Evolution of SARS-CoV-2 Viral Mutations in Individuals with Prolonged Infections.

OBJECTIVE: The world has been faced with the repeat rise of SARS-CoV-2 variants since late 2020, including Alpha, Beta, Gamma, Delta, and the latest simultaneous emergence of far-flung spawn of Omicron sub-lineages in different parts of the globe. This has brought us the challenge of determining what factor(s) have been the selective force behind these immune evasive and therapy resistant mutations. It is very possible that such variants evolved in limited host individuals with prolonged infections, or from a localized community of patients. METHODS: This study surveys the GISAID time capsule of mutations found in viral genomes from patients with prolonged same lineage viral infections. We analyzed 288 SARS-CoV-2 genomes representing 113 patients who had same lineage viral genomes in two or more samples stored in GISAID. RESULTS: Of these, thirty-five (30.9%) of the 113 patients developed mutations during their infections. Samples from patients whose viral genomes showed nucleotide changes(s) (n=35) versus those that showed no change (n=78) had a statistically significant difference (p=2.121x10-4) in duration of infection by a median of 13 days (range 0-109 days) versus 6 days (range 0-72 days), respectively. Five highly recognizable variant-defining mutations with immune evasion properties were identified in 5 cases infected by the B.1 lineages in late 2020 and early 2021. CONCLUSION: This suggests the duration of infection is a contributing factor that gives rise to mutations, but not the sole factor, and individual host conditions may play a critical role in driving viral evolution.

Phylogeny of Crataegus (Rosaceae) based on 257 nuclear loci and chloroplast genomes: evaluating the impact of hybridization.

BACKGROUND: Hawthorn species (Crataegus L.; Rosaceae tribe Maleae) form a well-defined clade comprising five subgeneric groups readily distinguished using either molecular or morphological data. While multiple subsidiary groups (taxonomic sections, series) are recognized within some subgenera, the number of and relationships among species in these groups are subject to disagreement. Gametophytic apomixis and polyploidy are prevalent in the genus, and disagreement concerns whether and how apomictic genotypes should be recognized taxonomically. Recent studies suggest that many polyploids arise from hybridization between members of different infrageneric groups. METHODS: We used target capture and high throughput sequencing to obtain nucleotide sequences for 257 nuclear loci and nearly complete chloroplast genomes from a sample of hawthorns representing all five currently recognized subgenera. Our sample is structured to include two examples of intersubgeneric hybrids and their putative diploid and tetraploid parents. We queried the alignment of nuclear loci directly for evidence of hybridization, and compared individual gene trees with each other, and with both the maximum likelihood plastome tree and the nuclear concatenated and multilocus coalescent-based trees. Tree comparisons provided a promising, if challenging (because of the number of comparisons involved) method for visualizing variation in tree topology. We found it useful to deploy comparisons based not only on tree-tree distances but also on a metric of tree-tree concordance that uses extrinsic information about the relatedness of the terminals in comparing tree topologies. RESULTS: We obtained well-supported phylogenies from plastome sequences and from a minimum of 244 low copy-number nuclear loci. These are consistent with a previous morphology-based subgeneric classification of the genus. Despite the high heterogeneity of individual gene trees, we corroborate earlier evidence for the importance of hybridization in the evolution of Crataegus. Hybridization between subgenus Americanae and subgenus Sanguineae was documented for the origin of Sanguineae tetraploids, but not for a tetraploid Americanae species. This is also the first application of target capture probes designed with apple genome sequence. We successfully assembled 95% of 257 loci in Crataegus, indicating their potential utility across the genera of the apple tribe.

Estimating the genetic diversity of Pacific salmon and trout using multigene eDNA metabarcoding.

Genetic diversity underpins species conservation and management goals, and ultimately determines a species' ability to adapt. Using freshwater environmental DNA (eDNA) samples, we examined mitochondrial genetic diversity using multigene metabarcode sequence data from four Oncorhynchus species across 16 sites in Oregon and northern California. Our multigene metabarcode panel included targets commonly used in population genetic NADH dehydrogenase 2 (ND2), phylogenetic cytochrome c oxidase subunit 1 (COI) and eDNA (12S ribosomal DNA) screening. The ND2 locus showed the greatest within-species haplotype diversity for all species, followed by COI and then 12S rDNA for all species except Oncorhynchus kisutch. Sequences recovered for O. clarkii clarkii were either identical to, or one mutation different from, previously characterized haplotypes (95.3% and 4.5% of reads, respectively). The greatest diversity in O. c. clarkii was among coastal watersheds, and subsets of this diversity were shared with fish in inland watersheds. However, coastal streams and the Umpqua River watershed appear to harbour unique haplotypes. Sequences from O. mykiss revealed a disjunction between the Willamette watershed and southern watersheds suggesting divergent histories. We also identified similarities between populations in the northern Deschutes and southern Klamath watersheds, consistent with previously hypothesized connections between the two via inland basins. Oncorhynchus kisutch was only identified in coastal streams and the Klamath River watershed, with most diversity concentrated in the coastal Coquille watershed. Oncorhynchus tshawytscha was only observed at one site, but contained multiple haplotypes at each locus. The characterization of genetic diversity at multiple loci expands the knowledge gained from eDNA sampling and provides crucial information for conservation actions and genetic management.

A taxonomic monograph of Ipomoea integrated across phylogenetic scales.

Taxonomic monographs have the potential to make a unique contribution to the understanding of global biodiversity. However, such studies, now rare, are often considered too daunting to undertake within a realistic time frame, especially as the world's collections have doubled in size in recent times. Here, we report a global-scale monographic study of morning glories (Ipomoea) that integrated DNA barcodes and high-throughput sequencing with the morphological study of herbarium specimens. Our approach overhauled the taxonomy of this megadiverse group, described 63 new species and uncovered significant increases in net diversification rates comparable to the most iconic evolutionary radiations in the plant kingdom. Finally, we show that more than 60 species of Ipomoea, including sweet potato, independently evolved storage roots in pre-human times, indicating that the storage root is not solely a product of human domestication but a trait that predisposed the species for cultivation. This study demonstrates how the world's natural history collections can contribute to global challenges in the Anthropocene.

A draft genome and transcriptome of common milkweed (Asclepias syriaca) as resources for evolutionary, ecological, and molecular studies in milkweeds and Apocynaceae.

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present a transcriptome and draft nuclear genome assembly of the common milkweed, Asclepias syriaca. This reconstruction of the nuclear genome is augmented by linkage group information, adding to existing chloroplast and mitochondrial genomic resources for this member of the Apocynaceae subfamily Asclepiadoideae. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3,415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Scaffolds covering 75 Mbp of the Asclepias assembly formed 11 linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The progesterone 5ß-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family.

eDNA as a tool for identifying freshwater species in sustainable forestry: A critical review and potential future applications.

Environmental DNA (eDNA) is an emerging biological monitoring tool that can aid in assessing the effects of forestry and forest manufacturing activities on biota. Monitoring taxa across broad spatial and temporal scales is necessary to ensure forest management and forest manufacturing activities meet their environmental goals of maintaining biodiversity. Our objectives are to describe potential applications of eDNA across the wood products supply chain extending from regenerating forests, harvesting, and wood transport, to manufacturing facilities, and to review the current state of the science in this context. To meet our second objective, we summarize the taxa examined with targeted (PCR, qPCR or ddPCR) or metagenomic eDNA methods (eDNA metabarcoding), evaluate how estimated species richness compares between traditional field sampling and eDNA metabarcoding approaches, and compare the geographical representation of prior eDNA studies in freshwater ecosystems to global wood baskets. Potential applications of eDNA include evaluating the effects of forestry and forest manufacturing activities on aquatic biota, delineating fish-bearing versus non fish-bearing reaches, evaluating effectiveness of constructed road crossings for freshwater organism passage, and determining the presence of at-risk species. Studies using targeted eDNA approaches focused on fish, amphibians, and invertebrates, while metagenomic studies focused on fish, invertebrates, and microorganisms. Rare, threatened, or endangered species received the least attention in targeted eDNA research, but are arguably of greatest interest to sustainable forestry and forest manufacturing that seek to preserve freshwater biodiversity. Ultimately, using eDNA methods will enable forestry and forest manufacturing managers to have data-driven prioritization for conservation actions for all freshwater species.

Reconciling Conflicting Phylogenies in the Origin of Sweet Potato and Dispersal to Polynesia.

The sweet potato is one of the world's most widely consumed crops, yet its evolutionary history is poorly understood. In this paper, we present a comprehensive phylogenetic study of all species closely related to the sweet potato and address several questions pertaining to the sweet potato that remained unanswered. Our research combined genome skimming and target DNA capture to sequence whole chloroplasts and 605 single-copy nuclear regions from 199 specimens representing the sweet potato and all of its crop wild relatives (CWRs). We present strongly supported nuclear and chloroplast phylogenies demonstrating that the sweet potato had an autopolyploid origin and that Ipomoea trifida is its closest relative, confirming that no other extant species were involved in its origin. Phylogenetic analysis of nuclear and chloroplast genomes shows conflicting topologies regarding the monophyly of the sweet potato. The process of chloroplast capture explains these conflicting patterns, showing that I. trifida had a dual role in the origin of the sweet potato, first as its progenitor and second as the species with which the sweet potato introgressed so one of its lineages could capture an I. trifida chloroplast. In addition, we provide evidence that the sweet potato was present in Polynesia in pre-human times. This, together with several other examples of long-distance dispersal in Ipomoea, negates the need to invoke ancient human-mediated transport as an explanation for its presence in Polynesia. These results have important implications for understanding the origin and evolution of a major global food crop and question the existence of pre-Columbian contacts between Polynesia and the American continent.

Evolution of pyrrolizidine alkaloid biosynthesis in Apocynaceae: revisiting the defence de-escalation hypothesis.

Plants produce specialized metabolites for their defence. However, specialist herbivores adapt to these compounds and use them for their own benefit. Plants attacked predominantly by specialists may be under selection to reduce or eliminate production of co-opted chemicals: the defence de-escalation hypothesis. We studied the evolution of pyrrolizidine alkaloids (PAs) in Apocynaceae, larval host plants for PA-adapted butterflies (Danainae, milkweed and clearwing butterflies), to test if the evolutionary pattern is consistent with de-escalation. We used the first PA biosynthesis specific enzyme (homospermidine synthase, HSS) as tool for reconstructing PA evolution. We found hss orthologues in diverse Apocynaceae species, not all of them known to produce PAs. The phylogenetic analysis showed a monophyletic origin of the putative hss sequences early in the evolution of one Apocynaceae lineage (the APSA clade). We found an hss pseudogene in Asclepias syriaca, a species known to produce cardiac glycosides but no PAs, and four losses of an HSS amino acid motif. APSA clade species are significantly more likely to be Danainae larval host plants than expected if all Apocynaceae species were equally likely to be exploited. Our findings are consistent with PA de-escalation as an adaptive response to specialist attack.

Phylogenetic marker development for target enrichment from transcriptome and genome skim data: the pipeline and its application in southern African Oxalis (Oxalidaceae).

Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low-copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds of LCN loci by a comparison between transcriptome and genome skim data. We used our script to obtain LCN genes for southern African Oxalis (Oxalidaceae), a speciose plant lineage in the Greater Cape Floristic Region. This resulted in 1164 LCN genes greater than 600 bp. Using target enrichment combined with genome skimming (Hyb-Seq), we obtained on average 1141 LCN loci, nearly the whole plastid genome and the nrDNA cistron from 23 southern African Oxalis species. Despite a wide range of gene trees, the phylogeny based on the LCN genes was very robust, as retrieved through various gene and species tree reconstruction methods as well as concatenation. Cytonuclear discordance was strong. This indicates that organellar phylogenies alone are unlikely to represent the species tree and stresses the utility of Hyb-Seq in phylogenetics.

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae).

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual's consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the "noncoding" ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming).

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics.

PREMISE OF THE STUDY: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • METHODS AND RESULTS: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • CONCLUSIONS: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PREMISE OF THE STUDY: Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. METHODS: Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). KEY RESULTS: Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. CONCLUSIONS: Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

BACKGROUND: Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. RESULTS: A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. CONCLUSIONS: The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.