The genome and population genomics of allopolyploid Coffea arabica reveal the diversification history of modern coffee cultivars.

Coffea arabica, an allotetraploid hybrid of Coffea eugenioides and Coffea canephora, is the source of approximately 60% of coffee products worldwide, and its cultivated accessions have undergone several population bottlenecks. We present chromosome-level assemblies of a di-haploid C. arabica accession and modern representatives of its diploid progenitors, C. eugenioides and C. canephora. The three species exhibit largely conserved genome structures between diploid parents and descendant subgenomes, with no obvious global subgenome dominance. We find evidence for a founding polyploidy event 350,000-610,000 years ago, followed by several pre-domestication bottlenecks, resulting in narrow genetic variation. A split between wild accessions and cultivar progenitors occurred ~30.5 thousand years ago, followed by a period of migration between the two populations. Analysis of modern varieties, including lines historically introgressed with C. canephora, highlights their breeding histories and loci that may contribute to pathogen resistance, laying the groundwork for future genomics-based breeding of C. arabica.

Recent advances in the culture-independent discovery of natural products using metagenomic approaches.

Natural products derived from bacterial sources have long been pivotal in the discovery of drug leads. However, the cultivation of only about 1% of bacteria in laboratory settings has left a significant portion of biosynthetic diversity hidden within the genomes of uncultured bacteria. Advances in sequencing technologies now enable the exploration of genetic material from these metagenomes through culture-independent methods. This approach involves extracting genetic sequences from environmental DNA and applying a hybrid methodology that combines functional screening, sequence tag-based homology screening, and bioinformatic-assisted chemical synthesis. Through this process, numerous valuable natural products have been identified and synthesized from previously uncharted metagenomic territories. This paper provides an overview of the recent advancements in the utilization of culture-independent techniques for the discovery of novel biosynthetic gene clusters and bioactive small molecules within metagenomic libraries.

Population genomics of post-glacial western Eurasia.

Western Eurasia witnessed several large-scale human migrations during the Holocene1-5. Here, to investigate the cross-continental effects of these migrations, we shotgun-sequenced 317 genomes-mainly from the Mesolithic and Neolithic periods-from across northern and western Eurasia. These were imputed alongside published data to obtain diploid genotypes from more than 1,600 ancient humans. Our analyses revealed a 'great divide' genomic boundary extending from the Black Sea to the Baltic. Mesolithic hunter-gatherers were highly genetically differentiated east and west of this zone, and the effect of the neolithization was equally disparate. Large-scale ancestry shifts occurred in the west as farming was introduced, including near-total replacement of hunter-gatherers in many areas, whereas no substantial ancestry shifts happened east of the zone during the same period. Similarly, relatedness decreased in the west from the Neolithic transition onwards, whereas, east of the Urals, relatedness remained high until around 4,000 BP, consistent with the persistence of localized groups of hunter-gatherers. The boundary dissolved when Yamnaya-related ancestry spread across western Eurasia around 5,000 BP, resulting in a second major turnover that reached most parts of Europe within a 1,000-year span. The genetic origin and fate of the Yamnaya have remained elusive, but we show that hunter-gatherers from the Middle Don region contributed ancestry to them. Yamnaya groups later admixed with individuals associated with the Globular Amphora culture before expanding into Europe. Similar turnovers occurred in western Siberia, where we report new genomic data from a 'Neolithic steppe' cline spanning the Siberian forest steppe to Lake Baikal. These prehistoric migrations had profound and lasting effects on the genetic diversity of Eurasian populations.

The first ITS2 sequence data set of eDNA from honey of Malaysian giant honeybees (Apis dorsata) and stingless bees (Heterotrigona itama) reveals plant species diversity.

OBJECTIVES: Pollen is a useful tool for identifying the provenance and complex ecosystems surrounding honey production in Malaysian forests. As native key pollinators in Malaysia, Apis dorsata and Heterotrigona itama forage on various plant/pollen species to collect honey. This study aims to generate a dataset that uncovers the presence of these plant/pollen species and their relative abundance in the honey of A. dorsata and H. itama. The information gathered from this study can be used to determine the geographical and botanical origin and authenticity of the honey produced by these two species. RESULTS: Sequence data were obtained for both A. dorsata and H. itama. The raw sequence data for A. dorsata was 5 Mb, which was assembled into 5 contigs with a size of 6,098,728 bp, an N50 of 15,534, and a GC average of 57.42. Similarly, the raw sequence data for H. itama was 6.3 Mb, which was assembled into 11 contigs with a size of 7,642,048 bp, an N50 of 17,180, and a GC average of 55.38. In the honey sample of A. dorsata, we identified five different plant/pollen species, with only one of the five species exhibiting a relative abundance of less than 1%. For H. itama, we identified seven different plant/pollen species, with only three of the species exhibiting a relative abundance of less than 1%. All of the identified plant species were native to Peninsular Malaysia, especially the East Coast area of Terengganu. DATA DESCRIPTION: Our data offers valuable insights into honey's geographical and botanical origin and authenticity. Metagenomic studies could help identify the plant species that honeybees forage and provide preliminary data for researchers studying the biological development of A. dorsata and H. itama. The identification of various flowers from the eDNA of honey that are known for their medicinal properties could aid in regional honey with accurate product origin labeling, which is crucial for guaranteeing product authenticity to consumers.

Comparative and population genomics of buckwheat species reveal key determinants of flavor and fertility.

Common buckwheat (Fagopyrum esculentum) is an ancient crop with a world-wide distribution. Due to its excellent nutritional quality and high economic and ecological value, common buckwheat is becoming increasingly important throughout the world. The availability of a high-quality reference genome sequence and population genomic data will accelerate the breeding of common buckwheat, but the high heterozygosity due to the outcrossing nature has greatly hindered the genome assembly. Here we report the assembly of a chromosome-scale high-quality reference genome of F. esculentum var. homotropicum, a homozygous self-pollinating variant of common buckwheat. Comparative genomics revealed that two cultivated buckwheat species, common buckwheat (F. esculentum) and Tartary buckwheat (F. tataricum), underwent metabolomic divergence and ecotype differentiation. The expansion of several gene families in common buckwheat, including FhFAR genes, is associated with its wider distribution than Tartary buckwheat. Copy number variation of genes involved in the metabolism of flavonoids is associated with the difference of rutin content between common and Tartary buckwheat. Furthermore, we present a comprehensive atlas of genomic variation based on whole-genome resequencing of 572 accessions of common buckwheat. Population and evolutionary genomics reveal genetic variation associated with environmental adaptability and floral development between Chinese and non-Chinese cultivated groups. Genome-wide association analyses of multi-year agronomic traits with the content of flavonoids revealed that Fh05G014970 is a potential major regulator of flowering period, a key agronomic trait controlling the yield of outcrossing crops, and that Fh06G015130 is a crucial gene underlying flavor-associated flavonoids. Intriguingly, we found that the gene translocation and sequence variation of FhS-ELF3 contribute to the homomorphic self-compatibility of common buckwheat. Collectively, our results elucidate the genetic basis of speciation, ecological adaptation, fertility, and unique flavor of common buckwheat, and provide new resources for future genomics-assisted breeding of this economically important crop.

Enhanced biological phosphorus removal by high concentration powder carrier bio-fluidized bed (HPB): Phosphorus distribution, cyclone separation, and metagenomics.

This study provides a comparative investigation of phosphorus removal between anaerobic-anoxic-oxic (AAO) and high-concentration powder carrier bio-fluidized bed (HPB) in the same full-scale wastewater treatment plant. The results showed that the total phosphorus removal of HPB was 71.45%-96.71%. Compared with AAO, the total phosphorus removal of HPB can be increased by a maximum of 15.73%. The mechanisms of enhanced phosphorus removal by HPB include the followings. Biological phosphorus removal was significant. The anaerobic phosphorus release capacity of HPB was enhanced and polyphosphate (Poly-P) in the excess sludge of HPB was 1.5 times higher than that of AAO. The relative abundance of Candidatus Accumulibacter was 5 times higher than that of AAO, and oxidative phosphorylation and butanoate metabolism were enhanced. The analysis of phosphorus distribution showed that cyclone separation increased the chemical phosphorus precipitation (Chem-P) in the excess sludge by 16.96% to avoid accumulation in the biochemical tank. The phosphorus adsorbed by extracellular polymeric substance (EPS) in the recycled sludge was stripped, and the EPS bound-P in the excess sludge increased by 1.5 times. This study demonstrated the feasibility of HPB to improve the phosphorus removal efficiency for domestic wastewater.

Chromosome-level genome assembly and population genomics of Robinia pseudoacacia reveal the genetic basis for its wide cultivation.

Urban greening provides important ecosystem services and ideal places for urban recreation and is a serious consideration for municipal decision-makers. Among the tree species cultivated in urban green spaces, Robinia pseudoacacia stands out due to its attractive flowers, fragrances, high trunks, wide adaptability, and essential ecosystem services. However, the genomic basis and consequences of its wide-planting in urban green spaces remains unknown. Here, we report the chromosome-level genome assembly of R. pseudoacacia, revealing a genome size of 682.4 Mb and 33,187 protein-coding genes. More than 99.3% of the assembly is anchored to 11 chromosomes with an N50 of 59.9 Mb. Comparative genomic analyses among 17 species reveal that gene families related to traits favoured by urbanites, such as wood formation, biosynthesis, and drought tolerance, are notably expanded in R. pseudoacacia. Our population genomic analyses further recover 11 genes that are under recent selection. Ultimately, these genes play important roles in the biological processes related to flower development, water retention, and immunization. Altogether, our results reveal the evolutionary forces that shape R. pseudoacacia cultivated for urban greening. These findings also present a valuable foundation for the future development of agronomic traits and molecular breeding strategies for R. pseudoacacia.

Anaerobic selenite-reducing bacteria and their metabolic potentials in Se-rich sediment revealed by the combination of DNA-stable isotope probing, metagenomic binning, and metatranscriptomics.

Microorganisms play a critical role in the biogeochemical cycling of selenium (Se) in aquatic environments, particularly in reducing the toxicity and bioavailability of selenite (Se(IV)). This study aimed to identify putative Se(IV)-reducing bacteria (SeIVRB) and investigate the genetic mechanisms underlying Se(IV) reduction in anoxic Se-rich sediment. Initial microcosm incubation confirmed that Se(IV) reduction was driven by heterotrophic microorganisms. DNA stable-isotope probing (DNA-SIP) analysis identified Pseudomonas, Geobacter, Comamonas, and Anaeromyxobacter as putative SeIVRB. High-quality metagenome-assembled genomes (MAGs) affiliated with these four putative SeIVRB were retrieved. Annotation of functional gene indicated that these MAGs contained putative Se(IV)-reducing genes such as DMSO reductase family, fumarate and sulfite reductases. Metatranscriptomic analysis of active Se(IV)-reducing cultures revealed significantly higher transcriptional levels of genes associated with DMSO reductase (serA/PHGDH), fumarate reductase (sdhCD/frdCD), and sulfite reductase (cysDIH) compared to those in cultures not amended with Se(IV), suggesting that these genes played important roles in Se(IV) reduction. The current study expands our knowledge of the genetic mechanisms involved in less-understood anaerobic Se(IV) bio-reduction. Additinally, the complementary abilities of DNA-SIP, metagenomics, and metatranscriptomics analyses are demonstrated in elucidating the microbial mechanisms of biogeochemical processes in anoxic sediment.

Metagenomics-metabolomics analysis of microbial function and metabolism in petroleum-contaminated soil.

Contamination of soil by petroleum is becoming increasingly serious in the world today. However, the research on gene functional characteristics, metabolites and distribution of microbial genomes in oil-contaminated soil is limited. Considering that, metagenomic and metabonomic were used to detect microbes and metabolites in oil-contaminated soil, and the changes of functional pathways were analyzed. We found that oil pollution significantly changed the composition of soil microorganisms and metabolites, and promoted the relative abundance of Pseudoxanthomonas, Pseudomonas, Mycobacterium, Immundisolibacter, etc. The degradation of toluene, xylene, polycyclic aromatic hydrocarbon and fluorobenzoate increased in Xenobiotics biodegradation and metabolism. Key monooxygenases and dioxygenase systems were regulated to promote ring opening and degradation of aromatic hydrocarbons. Metabolite contents of polycyclic aromatic hydrocarbons (PAHs) such as 9-fluoronone and gentisic acid increased significantly. The soil microbiome degraded petroleum pollutants into small molecular substances and promoted the bioremediation of petroleum-contaminated soil. Besides, we discovered the complete degradation pathway of petroleum-contaminated soil microorganisms to generate gentisic acid from the hydroxylation of naphthalene in PAHs by salicylic acid. This study offers important insights into bioremediation of oil-contaminated soil from the aspect of molecular regulation mechanism and provides a theoretical basis for the screening of new oil degrading bacteria.

A community genomics approach to natural hybridization.

Hybridization is a complicated, oft-misunderstood process. Once deemed unnatural and uncommon, hybridization is now recognized as ubiquitous among species. But hybridization rates within and among communities are poorly understood despite the relevance to ecology, evolution and conservation. To clarify, we examined hybridization across 75 freshwater fish communities within the Ozarks of the North American Interior Highlands (USA) by single nucleotide polymorphism (SNP) genotyping 33 species (N = 2865 individuals; double-digest restriction site-associated DNA sequencing (ddRAD)). We found evidence of hybridization (70 putative hybrids; 2.4% of individuals) among 18 species-pairs involving 73% (24/33) of study species, with the majority being concentrated within one family (Leuciscidae/minnows; 15 species; 66 hybrids). Interspecific genetic exchange-or introgression-was evident from 24 backcrossed individuals (10/18 species-pairs). Hybrids occurred within 42 of 75 communities (56%). Four selected environmental variables (species richness, protected area extent, precipitation (May and annually)) exhibited 73-78% accuracy in predicting hybrid occurrence via random forest classification. Our community-level assessment identified hybridization as spatially widespread and environmentally dependent (albeit predominantly within one diverse, omnipresent family). Our approach provides a more holistic survey of natural hybridization by testing a wide range of species-pairs, thus contrasting with more conventional evaluations.

16S rRNA and Metagenomics Combined with UPLC-Q/TOF-MS Metabolomics Analysis Reveals the Potential Mechanism of Radix Astragali Against Hyperuricemia in Mice.

Purpose: This study aimed to investigate the underlying treatment mechanism of Radix Astragali (RA) in hyperuricemia from the perspective of microbiota and metabolomics. Methods: We used potassium oxyazinate (PO) to induce hyperuricemia mice, and we determined serum alanine aminotransferase/aspartate aminotransferase (ALT/AST), xanthine oxidase (XOD), creatinine (CRE), uric acid (UA), blood urea nitrogen (BUN) levels, liver XOD levels and assessed the kidney tissue histopathology. The therapeutic mechanism of RA in hyperuricemic mice was studied by 16S rRNA, metagenomic sequencing and metabolomics. Results: Our research showed that RA has therapeutic effect in hyperuricemia mice, such as slow the weight loss, repair kidney damage, and downregulate serum UA, XOD, CRE, ALT/AST, BUN, and liver XOD levels. RA restored the disturbance structure of the microbiota in hyperuricemia mice by increasing the relative abundances of beneficial bacteria (Lactobacillaceae and Lactobacillus murine) but decreasing the relative abundances of pathogenic bacteria (Prevotellaceae, Rikenellaceae and Bacteroidaceae). Meanwhile, we found that RA directly regulated the metabolic pathway (such as linoleic acid metabolism and glycerophospholipid metabolism) and indirectly regulated bile acid metabolism by mediating microbiota to ameliorate metabolic disorders. Subsequently, there was a robust correlation between specific microbiota, metabolites and the disease index. Conclusion: The ability of RA to protect mice against hyperuricemia is strongly linked to the microbiome-metabolite axis, which would provide evidence for RA as a medicine to prevent or treat hyperuricemia.

Integrating metabonomics and metagenomics sequencing to study the anti-liver fibrosis effects of palmatine in Corydalis saxicola Bunting.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis saxicola Bunting (CS), a traditional Chinese folk medicine, has been effectively used for treating liver disease in Zhuang nationality in South China. However, the main anti-liver fibrosis ingredients in CS are incompletely understood. AIM OF THE STUDY: To elucidate the main anti-liver fibrosis ingredients in CS and its underlying mechanism. MATERIAL AND METHODS: Firstly, spectrum-effect relationship (SER) strategy was applied to identify the major ingredients against liver fibrosis in CS. Subsequently, 1H NMR metabonomics and metagenomics sequencing techniques were used to clarify the intervention of palmatine (PAL) on liver fibrosis. Furthermore, the expression of tight junction proteins and the levels of liver inflammation factors were examined, the effect of PAL on microbiota was verified by FMT. RESULTS: The SER model revealed that PAL was the most important active ingredient in CS. 1H NMR fecal metabonomics showed that PAL could reserve the abnormal levels of gut microbial-mediated metabolites of liver fibrosis, such as isoleucine, taurine, butyrate, propionate, lactate, glucose, which mainly involved in amino acid metabolism, intestinal flora metabolism and energy metabolism. Metagenomics sequencing found that PAL could callback the abundance of s__Lactobacillus_murinus, s__Lactobacillus_reuteri, s__Lactobacillus_johnsonii, s__Lactobacillus_acidophilus and s__Faecalibaculum_rodentium to varying degree. Furthermore, the intestinal barrier function and the levels of hepatic inflammation factors were significantly ameliorated by PAL. FMT demonstrated that the therapeutic efficiency of PAL was closely associated with gut microbiota. CONCLUSION: The effects of CS on liver fibrosis were attributed in part to PAL by alleviating metabolic disorders and rebalancing gut microbiota. The SER strategy may be a useful method for the discovery of active constituents in natural plants.

Metagenomics insights into responses of rhizobacteria and their alleviation role in licorice allelopathy.

BACKGROUND: Allelopathy is closely associated with rhizosphere biological processes, and rhizosphere microbial communities are essential for plant development. However, our understanding of rhizobacterial communities under influence of allelochemicals in licorice remains limited. In the present study, the responses and effects of rhizobacterial communities on licorice allelopathy were investigated using a combination of multi-omics sequencing and pot experiments, under allelochemical addition and rhizobacterial inoculation treatments. RESULTS: Here, we demonstrated that exogenous glycyrrhizin inhibits licorice development, and reshapes and enriches specific rhizobacteria and corresponding functions related to glycyrrhizin degradation. Moreover, the Novosphingobium genus accounted for a relatively high proportion of the enriched taxa and appeared in metagenomic assembly genomes. We further characterized the different capacities of single and synthetic inoculants to degrade glycyrrhizin and elucidated their distinct potency for alleviating licorice allelopathy. Notably, the single replenished N (Novosphingobium resinovorum) inoculant had the greatest allelopathy alleviation effects in licorice seedlings. CONCLUSIONS: Altogether, the findings highlight that exogenous glycyrrhizin simulates the allelopathic autotoxicity effects of licorice, and indigenous single rhizobacteria had greater effects than synthetic inoculants in protecting licorice growth from allelopathy. The results of the present study enhance our understanding of rhizobacterial community dynamics during licorice allelopathy, with potential implications for resolving continuous cropping obstacle in medicinal plant agriculture using rhizobacterial biofertilizers. Video Abstract.

Microbiological diagnostic performance of metagenomic next-generation sequencing compared with conventional culture for patients with community-acquired pneumonia.

Background: Community-acquired pneumonia (CAP) is an extraordinarily heterogeneous illness, both in the range of responsible pathogens and the host response. Metagenomic next-generation sequencing (mNGS) is a promising technology for pathogen detection. However, the clinical application of mNGS for pathogen detection remains challenging. Methods: A total of 205 patients with CAP admitted to the intensive care unit were recruited, and broncho alveolar lavage fluids (BALFs) from 83 patients, sputum samples from 33 cases, and blood from 89 cases were collected for pathogen detection by mNGS. At the same time, multiple samples of each patient were tested by culture. The diagnostic performance was compared between mNGS and culture for pathogen detection. Results: The positive rate of pathogen detection by mNGS in BALF and sputum samples was 89.2% and 97.0%, which was significantly higher (P < 0.001) than that (67.4%) of blood samples. The positive rate of mNGS was significantly higher than that of culture (81.0% vs. 56.1%, P = 1.052e-07). A group of pathogens including Mycobacterium abscessus, Chlamydia psittaci, Pneumocystis jirovecii, Orientia tsutsugamushi, and all viruses were only detected by mNGS. Based on mNGS results, Escherichia coli was the most common pathogen (15/61, 24.59%) of non-severe patients with CAP, and Mycobacterium tuberculosis was the most common pathogen (21/144, 14.58%) leading to severe pneumonia. Pneumocystis jirovecii was the most common pathogen (26.09%) in severe CAP patients with an immunocompromised status, which was all detected by mNGS only. Conclusion: mNGS has higher overall sensitivity for pathogen detection than culture, BALF, and sputum mNGS are more sensitive than blood mNGS. mNGS is a necessary supplement of conventional microbiological tests for the pathogen detection of pulmonary infection.

Nitrite-resistance mechanisms on wastewater treatment in denitrifying phosphorus removal process revealed by machine learning, co-occurrence, and metagenomics analysis.

Nitrite is a key intermediate in nitrogen metabolism that determines microbial transformations of N and P, greenhouse gas (N2O) emissions, and system nutrient removal efficiency. However, nitrite also exerts toxic effects on microorganisms. A lack of understanding of high nitrite-resistance mechanisms at community- and genome-scale resolutions hinders the optimization for robustness of wastewater treatment systems. Here, we established nitrite-dependent denitrifying and phosphorus removal (DPR) systems under a gradient concentration of nitrite (0, 5, 10, 15, 20, and 25 mg N/L), relying on 16S rRNA gene amplicon and metagenomics to explore high nitrite-resistance mechanism. The results demonstrated that specific taxa were adopted to change the metabolic relationship of the community through phenotypic evolution to resist toxic nitrite contributing to the enhancement of denitrification and inhibition of nitrification and phosphorus removal. The key specific species, Thauera enhanced denitrification, whereas Candidatus Nitrotoga decreased in abundance to maintain partial nitrification. The extinction of Candidatus Nitrotoga induced a simpler restructuring-community, forcing high nitrite-stimulating microbiome to establish a more focused denitrification rather than nitrification or P metabolism in response to nitrite toxicity. Our work provides insights for understanding microbiome adaptation to toxic nitrite and giving theoretical support for operation strategy of nitrite-based wastewater treatment technology.

Incorporating plasmid biology and metagenomics into a holistic model of the human gut microbiome.

The human gut microbiome is often described as the collection of bacteria, archaea, fungi, protists, and viruses associated with an individual, with no acknowledgement of the plasmid constituents. However, like viruses, plasmids are autonomous intracellular replicating entities that can influence the genotype and phenotype of their host and mediate trans-kingdom interactions. Plasmids are frequently noted as vehicles for horizontal gene transfer and for spreading antibiotic resistance, yet their multifaceted contribution to mutualistic and antagonistic interactions within the human microbiome and impact on human health is overlooked. In this review, we highlight the importance of plasmids and their biological properties as overlooked components of microbiomes. Subsequent human microbiome studies should include dedicated analyses of plasmids, particularly as a holistic understanding of human-microbial interactions is required before effective and safe interventions can be implemented to improve human well-being.

CRISPR detection in metagenome-assembled genomes (MAGs) of coal mine.

Bacterial and archaeal CRISPR-Cas systems provide adaptive immune protection against foreign mobile genetic elements. When viruses infect bacteria, a small portion of the viral DNA is inserted into the bacterial DNA in a specific pattern to produce segments known as CRISPR arrays. Metagenome assembled genomes (MAGs) were used in our study to identify the CRISPR sequence for determining the interacted phage. Metagenomic data from a coal mine was used to perform a computational study. From raw reads, 206151 contigs were assembled. Then contigs were clustered into 150 Metagenome assembled genomes from which 78 non-redundant MAGs were selected. Using the CHECKM standard, seven MAGs were found to have >80 completeness and <20 contaminations. Those MAGs were analyzed for the presence of CRISPR elements. Out of seven MAGs, four MAGs have the CRISPR elements and are searched against the VIROblast database. CRISPR arrays have 4, 1, 3, and 7 spacer sequences in the MAGs of Burkholderia, Acinetobacter, Oxalobacteraceae, and Burkholderia multivorans respectively. The uncultured Caudovirales phage genomic regions were present in the genomes of Burkholderia, Oxalobacteriaceae, and Burkholderia multivorans. This study follows the unconventional metagenomics workflow to provide a better understanding of bacteria and phage interactions.

Virome comparison (VC): A novel approach to comparing viromes based on virus species specificity and virome specificity diversity.

The human virome, or the viral communities distributed on or in our body, is estimated to contain about 380 trillion of viruses (individuals), which has far reaching influences on our health and diseases. Obviously, the sheer numbers of viruses alone make the comparisons of two or multiple viromes extremely challenging. In fact, the theory of computation in computer science for so-termed NP-hard problems stipulates that the problem is unsolvable when the size of virome is sufficiently large even with fastest supercomputers. Practically, one has to develop heuristic and approximate algorithms to obtain practically satisfactory solutions for NP-hard problems. Here, we extend the species-specificity and specificity-diversity framework to develop a method for virome comparison (VC). The VC method consists of a pair of metrics: virus species specificity (VS) and virome specificity diversity (VSD) and corresponding pair of random search algorithms. Specifically, the VS and VS permutation (VSP) test can detect unique virus species (US) or enriched virus species (ES) in each virome (treatment), and the VSD and VSD permutation (VSDP) test can further determine holistic differences between two viromes or their subsets (assemblages of viruses). The test with four virome data sets demonstrated that the VC method is effective, efficient, and robust.

QMRA of beach water by Nanopore sequencing-based viability-metagenomics absolute quantification.

The majority of the current regulatory practices for routine monitoring of beach water quality rely on the culture-based enumeration of faecal indicator bacteria (FIB) to develop criteria for promoting the general public's health. To address the limitations of culture methods and the arguable reliability of FIB in indicating health risks, we developed a Nanopore metagenomic sequencing-based viable cell absolute quantification workflow to rapidly and accurately estimate a broad range of microbes in beach waters by a combination of propidium monoazide (PMA) and cellular spike-ins. Using the simple synthetic bacterial communities mixed with viable and heat-killed cells, we observed near-complete relic DNA removal by PMA with minimal disturbance to the composition of viable cells, demonstrating the feasibility of PMA treatment in profiling viable cells by Nanopore sequencing. On a simple mock community comprised of 15 prokaryotic species, our results showed high accordance between the expected and estimated concentrations, suggesting the accuracy of our method in absolute quantification. We then further assessed the accuracy of our method for counting viable Escherichia coli and Vibrio spp. in beach waters by comparing to culture-based method, which were also in high agreement. Furthermore, we demonstrated that 1 Gb sequences obtained within 2 h would be sufficient to quantify a species having a concentration of ≥ 10 cells/mL in beach waters. Using our viability-resolved quantification workflow to assess the microbial risk of the beach water, we conducted (1) screening-level quantitative microbial risk assessment (QMRA) to investigate human illness risk and site-specific risk patterns that might guide risk management efforts and (2) metagenomics-based resistome risk assessment to evaluate another layer of risk caused by difficult illness treatment due to antimicrobial resistance (AMR). In summary, our metagenomic workflow for the rapid absolute quantification of viable bacteria demonstrated its great potential in paving new avenues toward holistic microbial risk assessment.

The hybridization origin of the Chinese endemic herb genus Notopterygium (Apiaceae): Evidence from population genomics and ecological niche analysis.

Hybridization is recognized as a major force in species evolution and biodiversity formation, generally leading to the origin and differentiation of new species. Multiple hybridization events cannot easily be reconstructed, yet they offer the potential to study a number of evolutionary processes. Here, we used nuclear expressed sequence tag-simple sequence repeat and large-scale single nucleotide polymorphism variation data, combined with niche analysis, to investigate the putative independent hybridization events in Notopterygium, a group of perennial herb plants endemic to China. Population genomic analysis indicated that the four studied species are genetically well-delimited and that N. forrestii and N. oviforme have originated by hybridization. According to Approximate Bayesian Computation, the best-fit model involved the formation of N. forrestii from the crossing of N. franchetii and N. incisum, with N. forrestii further backcrossing to N. franchetii to form N. oviforme. The niche analyses indicated that niche divergence [likely triggered by the regional climate changes, particularly the intensification of East Asian winter monsoon, and tectonic movements (affecting both Qinghai-Tibetan Plateau and Qinling Mountains)] may have promoted and maintained the reproductive isolation among hybrid species. N. forrestii shows ecological specialization with respect to their parental species, whereas N. oviforme has completely shifted its niche. These results suggested that the climate and environmental factors together triggered the two-step hybridization of the East Asia herb plants. Our study also emphasizes the power of genome-wide SNPs for investigating suspected cases of hybridization, particularly unravelling old hybridization events.