Evolution of mitochondrial RNA editing in extant gymnosperms.

To unveil the evolution of mitochondrial RNA editing in gymnosperms, we characterized mitochondrial genomes (mitogenomes), plastid genomes, RNA editing sites, and pentatricopeptide repeat (PPR) proteins from 10 key taxa representing four of the five extant gymnosperm clades. The assembled mitogenomes vary in gene content due to massive gene losses in Gnetum and Conifer II clades. Mitochondrial gene expression levels also vary according to protein function, with the most highly expressed genes involved in the respiratory complex. We identified 9132 mitochondrial C-to-U editing sites, as well as 2846 P-class and 8530 PLS-class PPR proteins. Regains of editing sites were demonstrated in Conifer II rps3 transcripts whose corresponding mitogenomic sequences lack introns due to retroprocessing. Our analyses reveal that non-synonymous editing is efficient and results in more codons encoding hydrophobic amino acids. In contrast, synonymous editing, although performed with variable efficiency, can increase the number of U-ending codons that are preferentially utilized in gymnosperm mitochondria. The inferred loss-to-gain ratio of mitochondrial editing sites in gymnosperms is 2.1:1, of which losses of non-synonymous editing are mainly due to genomic C-to-T substitutions. However, such substitutions only explain a small fraction of synonymous editing site losses, indicating distinct evolutionary mechanisms. We show that gymnosperms have experienced multiple lineage-specific duplications in PLS-class PPR proteins. These duplications likely contribute to accumulated RNA editing sites, as a mechanistic correlation between RNA editing and PLS-class PPR proteins is statistically supported.

Plastid phylogenomics and plastome evolution in the morning glory family (Convolvulaceae).

Convolvulaceae, the morning glories or bindweeds, is a large family containing species of economic value, including crops, traditional medicines, ornamentals, and vegetables. However, not only are the phylogenetic relationships within this group still debated at the intertribal and intergeneric levels, but also plastid genome (plastome) complexity within Convolvulaceae is not well surveyed. We gathered 78 plastomes representing 17 genera across nine of the 12 Convolvulaceae tribes. Our plastid phylogenomic trees confirm the monophyly of Convolvulaceae, place the genus Jacquemontia within the subfamily Dicranostyloideae, and suggest that the tribe Merremieae is paraphyletic. In contrast, positions of the two genera Cuscuta and Erycibe are uncertain as the bootstrap support of the branches leading to them is moderate to weak. We show that nucleotide substitution rates are extremely variable among Convolvulaceae taxa and likely responsible for the topological uncertainty. Numerous plastomic rearrangements are detected in Convolvulaceae, including inversions, duplications, contraction and expansion of inverted repeats (IRs), and losses of genes and introns. Moreover, integrated foreign DNA of mitochondrial origin was found in the Jacquemontia plastome, adding a rare example of gene transfer from mitochondria to plastids in angiosperms. In the IR of Dichondra, we discovered an extra copy of rpl16 containing a direct repeat of ca. 200 bp long. This repeat was experimentally demonstrated to trigger effective homologous recombination, resulting in the coexistence of intron-containing and -lacking rpl16 duplicates. Therefore, we propose a hypothetical model to interpret intron loss accompanied by invasion of direct repeats at appropriate positions. Our model complements the intron loss model driven by retroprocessing when genes have lost introns but contain abundant RNA editing sites adjacent to former splicing sites.

The complete plastid genome of Selaginella erythropus (Selaginellaceae), a species with distinctive giant chloroplasts.

The plastid genome of the deep-shade plant Selaginella erythropus, which has highly unusual chloroplasts, was characterized using Illumina pair-end sequencing. This plastome is 140,151 bp in length with a large single-copy region (LSC) of 56,133 bp, a small single-copy region (SSC) of 61,268 bp, and two direct repeats (DRs) of 11,375 bp. The overall GC content is 50.68%, while those of LSC, SSC, and DR are 48.96%, 50.3%, and 55.96%, respectively. The plastome contains 102 genes, including 76 protein-coding, 15 tRNA (12 tRNA species), and 8 rRNA genes (4 rRNA species). The phylogenetic analysis shows that S. erythropus is closely related to S. moellendorffii and S. doederleinii. This result is consistent with the previous phylogenetic relationship inferred from multiple plastid and nuclear loci. However, only S. erythropus has the two-zoned giant chloroplast, the bizonoplast. The plastome provides an excellent reference for understanding the unique chloroplast differentiation in Selaginellaceae.

Corrigendum: Reassessing Banana Phylogeny and Organelle Inheritance Modes Using Genome Skimming Data.

[This corrects the article DOI: 10.3389/fpls.2021.713216.].

Reassessing Banana Phylogeny and Organelle Inheritance Modes Using Genome Skimming Data.

Bananas (Musa spp.) are some of the most important fruit crops in the world, contributing up to US$10 billion in export values annually. In this study, we use high-throughput sequencing to obtain genomic resources of high-copy DNA molecules in bananas. We sampled 13 wild species and eight cultivars that represent the three genera (Ensete, Musa, and Musella) of the banana family (Musaceae). Their plastomic, 45S rDNA, and mitochondrial scaffolds were recovered from genome skimming data. Two major clades (Clades I & II) within Musa are strongly supported by the three genomic compartment data. We document, for the first time, that the plastomes of Musaceae have expanded inverted repeats (IR) after they diverged from their two close relatives, Heliconiaceae (the lobster-claws) and Strelitziaceae (the traveler's bananas). The presence/absence of rps19 within IR regions reinforces the two intra-generic clades within Musa. Our comparisons of the bananas' plastomic and mitochondrial DNA sequence trees aid in identifying hybrid bananas' parentage. As the mitochondrial genes of Musa have elevated substitution rates, paternal inheritance likely plays an influential role on the Musa mitogenome evolution. We propose genome skimming as a useful method for reliable genealogy tracing and phylogenetics in bananas.

Tight association of genome rearrangements with gene expression in conifer plastomes.

BACKGROUND: Our understanding of plastid transcriptomes is limited to a few model plants whose plastid genomes (plastomes) have a highly conserved gene order. Consequently, little is known about how gene expression changes in response to genomic rearrangements in plastids. This is particularly important in the highly rearranged conifer plastomes. RESULTS: We sequenced and reported the plastomes and plastid transcriptomes of six conifer species, representing all six extant families. Strand-specific RNAseq data show a nearly full transcription of both plastomic strands and detect C-to-U RNA-editing sites at both sense and antisense transcripts. We demonstrate that the expression of plastid coding genes is strongly functionally dependent among conifer species. However, the strength of this association declines as the number of plastomic rearrangements increases. This finding indicates that plastomic rearrangement influences gene expression. CONCLUSIONS: Our data provide the first line of evidence that plastomic rearrangements not only complicate the plastomic architecture but also drive the dynamics of plastid transcriptomes in conifers.

Genome skimming and exploration of DNA barcodes for Taiwan endemic cypresses.

Cypresses are characterized by their longevity and valuable timber. In Taiwan, two endemic cypress species, Chamaecyparis formosensis and C. obtusa var. formosana, are threatened by prevalent illegal logging. A DNA barcode system is urgently needed for reforestation and conservation of these two cypresses. In this study, both plastomes and 35S rDNAs from 16, 10, and 6 individuals of C. formosensis, C. obtusa var. formosana, and C. obtusa var. obtusa were sequenced, respectively. We show that the loss of plastid trnT-GGU readily distinguishes C. formosensis from its congeneric species. We demonstrate that entire sequences of plastomes or 35S rDNAs are capable of correctly identifying cypress species and varieties, suggesting that they are effective super-barcodes. We also discover three short hypervariable loci (i.e., 3'ETS, ITS1, and trnH-psbA) that are promising barcodes for identifying cypress species and varieties. Moreover, nine species-specific indels of > 100 bp were detected in the cypress plastomes. These indels, together with the three aforementioned short barcodes, constitute an alternative and powerful barcode system crucial for identifying specimens that are fragmentary or contain degraded/poor DNA. Our sequenced data and barcode systems not only enrich the genetic reference for cypresses, but also contribute to future reforestation, conservation, and forensic investigations.

The Origin and Evolution of Plastid Genome Downsizing in Southern Hemispheric Cypresses (Cupressaceae).

Plastome downsizing is rare in photosynthetic seed plants. However, a large-scale study of five cupressophyte families (conifers II) indicated that the plastomes of some Cupressaceous genera are notably reduced and compact. Here, we enriched taxon sampling in Cupressaceae by adding plastomes of ten previously unreported genera to determine the origin, evolution, and consequences of plastome reduction in this family. We discovered that plastome downsizing is specific to Callitroideae (a Southern Hemispheric subfamily). Their plastomes are the smallest, encode the fewest plastid genes, and contain the fewest GC-end codons among Cupressaceae. We show that repeated tRNA losses and shrinkage of intergenic spacers together contributed to the plastome downsizing in Callitroideae. Moreover, our absolute nucleotide substitution rate analyses suggest relaxed functional constraints in translation-related plastid genes (clpP, infA, rpl, and rps), but not in photosynthesis- or transcription-related ones, of Callitris (the most diverse genus in Callitroideae). We hypothesize that the small and low-GC plastomes of Callitroideae emerged ca. 112-75 million years ago as an adaptation to increased competition with angiosperms on the Gondwana supercontinent. Our findings highlight Callitroideae as another case of plastome downsizing in photosynthetic seed plant lineages.

Prevalence of isomeric plastomes and effectiveness of plastome super-barcodes in yews (Taxus) worldwide.

Taxus (yew) is both the most species-rich and taxonomically difficult genus in Taxaceae. To date, no study has elucidated the complexities of the plastid genome (plastome) or examined the possibility of whole plastomes as super-barcodes across yew species worldwide. In this study, we sequenced plastomes from two to three individuals for each of the 16 recognized yew species (including three potential cryptics) and Pseudotaxus chienii. Our comparative analyses uncovered several gene loss events that independently occurred in yews, resulting in a lower plastid gene number than other Taxaceous genera. In Pseudotaxus and Taxus, we found two isomeric arrangements that differ by the orientation of a 35 kb fragment flanked by "trnQ-IRs". These two arrangements exist in different ratios within each sampled individual, and intraspecific shifts in major isomeric arrangements are first reported here in Taxus. Moreover, we demonstrate that entire plastomes can be used to successfully discriminate all Taxus species with 100% support, suggesting that they are useful as super-barcodes for species identification. We also propose that accD and rrn16-rrn23 are promising special barcodes to discriminate yew species. Our newly developed Taxus plastomic sequences provide a resource for super-barcodes and conservation genetics of several endangered yews and serve as comprehensive data to improve models of plastome complexity in Taxaceae as a whole and authenticate Taxus species.

Stout camphor tree genome fills gaps in understanding of flowering plant genome evolution.

We present reference-quality genome assembly and annotation for the stout camphor tree (Cinnamomum kanehirae (Laurales, Lauraceae)), the first sequenced member of the Magnoliidae comprising four orders (Laurales, Magnoliales, Canellales and Piperales) and over 9,000 species. Phylogenomic analysis of 13 representative seed plant genomes indicates that magnoliid and eudicot lineages share more recent common ancestry than monocots. Two whole-genome duplication events were inferred within the magnoliid lineage: one before divergence of Laurales and Magnoliales and the other within the Lauraceae. Small-scale segmental duplications and tandem duplications also contributed to innovation in the evolutionary history of Cinnamomum. For example, expansion of the terpenoid synthase gene subfamilies within the Laurales spawned the diversity of Cinnamomum monoterpenes and sesquiterpenes.

Enlarged and highly repetitive plastome of Lagarostrobos and plastid phylogenomics of Podocarpaceae.

Podocarpaceae is the largest family in cupressophytes (conifers II), but its plastid genomes (plastomes) are poorly studied, with plastome data currently existing for only four of the 19 Podocarpaceous genera. In this study, we sequenced and assembled the complete plastomes from representatives of eight additional genera, including Afrocarpus, Dacrydium, Lagarostrobos, Lepidothamnus, Pherosphaera, Phyllocladus, Prumnopitys, and Saxegothaea. We found that Lagarostrobos, a monotypic genus native to Tasmania, has the largest plastome (151,496 bp) among any cupressophytes studied to date. Plastome enlargement in Lagarostrobos coincides with increased intergenic spacers, repeats, and duplicated genes. Among the Podocarpaceae, Lagarostrobos has the most rearranged plastome, but its substitution rates are modest. Plastid phylogenomic analyses based on 81 plastid genes clarify the positions of previously conflicting Podocarpaceous genera. Tree topologies firmly support the division of Podocarpaceae into two sister clades: (1) the Prumnopityoid clade and (2) the clade containing Podocarpoid, Dacrydioid, Pherosphaera, and Saxegothaea. The Phyllocladus is nested within the Podocarpaceae, thus familial status of the monotypic Phyllocladaceae is not supported.

Photosynthetic gas exchange responses of Swietenia macrophylla King and Melia azedarach L. plantations under drought conditions.

BACKGROUND: The environmental stresses caused by climate change have become more severe in recent decades, affecting tree growth and physiology. Tropical forests have great potential for global carbon sequestration. However, they suffer from heavy rainfall and prolonged dry periods due to climate change. Swietenia macrophylla King and Melia azedarach L. are economically valuable trees that are widely planted in southern Taiwan. Plantations are exposed to either prolonged dry periods or heavy rainfall within the seasons of tropical monsoon areas. Photo-physiological comparisons may provide information that can improve management of S. macrophylla and M. azedarach plantations in tropical regions. RESULTS: Both species exhibited a midday depression in leaf photosynthesis regardless of the season. The net photosynthetic rate (P N), stomatal conductance (g s), and transpiration rate (E) in the dry season all significantly decreased in both tree species. In addition, M. azedarach used water more efficiently than did S. macrophylla during the dry season, but S. macrophylla had higher P N compared with that in M. azedarach during the wet season. Temperature and vapor pressure deficit influenced P N variation in S. macrophylla and M. azedarach, respectively. Our data suggested that the P N and g s of M. azedarach, but not of S. macrophylla, were linearly correlated during the dry season. The reduction of the leaf area was more sever in M. azedarach than in S. macrophylla, thus preventing water loss more efficiently. CONCLUSIONS: M. azedarach adapted to drought by reducing total leaf area and maintaining higher P N, g s, E, and WUE compared with those measured in S. macrophylla during the dry season. M. azedarach is more drought adaptation and more suitable for both humid and semi-humid areas than S. macrophylla, whereas the latter should be limited to more humid areas.

Plastome Evolution in the Sole Hemiparasitic Genus Laurel Dodder (Cassytha) and Insights into the Plastid Phylogenomics of Lauraceae.

To date, little is known about the evolution of plastid genomes (plastomes) in Lauraceae. As one of the top five largest families in tropical forests, the Lauraceae contain many species that are important ecologically and economically. Lauraceous species also provide wonderful materials to study the evolutionary trajectory in response to parasitism because they contain both nonparasitic and parasitic species. This study compared the plastomes of nine Lauraceous species, including the sole hemiparasitic and herbaceous genus Cassytha (laurel dodder; here represented by Cassytha filiformis). We found differential contractions of the canonical inverted repeat (IR), resulting in two IR types present in Lauraceae. These two IR types reinforce Cryptocaryeae and Neocinnamomum-Perseeae-Laureae as two separate clades. Our data reveal several traits unique to Cas. filiformis, including loss of IRs, loss or pseudogenization of 11 ndh and rpl23 genes, richness of repeats, and accelerated rates of nucleotide substitutions in protein-coding genes. Although Cas. filiformis is low in chlorophyll content, our analysis based on dN/dS ratios suggests that both its plastid house-keeping and photosynthetic genes are under strong selective constraints. Hence, we propose that short generation time and herbaceous lifestyle rather than reduced photosynthetic ability drive the accelerated rates of nucleotide substitutions in Cas. filiformis.

Insights into the Existence of Isomeric Plastomes in Cupressoideae (Cupressaceae).

The cypress family (Cupressaceae) possesses highly rearranged plastomes that lack a pair of large inverted repeats typically found in land plants. A few cypress species have been reported to contain isomeric plastomes, but whether the existence of isomeric plastomes is ubiquitous in the family remains to be investigated with a broader taxon sampling. In this study, we sequenced the complete plastomes of ten species in Cupressoideae, the largest cypress subfamily. Cupressoideae showed relatively accelerated rates of substitutions at both nonsynonymous and synonymous sites as compared with other subfamilies of Cupressaceae. Our PCR and read mapping analyses together suggested the existence of isomeric plastomes in eight of the ten sequenced Cupressoideae species. The isomeric plastomes were also detected in 176 individuals from nine wild populations of four Cupressoideae species. Within Calocedrus macrolepis, we discovered a new type of isomeric plastomes that was likely derived from homologous recombination mediated by an 11-bp repeat. We conclude that isomeric plastomes are commonly present in Cupressoideae, thereby contributing to increased plastomic complexity.

Revisiting the Plastid Phylogenomics of Pinaceae with Two Complete Plastomes of Pseudolarix and Tsuga.

Phylogeny of the ten Pinaceous genera has long been contentious. Plastid genomes (plastomes) provide an opportunity to resolve this problem because they contain rich evolutionary information. To comprehend the plastid phylogenomics of all ten Pinaceous genera, we sequenced the plastomes of two previously unavailable genera, Pseudolarix amabilis (122,234 bp) and Tsuga chinensis (120,859 bp). Both plastomes share similar gene repertoire and order. Here for the first time we report a unique insertion of tandem repeats in accD of T. chinensis From the 65 plastid protein-coding genes common to all Pinaceous genera, we re-examined the phylogenetic relationship among all Pinaceous genera. Our two phylogenetic trees are congruent in an identical tree topology, with the five genera of the Abietoideae subfamily constituting a monophyletic clade separate from the other three subfamilies: Pinoideae, Piceoideae, and Laricoideae. The five genera of Abietoideae were grouped into two sister clades consisting of (1) Cedrus alone and (2) two sister subclades of Pseudolarix-Tsuga and Abies-Keteleeria, with the former uniquely losing the gene psaM and the latter specifically excluding the 3 psbA from the residual inverted repeat.

Birth of Four Chimeric Plastid Gene Clusters in Japanese Umbrella Pine.

Many genes in the plastid genomes (plastomes) of plants are organized as gene clusters, in which genes are co-transcribed, resembling bacterial operons. These plastid operons are highly conserved, even among conifers, whose plastomes are highly rearranged relative to other seed plants. We have determined the complete plastome sequence of Sciadopitys verticillata (Japanese umbrella pine), the sole member of Sciadopityaceae. The Sciadopitys plastome is characterized by extensive inversions, pseudogenization of four tRNA genes after tandem duplications, and a unique pair of 370-bp inverted repeats involved in the formation of isomeric plastomes. We showed that plastomic inversions in Sciadopitys have led to shuffling of the remote conserved operons, resulting in the birth of four chimeric gene clusters. Our data also demonstrated that the relocated genes can be co-transcribed in these chimeric gene clusters. The plastome of Sciadopitys advances our current understanding of how the conifer plastomes have evolved toward increased diversity and complexity.

Large-Scale Comparative Analysis Reveals the Mechanisms Driving Plastomic Compaction, Reduction, and Inversions in Conifers II (Cupressophytes).

Conifers II (cupressophytes), comprising about 400 tree species in five families, are the most diverse group of living gymnosperms. Their plastid genomes (plastomes) are highly variable in size and organization, but such variation has never been systematically studied. In this study, we assessed the potential mechanisms underlying the evolution of cupressophyte plastomes. We analyzed the plastomes of 24 representative genera in all of the five cupressophyte families, focusing on their variation in size, noncoding DNA content, and nucleotide substitution rates. Using a tree-based method, we further inferred the ancestral plastomic organizations of internal nodes and evaluated the inversions across the evolutionary history of cupressophytes. Our data showed that variation in plastome size is statistically associated with the dynamics of noncoding DNA content, which results in different degrees of plastomic compactness among the cupressophyte families. The degrees of plastomic inversions also vary among the families, with the number of inversions per genus ranging from 0 in Araucariaceae to 1.27 in Cupressaceae. In addition, we demonstrated that synonymous substitution rates are significantly correlated with plastome size as well as degree of inversions. These data suggest that in cupressophytes, mutation rates play a critical role in driving the evolution of plastomic size while plastomic inversions evolve in a neutral manner.

The complete plastome sequence of Gnetum ula (Gnetales: Gnetaceae).

This study reports the complete plastome sequence of Gnetum ula, a gymnosperm species of Gnetaceae (Gnetophyta). The plastome is 113 249 bp long. It has a quadripartite structure containing a pair of large inverted repeat regions of 19 772 bp each, a large single-copy region of 64 914 bp, and a small single-copy region of 8791 bp. One hundred sixteen genes were predicted in the plastome, including 68 protein-coding genes, eight ribosomal RNA genes, and 40 transfer RNA genes. The gene density is 1.024 (genes/kb). Similar to other known Gnetum plastomes, the G.ula plastome has lost 20 protein-coding genes commonly present in other seed plant plastomes. Our phylogenetic analyses indicate that the four sampled Gnetum species are monophyletic and that G. ula is close to the two other lianas rather than the only small tree species, G. gnemon. Our phylogenetic trees also indicate that gnetophytes have the fastest evolutionary rates among gymnosperms.

Evolutionary Stasis in Cycad Plastomes and the First Case of Plastome GC-Biased Gene Conversion.

In angiosperms, gene conversion has been known to reduce the mutational load of plastid genomes (the plastomes). Particularly, more frequent gene conversions in inverted repeat (IR) than in single copy (SC) regions result in contrasting substitution rates between these two regions. However, little has been known about the effect of gene conversion in the evolution of gymnosperm plastomes. Cycads (Cycadophyta) are the second largest gymnosperm group. Evolutionary study of their plastomes is limited to the basal cycad genus, Cycas. In this study, we addressed three questions. 1) Do the plastomes of other cycad genera evolve slowly as previously observed in the plastome of Cycas taitungensis? 2) Do substitution rates differ between their SC and IR regions? And 3) Does gene conversion occur in the cycad plastomes? If yes, is it AT-biased or GC-biased? Plastomes of eight species from other eight genera of cycads were sequenced. These plastomes are highly conserved in genome organization. Excluding ginkgo, cycad plastomes have significantly lower synonymous and nonsynonymous substitution rates than other gymnosperms, reflecting their evolutionary stasis in nucleotide mutations. In the IRs of cycad plastomes, the reduced substitution rates and GC-biased mutations are associated with a GC-biased gene conversion (gBGC) mechanism. Further investigations suggest that in cycads, gBGC is able to rectify plastome-wide mutations. Therefore, this study is the first to uncover the plastomic gBGC in seed plants. We also propose a gBGC model to interpret the dissimilar evolutionary patterns as well as the compositionally biased mutations in the SC and IR regions of cycad plastomes.

Ancient nuclear plastid DNA in the yew family (taxaceae).

Plastid-to-nucleus DNA transfer provides a rich genetic resource to the complexity of plant nuclear genome architecture. To date, the evolutionary route of nuclear plastid DNA (nupt) remain unknown in conifers. We have sequenced the complete plastomes of two yews, Amentotaxus formosana and Taxus mairei (Taxaceae of coniferales). Our comparative genomic analyses recovered an evolutionary scenario for plastomic reorganization from ancestral to extant plastomes in the three sampled Taxaceae genera, Amentotaxus, Cephalotaxus, and Taxus. Specific primers were designed to amplify nonsyntenic regions between ancestral and extant plastomes, and 12.6 kb of nupts were identified based on phylogenetic analyses. These nupts have significantly accumulated GC-to-AT mutations, reflecting a nuclear mutational environment shaped by spontaneous deamination of 5-methylcytosin. The ancestral initial codon of rps8 is retained in the T. nupts, but its corresponding extant codon is mutated and requires C-to-U RNA-editing. These findings suggest that nupts can help recover scenarios of the nucleotide mutation process. We show that the Taxaceae nupts we retrieved may have been retained because the Cretaceous and they carry information of both ancestral genomic organization and nucleotide composition, which offer clues for understanding the plastome evolution in conifers.