Extensive Shifts from Cis- to Trans-splicing of Gymnosperm Mitochondrial Introns.

Hundreds of plant mitogenomes have been sequenced from angiosperms, but relatively few mitogenomes are available from its sister lineage, gymnosperms. To examine mitogenomic diversity among extant gymnosperms, we generated draft mitogenomes from 11 diverse species and compared them with four previously published mitogenomes. Examined mitogenomes from Pinaceae and cycads retained all 41 protein genes and 26 introns present in the common ancestor of seed plants, whereas gnetophyte and cupressophyte mitogenomes experienced extensive gene and intron loss. In Pinaceae and cupressophyte mitogenomes, an unprecedented number of exons are distantly dispersed, requiring trans-splicing of 50-70% of mitochondrial introns to generate mature transcripts. RNAseq data confirm trans-splicing of these dispersed exons in Pinus. The prevalence of trans-splicing in vascular plant lineages with recombinogenic mitogenomes suggests that genomic rearrangement is the primary cause of shifts from cis- to trans-splicing in plant mitochondria.

Plastomes from tribe Plantagineae (Plantaginaceae) reveal infrageneric structural synapormorphies and localized hypermutation for Plantago and functional loss of ndh genes from Littorella.

Tribe Plantagineae (Plantaginaceae) comprises ~ 270 species in three currently recognized genera (Aragoa, Littorella, Plantago), of which Plantago is most speciose. Plantago plastomes exhibit several atypical features including large inversions, expansions of the inverted repeat, increased repetitiveness, intron losses, and gene-specific increases in substitution rate, but the prevalence of these plastid features among species and subgenera is unknown. To assess phylogenetic relationships and plastomic evolutionary dynamics among Plantagineae genera and Plantago subgenera, we generated 25 complete plastome sequences and compared them with existing plastome sequences from Plantaginaceae. Using whole plastome and partitioned alignments, our phylogenomic analyses provided strong support for relationships among major Plantagineae lineages. General plastid features-including size, GC content, intron content, and indels-provided additional support that reinforced major Plantagineae subdivisions. Plastomes from Plantago subgenera Plantago and Coronopus have synapomorphic expansions and inversions affecting the size and gene order of the inverted repeats, and particular genes near the inversion breakpoints exhibit accelerated nucleotide substitution rates, suggesting localized hypermutation associated with rearrangements. The Littorella plastome lacks functional copies of ndh genes, which may be related to an amphibious lifestyle and partial reliance on CAM photosynthesis.

High and Variable Rates of Repeat-Mediated Mitochondrial Genome Rearrangement in a Genus of Plants.

For 30 years, it has been clear that angiosperm mitochondrial genomes evolve rapidly in sequence arrangement (i.e., synteny), yet absolute rates of rearrangement have not been measured in any plant group, nor is it known how much these rates vary. To investigate these issues, we sequenced and reconstructed the rearrangement history of seven mitochondrial genomes in Monsonia (Geraniaceae). We show that rearrangements (occurring mostly as inversions) not only take place at generally high rates in these genomes but also uncover significant variation in rearrangement rates. For example, the hyperactive mitochondrial genome of Monsonia ciliata has accumulated at least 30 rearrangements over the last million years, whereas the branch leading to M. ciliata and its sister species has sustained rearrangement at a rate that is at least ten times lower. Furthermore, our analysis of published data shows that rates of mitochondrial genome rearrangement in seed plants vary by at least 600-fold. We find that sites of rearrangement are highly preferentially located in very close proximity to repeated sequences in Monsonia. This provides strong support for the hypothesis that rearrangement in angiosperm mitochondrial genomes occurs largely through repeat-mediated recombination. Because there is little variation in the amount of repeat sequence among Monsonia genomes, the variable rates of rearrangement in Monsonia probably reflect variable rates of mitochondrial recombination itself. Finally, we show that mitochondrial synonymous substitutions occur in a clock-like manner in Monsonia; rates of mitochondrial substitutions and rearrangements are therefore highly uncoupled in this group.

Ginkgo and Welwitschia Mitogenomes Reveal Extreme Contrasts in Gymnosperm Mitochondrial Evolution.

Mitochondrial genomes (mitogenomes) of flowering plants are well known for their extreme diversity in size, structure, gene content, and rates of sequence evolution and recombination. In contrast, little is known about mitogenomic diversity and evolution within gymnosperms. Only a single complete genome sequence is available, from the cycad Cycas taitungensis, while limited information is available for the one draft sequence, from Norway spruce (Picea abies). To examine mitogenomic evolution in gymnosperms, we generated complete genome sequences for the ginkgo tree (Ginkgo biloba) and a gnetophyte (Welwitschia mirabilis). There is great disparity in size, sequence conservation, levels of shared DNA, and functional content among gymnosperm mitogenomes. The Cycas and Ginkgo mitogenomes are relatively small, have low substitution rates, and possess numerous genes, introns, and edit sites; we infer that these properties were present in the ancestral seed plant. By contrast, the Welwitschia mitogenome has an expanded size coupled with accelerated substitution rates and extensive loss of these functional features. The Picea genome has expanded further, to more than 4 Mb. With regard to structural evolution, the Cycas and Ginkgo mitogenomes share a remarkable amount of intergenic DNA, which may be related to the limited recombinational activity detected at repeats in Ginkgo Conversely, the Welwitschia mitogenome shares almost no intergenic DNA with any other seed plant. By conducting the first measurements of rates of DNA turnover in seed plant mitogenomes, we discovered that turnover rates vary by orders of magnitude among species.

Complete mitochondrial genomes from the ferns Ophioglossum californicum and Psilotum nudum are highly repetitive with the largest organellar introns.

Currently, complete mitochondrial genomes (mitogenomes) are available from all major land plant lineages except ferns. Sequencing of fern mitogenomes could shed light on the major evolutionary transitions that established mitogenomic diversity among extant lineages. In this study, we generated complete mitogenomes from the adder's tongue fern (Ophioglossum californicum) and the whisk fern (Psilotum nudum). The Psilotum mitogenome (628 kb) contains a rich complement of genes and introns, some of which are the largest of any green plant organellar genome. In the Ophioglossum mitogenome (372 kb), gene and intron content is slightly reduced, including the loss of all four mitochondrial ccm genes. Transcripts of nuclear Ccm genes also were not detected, suggesting loss of the entire mitochondrial cytochrome c maturation pathway from Ophioglossum. Both fern mitogenomes are highly repetitive, yet they show extremely low levels of active recombination. Transcriptomic sequencing uncovered ˜1000 sites of C-to-U RNA editing in both species, plus a small number (< 60) of U-to-C edit sites. Overall, the first mitochondrial genomes of ferns show a mix of features shared with lycophytes and/or seed plants and several novel genomic features, enabling a robust reconstruction of the mitogenome in the common ancestor of vascular plants.

Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates.

Rates of nucleotide substitution were previously shown to be several times slower in the plastid inverted repeat (IR) compared with single-copy (SC) regions, suggesting that the IR provides enhanced copy-correction activity. To examine the generality of this synonymous rate dependence on the IR, we compared plastomes from 69 pairs of closely related species representing 52 families of angiosperms, gymnosperms, and ferns. We explored the breadth of IR boundary shifts in land plants and demonstrate that synonymous substitution rates are, on average, 3.7 times slower in IR genes than in SC genes. In addition, genes moved from the SC into the IR exhibit lower synonymous rates consistent with other IR genes, while genes moved from the IR into the SC exhibit higher rates consistent with other SC genes. Surprisingly, however, several plastid genes from Pelargonium, Plantago, and Silene have highly accelerated synonymous rates despite their IR localization. Together, these results provide strong evidence that the duplicative nature of the IR reduces the substitution rate within this region. The anomalously fast-evolving genes in Pelargonium, Plantago, and Silene indicate localized hypermutation, potentially induced by a higher level of error-prone double-strand break repair in these regions, which generates substitutional rate variation.

Unprecedented heterogeneity in the synonymous substitution rate within a plant genome.

The synonymous substitution rate varies widely among species, but it is generally quite stable within a genome due to the absence of strong selective pressures. In plants, plastid genes tend to evolve faster than mitochondrial genes, rate variation among species generally correlates between the mitochondrial and plastid genomes, and few examples of intragenomic rate heterogeneity exist. To study the extent of substitution rate variation between and within plant organellar genomes, we sequenced the complete mitochondrial and plastid genomes from the bugleweed, Ajuga reptans, which was previously shown to exhibit rate heterogeneity for several mitochondrial genes. Substitution rates were accelerated specifically in the mitochondrial genome, which contrasts with correlated plastid and mitochondrial rate changes in most other angiosperms. Strikingly, we uncovered a 340-fold range of synonymous substitution rate variation among Ajuga mitochondrial genes. This is by far the largest amount of synonymous rate heterogeneity ever reported for a genome, but the evolutionary forces driving this phenomenon are unclear. Selective effects on synonymous sites in plant mitochondria are generally weak and thus unlikely to generate such unprecedented intragenomic rate heterogeneity. Quickly evolving genes are not clustered in the genome, arguing against localized hypermutation, although it is possible that they were clustered ancestrally given the high rate of genomic rearrangement in plant mitochondria. Mutagenic retroprocessing, involving error-prone reverse transcription and genomic integration of mature transcripts, is hypothesized as another potential explanation.

Complete plastid genomes from Ophioglossum californicum, Psilotum nudum, and Equisetum hyemale reveal an ancestral land plant genome structure and resolve the position of Equisetales among monilophytes.

BACKGROUND: Plastid genome structure and content is remarkably conserved in land plants. This widespread conservation has facilitated taxon-rich phylogenetic analyses that have resolved organismal relationships among many land plant groups. However, the relationships among major fern lineages, especially the placement of Equisetales, remain enigmatic. RESULTS: In order to understand the evolution of plastid genomes and to establish phylogenetic relationships among ferns, we sequenced the plastid genomes from three early diverging species: Equisetum hyemale (Equisetales), Ophioglossum californicum (Ophioglossales), and Psilotum nudum (Psilotales). A comparison of fern plastid genomes showed that some lineages have retained inverted repeat (IR) boundaries originating from the common ancestor of land plants, while other lineages have experienced multiple IR changes including expansions and inversions. Genome content has remained stable throughout ferns, except for a few lineage-specific losses of genes and introns. Notably, the losses of the rps16 gene and the rps12i346 intron are shared among Psilotales, Ophioglossales, and Equisetales, while the gain of a mitochondrial atp1 intron is shared between Marattiales and Polypodiopsida. These genomic structural changes support the placement of Equisetales as sister to Ophioglossales + Psilotales and Marattiales as sister to Polypodiopsida. This result is augmented by some molecular phylogenetic analyses that recover the same relationships, whereas others suggest a relationship between Equisetales and Polypodiopsida. CONCLUSIONS: Although molecular analyses were inconsistent with respect to the position of Marattiales and Equisetales, several genomic structural changes have for the first time provided a clear placement of these lineages within the ferns. These results further demonstrate the power of using rare genomic structural changes in cases where molecular data fail to provide strong phylogenetic resolution.

Evolution of plant mitochondrial intron-encoded maturases: frequent lineage-specific loss and recurrent intracellular transfer to the nucleus.

Among land plants, mitochondrial and plastid group II introns occasionally encode proteins called maturases that are important for splicing. Angiosperm nuclear genomes also encode maturases that are targeted to the organelles, but it is not known whether nucleus-encoded maturases exist in other land plant lineages. To examine the evolutionary diversity and history of this essential gene family, we searched for maturase homologs in recently sequenced nuclear and mitochondrial genomes from diverse land plants. We found that maturase content in mitochondrial genomes is highly lineage specific, such that orthologous maturases are rarely shared among major land plant groups. The presence of numerous mitochondrial pseudogenes in the mitochondrial genomes of several species implies that the sporadic maturase distribution is due to frequent inactivation and eventual loss over time. We also identified multiple maturase paralogs in the nuclear genomes of the lycophyte Selaginella moellendorffii, the moss Physcomitrella patens, and the representative angiosperm Vitis vinifera. Phylogenetic analyses of organelle- and nucleus-encoded maturases revealed that the nuclear maturase genes in angiosperms, lycophytes, and mosses arose by multiple shared and independent transfers of mitochondrial paralogs to the nuclear genome during land plant evolution. These findings indicate that plant mitochondrial maturases have experienced a surprisingly dynamic history due to a complex interaction of multiple evolutionary forces that affect the rates of maturase gain, retention, and loss.

Clinical application value of metagenomic next-generation sequencing in the diagnosis of central nervous system infections.

Objectives: The purpose of this study is to evaluate the clinical application value of metagenomic next-generation sequencing (mNGS) in central nervous system (CNS) infections. Methods: Both mNGS and routine examination of cerebrospinal fluid (CSF) samples from patients with CNS infections retrospectively analyzed the efficacy of mNGS in this cohort and were ultimately compared with a clinical diagnosis. Results: A total of 94 cases consistent with CNS infections were included in the analysis. The positive rate for mNGS is 60.6% (57/94), which is significantly higher than 20.2% (19/94; p < 0.01) detected using conventional methods. mNGS detected 21 pathogenic strains that could not be detected by routine testing. Routine tests were positive for two pathogens but negative for mNGS. The sensitivity and specificity of mNGS in the diagnosis of central nervous system infections were 89.5% and 44%, respectively, when compared with traditional tests. At discharge, 20 (21.3%) patients were cured, 55 (58.5%) patients showed improvements, five (5.3%) patients did not recover, and two (2.1%) patients died. Conclusion: mNGS has unique advantages in the diagnosis of central nervous system infections. mNGS tests can be performed when patients are clinically suspected of having a central nervous system infection but no pathogenic evidence.

Metagenomic next-generation sequencing provides prognostic warning by identifying mixed infections in nocardiosis.

Nocardia is an opportunistic pathogen that mainly involves immunosuppressed patients and causes a high mortality rate. As an emerging approach to detect infectious pathogens, metagenomic next-generation sequencing (mNGS) was reported in the detection of Nocardia. However, there is no evidence demonstrating the effect of mNGS on the prognosis of Nocardia infection. In this retrospective study, we included 18 nocardiosis patients. Nocardia species were detected by mNGS from their clinical samples. All the patients were diagnosed with nocardiosis by clinical experts through a comprehensive evaluation. Of these 18 patients, fever is the most frequent initial symptom. Compared to traditional culture methods, mNGS provides a faster turnaround time (TAT) and higher sensitivity. Pulmonary nocardiosis was the most common clinical presentation in the study. mNGS detected 13 types of Nocardia species, of which Nocardia abscessus and Nocardia cyriacigeorgica were the most common species. The study's most noteworthy discovery is that mNGS outperforms culture at detecting mixed infections (more than one pathogen detected in one clinical specimen, including bacteria, fungi, and excluding virus), and number of infectious species was an independent risk factor for nocardiosis patients' prognostics after adjusting age, ICU days, gender and underlying diseases (adjusted HR = 1.47, 95% CI: 1.09-1.98, p = 0.011). As a result, we believe that by detecting mixed infections (more than one pathogenic species), mNGS can provide a clinical risk warning for the prognosis of nocardiosis.

Molecular evolution of candidate sour taste receptor gene PKD1L3 in mammals.

The PKD1L3 gene encodes an ion channel protein that can interact with the PKD2L1 protein to form a candidate sour taste receptor. In the present study, we have analyzed the evolutionary patterns of PKD1L3 genes from 10 mammalian species. The results showed that PKD1L3 genes have evolved under a dominant purifying selection force. However, for some branches and sites, PKD1L3 genes were detected to have been operated by positive selection. Moreover, some of these positive evolutionary sites are likely to participate in acid stimulus recognition. In rodents, PKD1L3 genes evolved more rapidly than other mammalian lineages. Combined with other functional research reports, our results suggest that rodents may not be the most appropriate model for functional research on the PKD1L3 gene.

Molecular evolution of PKD2 gene family in mammals.

PKD2 gene encodes a critical cation channel protein that plays important roles in various developmental processes and is usually evolutionarily conserved. In the present study, we analyzed the evolutionary patterns of PKD2 and its homologous genes (PKD2L1, PKD2L2) from nine mammalian species. In this study, we demonstrated the orthologs of PKD2 gene family evolved under a dominant purifying selection force. Our results in combination with the reported evidences from functional researches suggested the entire PKD2 gene family are conserved and perform essential biological roles during mammalian evolution. In rodents, PKD2 gene family members appeared to have evolved more rapidly than other mammalian lineages, probably resulting from relaxation of purifying selection. However, positive selection imposed on synonymous sites also potentially contributed to this case. For the paralogs, our results implied that PKD2L2 genes evolved under a weaker purifying selection constraint than PKD2 and PKD2L1 genes. Interestingly, some loop regions of transmembrane domain of PKD2L2 exhibited higher P (N)/P (S) ratios than expected, suggesting these regions are more functional divergent in organisms and worthy of special attention.

Complete loss of RNA editing from the plastid genome and most highly expressed mitochondrial genes of Welwitschia mirabilis.

Comparative genomics among gymnosperms suggested extensive loss of mitochondrial RNA editing sites from Welwitschia mirabilis based on predictive analysis. However, empirical or transcriptome data to confirm this massive loss event are lacking, and the potential mechanisms of RNA site loss are unclear. By comparing genomic sequences with transcriptomic and reverse-transcription PCR sequencing data, we performed a comprehensive analysis of the pattern of RNA editing in the mitochondrial and plastid genomes (mitogenome and plastome, respectively) of W. mirabilis and a second gymnosperm, Ginkgo biloba. For W. mirabilis, we found only 99 editing sites located in 13 protein-coding genes in the mitogenome and a complete loss of RNA editing from the plastome. The few genes having high editing frequency in the Welwitschia mitogenome showed a strong negative correlation with gene expression level. Comparative analyses with G. biloba, containing 1,405 mitochondrial and 345 plastid editing sites, revealed that the editing loss from W. mirabilis is mainly due to the substitution of editable cytidines to thymidines at the genomic level, which could be caused by retroprocessing. Our result is the first study to uncover massive editing loss from both the mitogenome and plastome in a single genus. Furthermore, our results suggest that gene expression level and retroprocessing both contributed to the evolution of RNA editing in plant organellar genomes.

Multiple origins of endosymbionts in Chlorellaceae with no reductive effects on the plastid or mitochondrial genomes.

Ancient endosymbiotic relationships have led to extreme genomic reduction in many bacterial and eukaryotic algal endosymbionts. Endosymbionts in more recent and/or facultative relationships can also experience genomic reduction to a lesser extent, but little is known about the effects of the endosymbiotic transition on the organellar genomes of eukaryotes. To understand how the endosymbiotic lifestyle has affected the organellar genomes of photosynthetic green algae, we generated the complete plastid genome (plastome) and mitochondrial genome (mitogenome) sequences from three green algal endosymbionts (Chlorella heliozoae, Chlorella variabilis and Micractinium conductrix). The mitogenomes and plastomes of the three newly sequenced endosymbionts have a standard set of genes compared with free-living trebouxiophytes, providing no evidence for functional genomic reduction. Instead, their organellar genomes are generally larger and more intron rich. Intron content is highly variable among the members of Chlorella, suggesting very high rates of gain and/or loss of introns during evolution. Phylogenetic analysis of plastid and mitochondrial genes demonstrated that the three endosymbionts do not form a monophyletic group, indicating that the endosymbiotic lifestyle has evolved multiple times in Chlorellaceae. In addition, M. conductrix is deeply nested within the Chlorella clade, suggesting that taxonomic revision is needed for one or both genera.

Variable frequency of plastid RNA editing among ferns and repeated loss of uridine-to-cytidine editing from vascular plants.

The distinct distribution and abundance of C-to-U and U-to-C RNA editing among land plants suggest that these two processes originated and evolve independently, but the paucity of information from several key lineages limits our understanding of their evolution. To examine the evolutionary diversity of RNA editing among ferns, we sequenced the plastid transcriptomes from two early diverging species, Ophioglossum californicum and Psilotum nudum. Using a relaxed automated approach to minimize false negatives combined with manual inspection to eliminate false positives, we identified 297 C-to-U and three U-to-C edit sites in the O. californicum plastid transcriptome but only 27 C-to-U and no U-to-C edit sites in the P. nudum plastid transcriptome. A broader comparison of editing content with the leptosporangiate fern Adiantum capillus-veneris and the hornwort Anthoceros formosae uncovered large variance in the abundance of plastid editing, indicating that the frequency and type of RNA editing is highly labile in ferns. Edit sites that increase protein conservation among species are more abundant and more efficiently edited than silent and non-conservative sites, suggesting that selection maintains functionally important editing. The absence of U-to-C editing from P. nudum plastid transcripts and other vascular plants demonstrates that U-to-C editing loss is a recurrent phenomenon in vascular plant evolution.

Predominant and substoichiometric isomers of the plastid genome coexist within Juniperus plants and have shifted multiple times during cupressophyte evolution.

Most land plant plastomes contain two copies of a large inverted repeat (IR) that promote high-frequency homologous recombination to generate isomeric genomic forms. Among conifer plastomes, this canonical IR is highly reduced in Pinaceae and completely lost from cupressophytes. However, both lineages have acquired short, novel IRs, some of which also exhibit recombinational activity to generate genomic structural diversity. This diversity has been shown to exist between, and occasionally within, cupressophyte species, but it is not known whether multiple genomic forms coexist within individual plants. To examine the recombinational potential of the novel cupressophyte IRs within individuals and between species, we sequenced the plastomes of four closely related species of Juniperus. The four plastomes have identical gene content and genome organization except for a large 36 kb inversion between approximately 250 bp IR containing trnQ-UUG. Southern blotting showed that different isomeric versions of the plastome predominate among individual junipers, whereas polymerase chain reaction and high-throughput read-pair mapping revealed the substoichiometric presence of the alternative isomeric form within each individual plant. Furthermore, our comparative genomic studies demonstrate that the predominant and substoichiometric arrangements of this IR have changed several times in other cupressophytes as well. These results provide compelling evidence for substoichiometric shifting of plastomic forms during cupressophyte evolution and suggest that substoichiometric shifting activity in plastid genomes may be adaptive.