Integration of large and diverse angiosperm DNA fragments into Asian Gnetum mitogenomes.

BACKGROUND: Horizontal gene transfer (HGT) events have rarely been reported in gymnosperms. Gnetum is a gymnosperm genus comprising 25‒35 species sympatric with angiosperms in West African, South American, and Southeast Asian rainforests. Only a single acquisition of an angiosperm mitochondrial intron has been documented to date in Asian Gnetum mitogenomes. We wanted to develop a more comprehensive understanding of frequency and fragment length distribution of such events as well as their evolutionary history in this genus. RESULTS: We sequenced and assembled mitogenomes from five Asian Gnetum species. These genomes vary remarkably in size and foreign DNA content. We identified 15 mitochondrion-derived and five plastid-derived (MTPT) foreign genes. Our phylogenetic analyses strongly indicate that these foreign genes were transferred from diverse eudicots-mostly from the Rubiaceae genus Coptosapelta and ten genera of Malpighiales. This indicates that Asian Gnetum has experienced multiple independent HGT events. Patterns of sequence evolution strongly suggest DNA-mediated transfer between mitochondria as the primary mechanism giving rise to these HGT events. Most Asian Gnetum species are lianas and often entwined with sympatric angiosperms. We therefore propose that close apposition of Gnetum and angiosperm stems presents opportunities for interspecific cell-to-cell contact through friction and wounding, leading to HGT. CONCLUSIONS: Our study reveals that multiple HGT events have resulted in massive amounts of angiosperm mitochondrial DNA integrated into Asian Gnetum mitogenomes. Gnetum and its neighboring angiosperms are often entwined with each other, possibly accounting for frequent HGT between these two phylogenetically remote lineages.

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.

Contrasting Patterns in the Early Stage of SARS-CoV-2 Evolution between Humans and Minks.

One of the unique features of SARS-CoV-2 is its apparent neutral evolution during the early pandemic (before February 2020). This contrasts with the preceding SARS-CoV epidemics, where viruses evolved adaptively. SARS-CoV-2 may exhibit a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes before the onset of the current pandemic. To test the scenarios above, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parental humans. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratio per site in the spike protein is 2.93, indicating a selection process favoring adaptive amino acid changes. Mutations in the spike protein were concentrated within its receptor-binding domain and receptor-binding motif. An excess of high-frequency derived variants produced by genetic hitchhiking was found during the middle (June to July 2020) and late phase I (August to September 2020) of the mink epidemic. In contrast, the site frequency spectra of early SARS-CoV-2 in humans only show an excess of low-frequency mutations, consistent with the recent outbreak of the virus. Strong positive selection in the mink SARS-CoV-2 implies that the virus may not be preadapted to a wide range of hosts and illustrates how a virus evolves to establish a continuous infection in a new host. Therefore, the lack of positive selection signal during the early pandemic in humans deserves further investigation.

Detecting Genetic Ancestry and Adaptation in the Taiwanese Han People.

The Taiwanese people are composed of diverse indigenous populations and the Taiwanese Han. About 95% of the Taiwanese identify themselves as Taiwanese Han, but this may not be a homogeneous population because they migrated to the island from various regions of continental East Asia over a period of 400 years. Little is known about the underlying patterns of genetic ancestry, population admixture, and evolutionary adaptation in the Taiwanese Han people. Here, we analyzed the whole-genome single-nucleotide polymorphism genotyping data from 14,401 individuals of Taiwanese Han collected by the Taiwan Biobank and the whole-genome sequencing data for a subset of 772 people. We detected four major genetic ancestries with distinct geographic distributions (i.e., Northern, Southeastern, Japonic, and Island Southeast Asian ancestries) and signatures of population mixture contributing to the genomes of Taiwanese Han. We further scanned for signatures of positive natural selection that caused unusually long-range haplotypes and elevations of hitchhiked variants. As a result, we identified 16 candidate loci in which selection signals can be unambiguously localized at five single genes: CTNNA2, LRP1B, CSNK1G3, ASTN2, and NEO1. Statistical associations were examined in 16 metabolic-related traits to further elucidate the functional effects of each candidate gene. All five genes appear to have pleiotropic connections to various types of disease susceptibility and significant associations with at least one metabolic-related trait. Together, our results provide critical insights for understanding the evolutionary history and adaption of the Taiwanese Han population.

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.

Genetic Differentiation and Demographic Trajectory of the Insular Formosan and Orii's Flying Foxes.

Insular flying foxes are keystone species in island ecosystems due to their critical roles in plant pollination and seed dispersal. These species are vulnerable to population decline because of their small populations and low reproductive rates. The Formosan flying fox (Pteropus dasymallus formosus) is one of the 5 subspecies of the Ryukyu flying fox. Pteropus dasymallus formosus has suffered from a severe decline and is currently recognized as a critically endangered population in Taiwan. On the contrary, the Orii's flying fox (Pteropus dasymallus inopinatus) is a relatively stable population inhabiting Okinawa Island. Here, we applied a genomic approach called double digest restriction-site associated DNA sequencing to study these 2 subspecies for a total of 7 individuals. We detected significant genetic structure between the 2 populations. Despite their contrasting contemporary population sizes, both populations harbor very low degrees of genetic diversity. We further inferred their demographic history based on the joint folded site frequency spectrum and revealed that both P. d. formosus and P. d. inopinatus had maintained small population sizes for a long period of time after their divergence. Recently, these populations experienced distinct trajectories of demographic changes. While P. d. formosus suffered from a drastic ~10-fold population decline not long ago, P. d. inopinatus underwent a ~4.5-fold population expansion. Our results suggest separate conservation management for the 2 populations-population recovery is urgently needed for P. d. formosus while long-term monitoring for adverse genetic effects should be considered for P. d. inopinatus.

The origin and underlying driving forces of the SARS-CoV-2 outbreak.

BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

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.

Highly rearranged and size-variable chloroplast genomes in conifers II clade (cupressophytes): evolution towards shorter intergenic spacers.

Although conifers are of immense ecological and economic value, bioengineering of their chloroplasts remains undeveloped. Understanding the chloroplast genomic organization of conifers can facilitate their bioengineering. Members of the conifer II clade (or cupressophytes) are highly diverse in both morphologic features and chloroplast genomic organization. We compared six cupressophyte chloroplast genomes (cpDNAs) that represent four of the five cupressophyte families, including three genomes that are first reported here (Agathis dammara, Calocedrus formosana and Nageia nagi). The six cupressophyte cpDNAs have lost a pair of large inverted repeats (IRs) and vary greatly in size, organization and tRNA copies. We demonstrate that cupressophyte cpDNAs have evolved towards reduced size, largely due to shrunken intergenic spacers. In cupressophytes, cpDNA rearrangements are capable of extending intergenic spacers, and synonymous mutations are negatively associated with the size and frequency of rearrangements. The variable cpDNA sizes of cupressophytes may have been shaped by mutational burden and genomic rearrangements. On the basis of cpDNA organization, our analyses revealed that in gymnosperms, cpDNA rearrangements are phylogenetically informative, which supports the 'gnepines' clade. In addition, removal of a specific IR influences the minimal rearrangements required for the gnepines and cupressophyte clades, whereby Pinaceae favours the removal of IRB but cupressophytes exclusion of IRA. This result strongly suggests that different IR copies have been lost from conifers I and II. Our data help understand the complexity and evolution of cupressophyte cpDNAs.

Phylogenomics identifies parents of naturally occurring tetraploid bananas.

BACKGROUND: Triploid bananas are almost sterile. However, we succeeded in harvesting seeds from two edible triploid banana individuals (Genotype: ABB) in our conservation repository where various wild diploid bananas were also grown. The resulting rare offspring survived to seedling stages. DNA content analyses reveal that they are tetraploid. Since bananas contain maternally inherited plastids and paternally inherited mitochondria, we sequenced and assembled plastomes and mitogenomes of these seedlings to trace their hybridization history. RESULTS: The coding sequences of both organellar genomic scaffolds were extracted, aligned, and concatenated for constructing phylogenetic trees. Our results suggest that these tetraploid seedlings be derived from hybridization between edible triploid bananas and wild diploid Musa balbisiana (BB) individuals. We propose that generating female triploid gametes via apomeiosis may allow the triploid maternal bananas to produce viable seeds. CONCLUSIONS: Our study suggests a practical avenue towards expanding genetic recombination and increasing genetic diversity of banana breeding programs. Further cellular studies are needed to understand the fusion and developmental processes that lead to formation of hybrid embryos in banana reproduction, polyploidization, and evolution.

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.

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.

Editing site analysis in a gymnosperm mitochondrial genome reveals similarities with angiosperm mitochondrial genomes.

Sequence analysis of organelle genomes and comprehensive analysis of C-to-U editing sites from flowering and non-flowering plants have provided extensive sequence information from diverse taxa. This study includes the first comprehensive analysis of RNA editing sites from a gymnosperm mitochondrial genome, and utilizes informatics analyses to determine conserved features in the RNA sequence context around editing sites. We have identified 565 editing sites in 21 full-length and 4 partial cDNAs of the 39 protein-coding genes identified from the mitochondrial genome of Cycas taitungensis. The information profiles and RNA sequence context of C-to-U editing sites in the Cycas genome exhibit similarity in the immediate flanking nucleotides. Relative entropy analyses indicate that similar regions in the 5' flanking 20 nucleotides have information content compared to angiosperm mitochondrial genomes. These results suggest that evolutionary constraints exist on the nucleotide sequences immediately adjacent to C-to-U editing sites, and similar regions are utilized in editing site recognition.

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.

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 mitochondrial genome of the gymnosperm Cycas taitungensis contains a novel family of short interspersed elements, Bpu sequences, and abundant RNA editing sites.

The mtDNA of Cycas taitungensis is a circular molecule of 414,903 bp, making it 2- to 6-fold larger than the known mtDNAs of charophytes and bryophytes, but similar to the average of 7 elucidated angiosperm mtDNAs. It is characterized by abundant RNA editing sites (1,084), more than twice the number found in the angiosperm mtDNAs. The A + T content of Cycas mtDNA is 53.1%, the lowest among known land plants. About 5% of the Cycas mtDNA is composed of a novel family of mobile elements, which we designated as "Bpu sequences." They share a consensus sequence of 36 bp with 2 terminal direct repeats (AAGG) and a recognition site for the Bpu 10I restriction endonuclease (CCTGAAGC). Comparison of the Cycas mtDNA with other plant mtDNAs revealed many new insights into the biology and evolution of land plant mtDNAs. For example, the noncoding sequences in mtDNAs have drastically expanded as land plants have evolved, with abrupt increases appearing in the bryophytes, and then in the seed plants. As a result, the genomic organizations of seed plant mtDNAs are much less compact than in other plants. Also, the Cycas mtDNA appears to have been exempted from the frequent gene loss observed in angiosperm mtDNAs. Similar to the angiosperms, the 3 Cycas genes nad1, nad2, and nad5 are disrupted by 5 group II intron squences, which have brought the genes into trans-splicing arrangements. The evolutionary origin and invasion/duplication mechanism of the Bpu sequences in Cycas mtDNA are hypothesized and discussed.

Evolution of reduced and compact chloroplast genomes (cpDNAs) in gnetophytes: selection toward a lower-cost strategy.

The cpDNA of Welwitschia mirabilis (the only species of Welwitschiales) was recently reported to be the most reduced and compact among photosynthetic land plants. However, cpDNAs of the other two gnetophyte lineages (viz. Ephedrales and Gnetales) have not yet been studied. It remains unclear what underlining mechanisms have downsized the cpDNA. To pin down major factors for cpDNA reduction and compaction in gnetophytes, we have determined 4 complete cpDNAs, including one from each of the 3 gnetophyte orders, Ephedra equisetina, Gnetum parvifolium, and W. mirabilis, and one from the non-Pinus Pinaceae, Keteleeria davidiana. We report that the cpDNAs of E. equisetina (109,518bp) and G.parvifolium (114,914bp) are not only smaller but more compact than that of W. mirabilis (118,919bp). The gnetophyte cpDNAs have commonly lost at least 18 genes that are retained in other seed plants. Furthermore, they have significantly biased usages of AT-rich codons and shorter introns and intergenic spaces, which are largely due to more deletions at inter-operon than intra-operon spaces and removal of segment sequences rather than single-nucleotides. We show that the reduced gnetophyte cpDNAs clearly resulted from selection for economy by deletions of genes and non-coding sequences, which then led to the compactness and the accelerated substitution rates. The smallest C-values in gnetophyte nuclear DNAs and the competitive or resource-poor situations encountered by gnetophytes further suggest a critical need for an economic strategy.