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.

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.

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.

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.

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.

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.

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.

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.

Two Independent Plastid accD Transfers to the Nuclear Genome of Gnetum and Other Insights on Acetyl-CoA Carboxylase Evolution in Gymnosperms.

Acetyl-CoA carboxylase (ACCase) is the key regulator of fatty acid biosynthesis. In most plants, ACCase exists in two locations (cytosol and plastids) and in two forms (homomeric and heteromeric). Heteromeric ACCase comprises four subunits, three of them (ACCA-C) are nuclear encoded (nr) and the fourth (ACCD) is usually plastid encoded. Homomeric ACCase is encoded by a single nr-gene (ACC). We investigated the ACCase gene evolution in gymnosperms by examining the transcriptomes of newly sequenced Gnetum ula, combined with 75 transcriptomes and 110 plastomes of other gymnosperms. AccD-coding sequences are elongated through the insertion of repetitive DNA in four out of five cupressophyte families (except Sciadopityaceae) and were functionally transferred to the nucleus of gnetophytes and Sciadopitys. We discovered that, among the three genera of gnetophytes, only Gnetum has two copies of nr-accD. Furthermore, using protoplast transient expression assays, we experimentally verified that the nr-accD precursor proteins in Gnetum and Sciadopitys can be delivered to the plastids. Of the two nr-accD copies of Gnetum, one dually targets plastids and mitochondria, whereas the other potentially targets plastoglobuli. The distinct transit peptides, gene architectures, and flanking sequences between the two Gnetum accDs suggest that they have independent origins. Our findings are the first account of two distinctly targeted nr-accDs of any green plants and the most comprehensive analyses of ACCase evolution in gymnosperms to date.

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.

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.

Multiple measures could alleviate long-branch attraction in phylogenomic reconstruction of Cupressoideae (Cupressaceae).

Long-branch attraction (LBA) is a major obstacle in phylogenetic reconstruction. The phylogenetic relationships among Juniperus (J), Cupressus (C) and the Hesperocyparis-Callitropsis-Xanthocyparis (HCX) subclades of Cupressoideae are controversial. Our initial analyses of plastid protein-coding gene matrix revealed both J and C with much longer stem branches than those of HCX, so their sister relationships may be attributed to LBA. We used multiple measures including data filtering and modifying, evolutionary model selection and coalescent phylogenetic reconstruction to alleviate the LBA artifact. Data filtering by strictly removing unreliable aligned regions and removing substitution saturation genes and rapidly evolving sites could significantly reduce branch lengths of subclades J and C and recovered a relationship of J (C, HCX). In addition, using coalescent phylogenetic reconstruction could elucidate the LBA artifact and recovered J (C, HCX). However, some valid methods for other taxa were inefficient in alleviating the LBA artifact in J-C-HCX. Different strategies should be carefully considered and justified to reduce LBA in phylogenetic reconstruction of different groups. Three subclades of J-C-HCX were estimated to have experienced ancient rapid divergence within a short period, which could be another major obstacle in resolving relationships. Furthermore, our plastid phylogenomic analyses fully resolved the intergeneric relationships of Cupressoideae.