaceae), two families of root-feeding hemiparasites (Ximeniaceae and Olacaceae), and two families of uncertain parasitic status (Aptandraceae and Octoknemaceae). With data from these lineages we aimed to explore plastome evolution in relation to evolution of parasitism. METHODS: From 29 new samples we sequenced and annotated plastomes and the nuclear ribosomal cistron. We examined phylogenetic patterns, plastome evolution, and patterns of relaxed or intensified selection in plastid genes. Available transcriptome data were analyzed to investigate potential transfer of infA to the nuclear genome. RESULTS: Phylogenetic relationships indicate a single functional loss of all plastid ndh genes (ndhA-K) in a clade formed by confirmed parasites and Aptandraceae, and the loss coincides with major size and boundary shifts of the inverted repeat (IR) region. Depending on an autotrophic or heterotrophic lifestyle in Aptandraceae, plastome changes are either correlated with or predate evolution of parasitism. Phylogenetic patterns also indicate repeated loss of infA from the plastome, and based on presence of transcribed sequences with presequences corresponding to thylakoid luminal transit peptides, we infer that the genes were transferred to the nuclear genome. CONCLUSIONS: Except for the loss of the ndh complex, relatively few genes have been lost from the plastome in deep-branching root parasites in Santalales. Prior to loss of the ndh genes, they show signs of relaxed selection indicative of their dispensability. To firmly establish a potential correlation between ndh gene loss, plastome instability and evolution of parasitism, it is pertinent to refute or confirm a parasitic lifestyle all Santalales clades.

Plastome Evolution, Phylogenomics, and DNA Barcoding Investigation of Gastrochilus (Aeridinae, Orchidaceae), with a Focus on the Systematic Position of Haraella retrocalla.

Gastrochilus is an orchid genus containing about 70 species in tropical and subtropical Asia with high morphological diversity. The phylogenetic relationships among this genus have not been fully resolved, and the plastome evolution has not been investigated either. In this study, five plastomes of Gastrochilus were newly reported, and sixteen plastomes of Gastrochilus were used to conduct comparative and phylogenetic analyses. Our results showed that the Gastrochilus plastomes ranged from 146,183 to 148,666 bp, with a GC content of 36.7-36.9%. There were 120 genes annotated, consisting of 74 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. No contraction and expansion of IR borders, gene rearrangements, or inversions were detected. Additionally, the repeat sequences and codon usage bias of Gastrochilus plastomes were highly conserved. Twenty hypervariable regions were selected as potential DNA barcodes. The phylogenetic relationships within Gastrochilus were well resolved based on the whole plastome, especially among main clades. Furthermore, both molecular and morphological data strongly supported Haraella retrocalla as a member of Gastrochilus (G. retrocallus).

Evolution of Whirly1 in the angiosperms: sequence, splicing, and expression in a clade of early transitional mycoheterotrophic orchids.

The plastid-targeted transcription factor Whirly1 (WHY1) has been implicated in chloroplast biogenesis, plastid genome stability, and fungal defense response, which together represent characteristics of interest for the study of autotrophic losses across the angiosperms. While gene loss in the plastid and nuclear genomes has been well studied in mycoheterotrophic plants, the evolution of the molecular mechanisms impacting genome stability is completely unknown. Here, we characterize the evolution of WHY1 in four early transitional mycoheterotrophic orchid species in the genus Corallorhiza by synthesizing the results of phylogenetic, transcriptomic, and comparative genomic analyses with WHY1 genomic sequences sampled from 21 orders of angiosperms. We found an increased number of non-canonical WHY1 isoforms assembled from all but the greenest Corallorhiza species, including intron retention in some isoforms. Within Corallorhiza, phylotranscriptomic analyses revealed the presence of tissue-specific differential expression of WHY1 in only the most photosynthetically capable species and a coincident increase in the number of non-canonical WHY1 isoforms assembled from fully mycoheterotrophic species. Gene- and codon-level tests of WHY1 selective regimes did not infer significant signal of either relaxed selection or episodic diversifying selection in Corallorhiza but did so for relaxed selection in the late-stage full mycoheterotrophic orchids Epipogium aphyllum and Gastrodia elata. Additionally, nucleotide substitutions that most likely impact the function of WHY1, such as nonsense mutations, were only observed in late-stage mycoheterotrophs. We propose that our findings suggest that splicing and expression changes may precede the selective shifts we inferred for late-stage mycoheterotrophic species, which therefore does not support a primary role for WHY1 in the transition to mycoheterotrophy in the Orchidaceae. Taken together, this study provides the most comprehensive view of WHY1 evolution across the angiosperms to date.

Chloroplast genome of Ecbolium viride (Forssk.) Alston: plastome evolution and phylogenomics of Justiceae (Acanthaceae, Acanthoideae).

Justicieae is the most taxonomically complex tribe in Acanthaceae. Here, we sequenced the plastome of Ecbolium viride, a medicinally important species. The genome was analyzed with previously reported plastome of Justiceae. The plastome of E. viride has quadripartite structure with a length of 151 185 bp. The comparative genomic analyses revealed no structural inversion in Justiceae and some regions (rpoC2, ycf2, ycf1, ndhH rps16-trnQ-UGG, and trnL-CAA-ycf15) exhibiting a significant level of nucleotide divergence. The positive selection analyses revealed that some species in the tribe have undergone adaptive evolution. The visualization of the boundaries between the single copy and inverted repeat regions revealed that Justiceae chloroplast genome experienced some levels of variation, which give an insight into the evolution of the species. The longest genome was in the earliest diverged taxa of the tribe Pseuderanthemum haikangense and from this genome, a series of contraction and expansion occurred contributing to the evolution of other lineages. The plastome-based phylogeny revealed and confirmed the monophyly of Justiceae, polyphyly of Justicia and supported the tribal classification Graptophyllinae, Tetrameriinae, and Isoglossinae. We proposed that Declipterinae should be treated as subtribe and the status of Justiciinae can only be confirmed after the resolution of the polyphyletic Justicia.

Comprehensive Comparative Analyses of Aspidistra Chloroplast Genomes: Insights into Interspecific Plastid Diversity and Phylogeny.

Limestone karsts are renowned for extremely high species richness and endemism. Aspidistra (Asparagaceae) is among the highly diversified genera distributed in karst areas, making it an ideal group for studying the evolutionary mechanisms of karst plants. The taxonomy and identification of Aspidistra species are mainly based on their specialized and diverse floral structures. Aspidistra plants have inconspicuous flowers, and the similarity in vegetative morphology often leads to difficulties in species discrimination. Chloroplast genomes possess variable genetic information and offer the potential for interspecies identification. However, as yet there is little information about the interspecific diversity and evolution of the plastid genomes of Aspidistra. In this study, we reported chloroplast (cp) genomes of seven Aspidistra species (A. crassifila, A. dolichanthera, A. erecta, A. longgangensis, A. minutiflora, A. nankunshanensis, and A. retusa). These seven highly-conserved plastid genomes all have a typical quartile structure and include a total of 113 unique genes, comprising 79 protein-coding genes, 4 rRNA genes and 30 tRNA genes. Additionally, we conducted a comprehensive comparative analysis of Aspidistra cp genomes. We identified eight divergent hotspot regions (trnC-GCA-petN, trnE-UUC-psbD, accD-psaI, petA-psbJ, rpl20-rps12, rpl36-rps8, ccsA-ndhD and rps15-ycf1) that serve as potential molecular markers. Our newly generated Aspidistra plastomes enrich the resources of plastid genomes of karst plants, and an investigation into the plastome diversity offers novel perspectives on the taxonomy, phylogeny and evolution of Aspidistra species.

Plastome evolution and phylogenomics of Trichosporeae (Gesneriaceae) with its morphological characters appraisal.

Trichosporeae is the largest and most taxonomically difficult tribe of Gesneriaceae due to its diverse morphology. Previous studies have not clarified the phylogenetic relationships within this tribe on several DNA markers, including the generic relationships within subtribes. Recently, plastid phylogenomics have been successfully employed to resolve the phylogenetic relationships at different taxonomic levels. In this study, plastid phylogenomics were used to explore the relationships within Trichosporeae. Eleven plastomes of Hemiboea were newly reported. Comparative analyses, phylogeny and morphological character evolution within Trichosporeae were conducted on 79 species representing seven subtribes. The Hemiboea plastomes range from 152,742 bp to 153,695 bp in length. Within Trichosporeae, the sampled plastomes range from 152,196 bp to 156,614 bp and GC content from 37.2% to 37.8%. A total of 121-133 genes were annotated in each species, including 80-91 protein-coding genes, 34-37 tRNA genes, and 8 rRNA genes. The contraction and expansion of IR borders were not detected, and gene rearrangements and inversions did not occur. The 13 hypervariable regions were proposed as the potential molecular markers for species identification. A total of 24,299 SNPs and 3,378 indels were inferred, and most of the SNPs were functionally missense and silent variations. There were 1968 SSRs, 2055 tandem repeats and 2802 dispersed repeats. The RSCU and ENC values indicated that the codon usage pattern was conserved in Trichosporeae. Both the phylogenetic frameworks based on the whole plastome and 80 CDSs were basically concordant. The sister relationships between Loxocarpinae and Didymocarpinae were confirmed, and Oreocharis was a sister group of Hemiboea with high support. The morphological characters showed a complex evolutionary pattern of Trichosporeae. Our findings may contribute to future research on genetic diversity, morphological evolutionary patterns, and conservation of the tribe Trichosporeae.

New insights into the phylogenetic relationships among wild onions (Allium, Amaryllidaceae), with special emphasis on the subgenera Anguinum and Rhizirideum, as revealed by plastomes.

The genus Allium, with over 900 species, is one of the largest monocotyledonous genera and is widely accepted with 15 recognized subgenera and 72 sections. The robust subgeneric and sectional relationships within Allium have long been not resolved. Based on 76 species of Allium (a total of 84 accessions), we developed a highly resolved plastome phylogenetic framework by integrating 18 newly sequenced species (20 accessions) in this study and assessed their subgeneric and sectional relationships, with special emphasis on the two subgenera Anguinum and Rhizirideum. We retrieved the three major evolutionary lines within Allium and found that the two subgenera Anguinum and Rhizirideum are monophyletic whereas others are highly polyphyletic (e.g., Allium, Cepa, Polyprason, and Melanocrommyum). Within the subgenus Anguinum, two strongly supported sublineages in East Asian and Eurasian-American were found. Allium tricoccum in North America belonged to the Eurasian clade. The distinct taxonomic status of A. ulleungense and its sister taxon were further determined. In subg. Rhizirideum, the Ulleung Island endemic A. dumebuchum shared its most recent common ancestor with the species from Mongolia and the narrow Korean endemic A. minus. Two Ulleung Island endemics were estimated to originate independently during the Pleistocene. In addition, a separate monotypic sectional treatment of the east Asian A. macrostemon (subg. Allium) and sister relationship between A. condensatum and A. chinense was suggested.

Phylogenomics and phylogeography of Menispermum (Menispermaceae).

Introduction: Phylogenomics have been widely used to resolve ambiguous and controversial evolutionary relationships among plant species and genera, and the identification of unique indels in plastomes may even help to understand the evolution of some plant families. Menispermum L. (Menispermaceae) consists of three species, M. dauricum DC., M. canadense L., and M. mexicanum Rose, which are disjuncly distributed among East Asia, Eastern North America and Mexico. Taxonomists continue to debate whether M. mexicanum is a distinct species, a variety of M. dauricum, or simply a synonym of M. canadense. To date, no molecular systematics studies have included this doubtful species in phylogenetic analyses. Methods: In this study, we examined phylogenomics and phylogeography of Menispermum across its entire range using 29 whole plastomes of Menispermaceae and 18 ITS1&ITS2 sequences of Menispermeae. We reconstructed interspecific relationships of Menispermum and explored plastome evolution in Menispermaceae, revealing several genomic hotspot regions for the family. Results and discussion: Phylogenetic and network analyses based on whole plastome and ITS1&ITS2 sequences show that Menispermum clusters into two clades with high support values, Clade A (M. dauricum) and Clade B (M. canadense + M. mexicanum). However, M. mexicanum is nested within M. canadense and, as a result, we support that M. mexicanum is a synonym of M. canadense. We also identified important molecular variations in the plastomes of Menispermaceae. Several indels and consequently premature terminations of genes occur in Menispermaceae. A total of 54 regions were identified as the most highly variable plastome regions, with nucleotide diversity (Pi) values > 0.05, including two coding genes (matK, ycf1), four introns (trnK intron, rpl16 intron, rps16 intron, ndhA intron), and 48 intergenic spacer (IGS) regions. Of these, four informative hotspot regions (trnH-psbA, ndhF-rpl32, trnK-rps16, and trnP-psaJ) should be especially useful for future studies of phylogeny, phylogeography and conservation genetics of Menispermaceae.

The plastid genome of twenty-two species from Ferula, Talassia, and Soranthus: comparative analysis, phylogenetic implications, and adaptive evolution.

BACKGROUND: The Ferula genus encompasses 180-185 species and is one of the largest genera in Apiaceae, with many of Ferula species possessing important medical value. The previous studies provided more information for Ferula, but its infrageneric relationships are still confusing. In addition, its genetic basis of its adaptive evolution remains poorly understood. Plastid genomes with more variable sites have the potential to reconstruct robust phylogeny in plants and investigate the adaptive evolution of plants. Although chloroplast genomes have been reported within the Ferula genus, few studies have been conducted using chloroplast genomes, especially for endemic species in China. RESULTS: Comprehensively comparative analyses of 22 newly sequenced and assembled plastomes indicated that these plastomes had highly conserved genome structure, gene number, codon usage, and repeats type and distribution, but varied in plastomes size, GC content, and the SC/IR boundaries. Thirteen mutation hotspot regions were detected and they would serve as the promising DNA barcodes candidates for species identification in Ferula and related genera. Phylogenomic analyses with high supports and resolutions showed that Talassia transiliensis and Soranthus meyeri were nested in the Ferula genus, and thus they should be transferred into the Ferula genus. Our phylogenies also indicated the monophyly of subgenera Sinoferula and subgenera Narthex in Ferula genus. Twelve genes with significant posterior probabilities for codon sites were identified in the positively selective analysis, and their function may relate to the photosystem II, ATP subunit, and NADH dehydrogenase. Most of them might play an important role to help Ferula species adapt to high-temperatures, strong-light, and drought habitats. CONCLUSION: Plastome data is powerful and efficient to improve the support and resolution of the complicated Ferula phylogeny. Twelve genes with significant posterior probabilities for codon sites were helpful for Ferula to adapt to the harsh environment. Overall, our study supplies a new perspective for comprehending the phylogeny and evolution of Ferula.

Evidence from Phylogenomics and Morphology Provide Insights into the Phylogeny, Plastome Evolution, and Taxonomy of Kitagawia.

Kitagawia Pimenov is one of the segregate genera of Peucedanum sensu lato within the Apiaceae. The phylogenetic position and morphological delimitation of Kitagawia have been controversial. In this study, we used plastid genome (plastome) and nuclear ribosomal DNA (nrDNA) sequences to reconstruct the phylogeny of Kitagawia, along with comparative plastome and morphological analyses between Kitagawia and related taxa. The phylogenetic results identified that all examined Kitagawia species were divided into Subclade I and Subclade II within the tribe Selineae, and they were all distant from the representative members of Peucedanum sensu stricto. The plastomes of Kitagawia and related taxa showed visible differences in the LSC/IRa junction (JLA) and several hypervariable regions, which separated Subclade I and Subclade II from other taxa. Fruit anatomical and micromorphological characteristics, as well as general morphological characteristics, distinguished the four Kitagawia species within Subclade I from Subclade II and other related genera. This study supported the separation of Kitagawia from Peucedanum sensu lato, confirmed that Kitagawia belongs to Selineae, and two species (K. praeruptora and K. formosana) within Subclade II should be placed in a new genus. We believe that the "core" Kitagawia should be limited to Subclade I, and this genus can be distinguished by the association of a series of morphological characteristics. Overall, our study provides new insights into the phylogeny, plastome evolution, and taxonomy of Kitagawia.

Plastid Phylogenomics and Plastome Evolution of Nandinoideae (Berberidaceae).

Subfamily Nandinoideae Heintze (Berberidaceae), comprising four genera and ca. 19 species, is disjunctively distributed in eastern North America vs. Eurasia (eastern Asia, Central Asia, Middle East, and southeastern Europe), and represents an ideal taxon to explore plastid phylogenomics and plastome evolution in Berberidaceae. Many species of this subfamily have been listed as national or international rare and endangered plants. In this study, we sequenced and assembled 20 complete plastomes, representing three genera and 13 species of Nandinoideae. Together with six plastomes from GenBank, a total of 26 plastomes, representing all four genera and 16 species of Nandinoideae, were used for comparative genomic and phylogenomic analyses. These plastomes showed significant differences in overall size (156,626-161,406 bp), which is mainly due to the expansion in inverted repeat (IR) regions and/or insertion/deletion (indel) events in intergenic spacer (IGS) regions. A 75-bp deletion in the ndhF gene occurred in Leontice and Gymnospermium when compared with Nandina and Caulophyllum. We found a severe truncation at the 5' end of ycf1 in three G. altaicum plastomes, and a premature termination of ropC1 in G. microrrhynchum. Our phylogenomic results support the topology of {Nandina, [Caulophyllum, (Leontice, Gymnospermium)]}. Within the core genus Gymnospermium, we identified G. microrrhynchum from northeastern Asia (Clade A) as the earliest diverging species, followed by G. kiangnanense from eastern China (Clade B), while the rest species clustered into the two sister clades (C and D). Clade C included three species from West Tianshan (G. albertii, G. darwasicum, G. vitellinum). Clade D consisted of G. altaicum from northern Central Asia, plus one species from the Caucasus Mountains (G. smirnovii) and three from southeastern Europe (G. odessanum, G. peloponnesiacum, G. scipetarum). Overall, we identified 21 highly variable plastome regions, including two coding genes (rpl22, ycf1) and 19 intergenic spacer (IGS) regions, all with nucleotide diversity (Pi) values > 0.02. These molecular markers should serve as powerful tools (including DNA barcodes) for future phylogenetic, phylogeographic and conservation genetic studies.

Putting small and big pieces together: a genome assembly approach reveals the largest Lamiid plastome in a woody vine.

The plastid genome of flowering plants generally shows conserved structural organization, gene arrangement, and gene content. While structural reorganizations are uncommon, examples have been documented in the literature during the past years. Here we assembled the entire plastome of Bignonia magnifica and compared its structure and gene content with nine other Lamiid plastomes. The plastome of B. magnifica is composed of 183,052 bp and follows the canonical quadripartite structure, synteny, and gene composition of other angiosperms. Exceptionally large inverted repeat (IR) regions are responsible for the uncommon length of the genome. At least four events of IR expansion were observed among the seven Bignoniaceae species compared, suggesting multiple expansions of the IRs over the SC regions in the family. A comparison with 6,231 other complete plastomes of flowering plants available on GenBank revealed that the plastome of B. magnifica is the longest Lamiid plastome described to date. The newly generated plastid genome was used as a source of selected genes. These genes were combined with orthologous regions sampled from other species of Bignoniaceae and all gene alignments concatenated to infer a phylogeny of the family. The tree recovered is consistent with known relationships within the Bignoniaceae.

The complete plastomes of seven Peucedanum plants: comparative and phylogenetic analyses for the Peucedanum genus.

BACKGROUND: The Peucedanum genus is the backbone member of Apiaceae, with many economically and medically important plants. Although the previous studies on Peucedanum provide us with a good research basis, there are still unclear phylogenetic relationships and many taxonomic problems in Peucedanum, and a robust phylogenetic framework of this genus still has not been obtained, which severely hampers the improvement and revision of taxonomic system for this genus. The plastid genomes possessing more variable characters have potential for reconstructing a robust phylogeny in plants. RESULTS: In the current study, we newly sequenced and assembled seven Peucedanum plastid genomes. Together with five previously published plastid genomes of Peucedanum, we performed a comprehensively comparative analyses for this genus. Twelve Peucedanum plastomes were similar in terms of genome structure, codon bias, RNA editing sites, and SSRs, but varied in genome size, gene content and arrangement, and border of SC/IR. Fifteen mutation hotspot regions were identified among plastid genomes that can serve as candidate DNA barcodes for species identification in Peucedanum. Our phylogenetic analyses based on plastid genomes generated a phylogeny with high supports and resolutions for Peucedanum that robustly supported the non-monophyly of genus Peucedanum. CONCLUSION: The plastid genomes of Peucedanum showed both conservation and diversity. The plastid genome data were efficient and powerful for improving the supports and resolutions of phylogeny for the complex Peucedanum genus. In summary, our study provides new sights into the plastid genome evolution, taxonomy, and phylogeny for Peucedanum species.

Plastid Phylogenomic Analysis of Tordylieae Tribe (Apiaceae, Apioideae).

Based on the nrDNA ITS sequence data, the Tordylieae tribe is recognized as monophyletic with three major lineages: the subtribe Tordyliinae, the Cymbocarpum clade, and the Lefebvrea clade. Recent phylogenomic investigations showed incongruence between the nuclear and plastid genome evolution in the tribe. To assess phylogenetic relations and structure evolution of plastomes in Tordylieae, we generated eleven complete plastome sequences using the genome skimming approach and compared them with the available data from this tribe and close relatives. Newly assembled plastomes had lengths ranging from 141,148 to 150,103 base pairs and contained 122-127 genes, including 79-82 protein-coding genes, 35-37 tRNAs, and 8 rRNAs. We observed substantial differences in the inverted repeat length and gene content, accompanied by a complex picture of multiple JLA and JLB shifts. In concatenated phylogenetic analyses, Tordylieae plastomes formed at least three not closely related lineages with plastomes of the Lefebvrea clade as a sister group to plastomes from the Selineae tribe. The newly obtained data have increased our knowledge on the range of plastome variability in Apiaceae.

Towards the plastome evolution and phylogeny of Cycas L. (Cycadaceae): molecular-morphology discordance and gene tree space analysis.

BACKGROUND: Plastid genomes (plastomes) present great potential in resolving multiscale phylogenetic relationship but few studies have focused on the influence of genetic characteristics of plastid genes, such as genetic variation and phylogenetic discordance, in resolving the phylogeny within a lineage. Here we examine plastome characteristics of Cycas L., the most diverse genus among extant cycads, and investigate the deep phylogenetic relationships within Cycas by sampling 47 plastomes representing all major clades from six sections. RESULTS: All Cycas plastomes shared consistent gene content and structure with only one gene loss detected in Philippine species C. wadei. Three novel plastome regions (psbA-matK, trnN-ndhF, chlL-trnN) were identified as containing the highest nucleotide variability. Molecular evolutionary analysis showed most of the plastid protein-coding genes have been under purifying selection except ndhB. Phylogenomic analyses that alternatively included concatenated and coalescent methods, both identified four clades but with conflicting topologies at shallow nodes. Specifically, we found three species-rich Cycas sections, namely Stangerioides, Indosinenses and Cycas, were not or only weakly supported as monophyly based on plastomic phylogeny. Tree space analyses based on different tree-inference methods both revealed three gene clusters, of which the cluster with moderate genetic properties showed the best congruence with the favored phylogeny. CONCLUSIONS: Our exploration in plastomic data for Cycas supports the idea that plastid protein-coding genes may exhibit discordance in phylogenetic signals. The incongruence between molecular phylogeny and morphological classification reported here may largely be attributed to the uniparental attribute of plastid, which cannot offer sufficient information to resolve the phylogeny. Contrasting to a previous consensus that genes with longer sequences and a higher proportion of variances are superior for phylogeny reconstruction, our result implies that the most effective phylogenetic signals could come from loci that own moderate variation, GC content, sequence length, and underwent modest selection.

Comparative analysis of 84 chloroplast genomes of Tylosema esculentum reveals two distinct cytotypes.

Tylosema esculentum (marama bean) is an important orphan legume from southern Africa that has long been considered to have the potential to be domesticated as a crop. The chloroplast genomes of 84 marama samples collected from various geographical locations in Namibia and Pretoria were compared in this study. The cp genomes were analyzed for diversity, including SNPs, indels, structural alterations, and heteroplasmy. The marama cp genomes ranged in length from 161,537 bp to 161,580 bp and contained the same sets of genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The genes rpoC2 and rpoB, and the intergenic spacers trnT-trnL and ndhG-ndhI were found to be more diverse than other regions of the marama plastome. 15 haplotypes were found to be divided into two groups, differing at 122 loci and at a 230 bp inversion. One type appears to have greater variability within the major genome present, and variations amongst individuals with this type of chloroplast genome seems to be distributed within specific geographic regions but with very limited sampling for some regions. However, deep sequencing has identified that within most of the individuals, both types of chloroplast genomes are present, albeit one is generally at a very low frequency. The inheritance of this complex of chloroplast genomes appears to be fairly constant, providing a conundrum of how the two genomes co-exist and are propagated through generations. The possible consequences for adaptation to the harsh environment in which T. esculentum survives are considered. The results pave the way for marama variety identification, as well as for understanding the origin and evolution of the bean.

Extensive genomic rearrangements mediated by repetitive sequences in plastomes of Medicago and its relatives.

BACKGROUND: Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC. RESULTS: We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences. CONCLUSIONS: Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.

Integrated Genomic Analyses From Low-Depth Sequencing Help Resolve Phylogenetic Incongruence in the Bamboos (Poaceae: Bambusoideae).

The bamboos (Bambusoideae, Poaceae) comprise a major grass lineage with a complex evolutionary history involving ancient hybridization and allopolyploidy. About 1700 described species are classified into three tribes, Olyreae (herbaceous bamboos), Bambuseae (tropical woody bamboos), and Arundinarieae (temperate woody bamboos). Nuclear analyses strongly support monophyly of the woody tribes, whereas plastome analyses strongly support paraphyly, with Bambuseae sister to Olyreae. Our objectives were to clarify the origin(s) of the woody bamboo tribes and resolve the nuclear vs. plastid conflict using genomic tools. For the first time, plastid and nuclear genomic information from the same bamboo species were combined in a single study. We sampled 51 species of bamboos representing the three tribes, estimated their genome sizes and generated low-depth sample sequence data, from which plastomes were assembled and nuclear repeats were analyzed. The distribution of repeat families was found to agree with nuclear gene phylogenies, but also provides novel insights into nuclear evolutionary history. We infer two early, independent hybridization events, one between an Olyreae ancestor and a woody ancestor giving rise to the two Bambuseae lineages, and another between two woody ancestors giving rise to the Arundinarieae. Retention of the Olyreae plastome associated with differential dominance of nuclear genomes and subsequent diploidization in some lineages explains the paraphyly observed in plastome phylogenetic estimations. We confirm ancient hybridization and allopolyploidy in the origins of the extant woody bamboo lineages and propose biased fractionation and diploidization as important factors in their evolution.

Highly Diverse Shrub Willows (Salix L.) Share Highly Similar Plastomes.

Plastome phylogenomics is used in a broad range of studies where single markers do not bear enough information. Phylogenetic reconstruction in the genus Salix is difficult due to the lack of informative characters and reticulate evolution. Here, we use a genome skimming approach to reconstruct 41 complete plastomes of 32 Eurasian and North American Salix species representing different lineages, different ploidy levels, and separate geographic regions. We combined our plastomes with published data from Genbank to build a comprehensive phylogeny of 61 samples (50 species) using RAxML (Randomized Axelerated Maximum Likelihood). Additionally, haplotype networks for two observed subclades were calculated, and 72 genes were tested to be under selection. The results revealed a highly conserved structure of the observed plastomes. Within the genus, we observed a variation of 1.68%, most of which separated subg. Salix from the subgeneric Chamaetia/Vetrix clade. Our data generally confirm previous plastid phylogenies, however, within Chamaetia/Vetrix phylogenetic results represented neither taxonomical classifications nor geographical regions. Non-coding DNA regions were responsible for most of the observed variation within subclades and 5.6% of the analyzed genes showed signals of diversifying selection. A comparison of nuclear restriction site associated DNA (RAD) sequencing and plastome data on a subset of 10 species showed discrepancies in topology and resolution. We assume that a combination of (i) a very low mutation rate due to efficient mechanisms preventing mutagenesis, (ii) reticulate evolution, including ancient and ongoing hybridization, and (iii) homoplasy has shaped plastome evolution in willows.