Environmental DNA as an emerging tool in botanical research.

Over the past quarter century, environmental DNA (eDNA) has been ascendant as a tool to detect, measure, and monitor biodiversity (species and communities), as a means of elucidating biological interaction networks, and as a window into understanding past patterns of biodiversity. However, only recently has the potential of eDNA been realized in the botanical world. Here we synthesize the state of eDNA applications in botanical systems with emphases on aquatic, ancient, contemporary sediment, and airborne systems, and focusing on both single-species approaches and multispecies community metabarcoding. Further, we describe how abiotic and biotic factors, taxonomic resolution, primer choice, spatiotemporal scales, and relative abundance influence the utilization and interpretation of airborne eDNA results. Lastly, we explore several areas and opportunities for further development of eDNA tools for plants, advancing our knowledge and understanding of the efficacy, utility, and cost-effectiveness, and ultimately facilitating increased adoption of eDNA analyses in botanical systems.

Chromosome-level reference genome of stinkwort, Dittrichia graveolens (L.) Greuter: A resource for studies on invasion, range expansion, and evolutionary adaptation under global change.

Dittrichia graveolens (L.) Greuter, or stinkwort, is a weedy annual plant within the family Asteraceae. The species is recognized for the rapid expansion of both its native and introduced ranges: in Europe, it has expanded its native distribution northward from the Mediterranean basin by nearly 7 °C latitude since the mid-20th century, while in California and Australia the plant is an invasive weed of concern. Here, we present the first de novo D. graveolens genome assembly (1N = 9 chromosomes), including complete chloroplast (151,013 bp) and partial mitochondrial genomes (22,084 bp), created using Pacific Biosciences HiFi reads and Dovetail Omni-C data. The final primary assembly is 835 Mbp in length, of which 98.1% are represented by 9 scaffolds ranging from 66 to 119 Mbp. The contig N50 is 74.9 Mbp and the scaffold N50 is 96.9 Mbp, which, together with a 98.8% completeness based on the BUSCO embryophyta10 database containing 1,614 orthologs, underscores the high quality of this assembly. This pseudo-molecule-scale genome assembly is a valuable resource for our fundamental understanding of the genomic consequences of range expansion under global change, as well as comparative genomic studies in the Asteraceae.

Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing.

Long-read sequencing facilitates assembly of complex genomic regions. In plants, loci containing nucleotide-binding, leucine-rich repeat (NLR) disease resistance genes are an important example of such regions. NLR genes constitute one of the largest gene families in plants and are often clustered, evolving via duplication, contraction, and transposition. We recently mapped the Xo1 locus for resistance to bacterial blight and bacterial leaf streak, found in the American heirloom rice variety Carolina Gold Select, to a region that in the Nipponbare reference genome is NLR gene-rich. Here, toward identification of the Xo1 gene, we combined Nanopore and Illumina reads and generated a high-quality Carolina Gold Select genome assembly. We identified 529 complete or partial NLR genes and discovered, relative to Nipponbare, an expansion of NLR genes at the Xo1 locus. One of these has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain, and the repeats within each protein are nearly perfect. Across diverse Oryzeae, we identified two sub-clades of NLR genes with these features, varying in the presence of the zfBED domain and the number of repeats. The Carolina Gold Select genome assembly also uncovered at the Xo1 locus a rice blast resistance gene and a gene encoding a polyphenol oxidase (PPO). PPO activity has been used as a marker for blast resistance at the locus in some varieties; however, the Carolina Gold Select sequence revealed a loss-of-function mutation in the PPO gene that breaks this association. Our results demonstrate that whole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Finally, the Carolina Gold Select genome assembly will facilitate identification of other useful traits in this historically important variety.

Landscape analyses using eDNA metabarcoding and Earth observation predict community biodiversity in California.

Ecosystems globally are under threat from ongoing anthropogenic environmental change. Effective conservation management requires more thorough biodiversity surveys that can reveal system-level patterns and that can be applied rapidly across space and time. Using modern ecological models and community science, we integrate environmental DNA and Earth observations to produce a time snapshot of regional biodiversity patterns and provide multi-scalar community-level characterization. We collected 278 samples in spring 2017 from coastal, shrub, and lowland forest sites in California, a complex ecosystem and biodiversity hotspot. We recovered 16,118 taxonomic entries from eDNA analyses and compiled associated traditional observations and environmental data to assess how well they predicted alpha, beta, and zeta diversity. We found that local habitat classification was diagnostic of community composition and distinct communities and organisms in different kingdoms are predicted by different environmental variables. Nonetheless, gradient forest models of 915 families recovered by eDNA analysis and using BIOCLIM variables, Sentinel-2 satellite data, human impact, and topographical features as predictors, explained 35% of the variance in community turnover. Elevation, sand percentage, and photosynthetic activities (NDVI32) were the top predictors. In addition to this signal of environmental filtering, we found a positive relationship between environmentally predicted families and their numbers of biotic interactions, suggesting environmental change could have a disproportionate effect on community networks. Together, these analyses show that coupling eDNA with environmental predictors including remote sensing data has capacity to test proposed Essential Biodiversity Variables and create new landscape biodiversity baselines that span the tree of life.

Eggplant Domestication: Pervasive Gene Flow, Feralization, and Transcriptomic Divergence.

In the context of food security, examining the genomics of domestication will help identify genes underlying adaptive and economically important phenotypes, for example, larger fruit, improved taste, and loss of agronomically inferior phenotypes. Examination of genome-scale single nucleotide polymorphisms demonstrates the relationships between wild ancestors of eggplant (Solanum melongena L.), confirming that Solanum insanum L. is the wild progenitor. This species is split roughly into an Eastern (Malaysian, Thai, and Vietnamese) and Western (Indian, Madagascan, and Sri Lankan) group, with domesticates derived from the former. Additional "wild" accessions from India appear to be feral escapes, derived multiple times from domesticated varieties through admixture. Accessions with small egg-shaped fruit are generally found intermixed with East Asian Solanum insanum confirming they are primitive relative to the large-fruited domesticates. Comparative transcriptomics was used to track the loci under selection. Sequence analysis revealed a genetic bottleneck reducing variation by almost 50% in the primitive accessions relative to the wild species and a further 10% in the landraces. We also show evidence for selection on genes with a role in response to wounding and apoptosis. Genes showing a significant difference in expression between wild and primitive or between primitive and landrace genepools were mostly (>75%) downregulated in the derived populations and enriched for gene ontologies related to defense, flowering, signaling, and response to biotic and abiotic stimuli. This work reveals genomic changes involved in crop domestication and improvement, and the population genetics work explains why defining the eggplant domestication trajectory has been so challenging.

A novel hydroxycinnamoyl transferase for synthesis of hydroxycinnamoyl spermine conjugates in plants.

BACKGROUND: Hydroxycinnamoyl-spermine conjugates (HCSpm) are a class of hydroxycinnamic acid amides (HCAAs), which not only are instrumental in plant development and stress response, but also benefit human health. However, HCSpm are not commonly produced in plants, and the mechanism of their biosynthesis remains unclear. In previous investigations of phenolics in Solanum fruits related to eggplant (Solanum melongena L.), we discovered that Solanum richardii, an African wild relative of eggplant, was rich in HCSpms in fruits. RESULTS: The putative spermine hydroxycinnamoyl transferase (HT) SpmHT was isolated from S. richardii and eggplant. SrSpmHT expression was high in flowers and fruit, and was associated with HCSpm accumulation in S. richardii; however, SpmHT was hardly detected in eggplant cultivars and other wild relatives. Recombinant SpmHT exclusively selected spermine as the acyl acceptor substrate, while showing donor substrate preference in the following order: caffeoyl-CoA, feruloyl-CoA, and p-coumaroyl-CoA. Molecular docking revealed that substrate binding pockets of SpmHT could properly accommodate spermine but not the shorter, more common spermidine. CONCLUSION: SrSpmHT is a novel spermine hydroxycinnamoyl transferase that uses Spm exclusively as the acyl acceptor substrate to produce HCSpms. Our findings shed light on the HCSpm biosynthetic pathway that may allow an increase of health beneficial metabolites in Solanum crops via methods such as introgression or engineering HCAA metabolism.

Evolution of crop species: genetics of domestication and diversification.

Domestication is a good model for the study of evolutionary processes because of the recent evolution of crop species (<12,000 years ago), the key role of selection in their origins, and good archaeological and historical data on their spread and diversification. Recent studies, such as quantitative trait locus mapping, genome-wide association studies and whole-genome resequencing studies, have identified genes that are associated with the initial domestication and subsequent diversification of crops. Together, these studies reveal the functions of genes that are involved in the evolution of crops that are under domestication, the types of mutations that occur during this process and the parallelism of mutations that occur in the same pathways and proteins, as well as the selective forces that are acting on these mutations and that are associated with geographical adaptation of crop species.

Possible loss of the chloroplast genome in the parasitic flowering plant Rafflesia lagascae (Rafflesiaceae).

Rafflesia is a genus of holoparasitic plants endemic to Southeast Asia that has lost the ability to undertake photosynthesis. With short-read sequencing technology, we assembled a draft sequence of the mitochondrial genome of Rafflesia lagascae Blanco, a species endemic to the Philippine island of Luzon, with ∼350× sequencing depth coverage. Using multiple approaches, however, we were only able to identify small fragments of plastid sequences at low coverage depth (<2×) and could not recover any substantial portion of a chloroplast genome. The gene fragments we identified included photosynthesis and energy production genes (atp, ndh, pet, psa, psb, rbcL), ribosomal RNA genes (rrn16, rrn23), ribosomal protein genes (rps7, rps11, rps16), transfer RNA genes, as well as matK, accD, ycf2, and multiple nongenic regions from the inverted repeats. None of the identified plastid gene sequences had intact reading frames. Phylogenetic analysis suggests that ∼33% of these remnant plastid genes may have been horizontally transferred from the host plant genus Tetrastigma with the rest having ambiguous phylogenetic positions (<50% bootstrap support), except for psaB that was strongly allied with the plastid homolog in Nicotiana. Our inability to identify substantial plastid genome sequences from R. lagascae using multiple approaches--despite success in identifying and developing a draft assembly of the much larger mitochondrial genome--suggests that the parasitic plant genus Rafflesia may be the first plant group for which there is no recognizable plastid genome, or if present is found in cryptic form at very low levels.

Location of chlorogenic acid biosynthesis pathway and polyphenol oxidase genes in a new interspecific anchored linkage map of eggplant.

BACKGROUND: Eggplant is a powerful source of polyphenols which seems to play a key role in the prevention of several human diseases, such as cancer and diabetes. Chlorogenic acid is the polyphenol most present in eggplant, comprising between the 70% and 90% of the total polyphenol content. Introduction of the high chlorogenic acid content of wild relatives, such as S. incanum, into eggplant varieties will be of great interest. A potential side effect of the increased level polyphenols could be a decrease on apparent quality due to browning caused by the polyphenol oxidase enzymes mediated oxidation of polyphenols. We report the development of a new interspecific S. melongena × S. incanum linkage map based on a first backcross generation (BC1) towards the cultivated S. melongena as a tool for introgressing S. incanum alleles involved in the biosynthesis of chlorogenic acid in the genetic background of S. melongena. RESULTS: The interspecific genetic linkage map of eggplant developed in this work anchor the most informative previously published genetic maps of eggplant using common markers. The 91 BC1 plants of the mapping population were genotyped with 42 COSII, 99 SSRs, 88 AFLPs, 9 CAPS, 4 SNPs and one morphological polymorphic markers. Segregation marker data resulted in a map encompassing 1085 cM distributed in 12 linkage groups. Based on the syntheny with tomato, the candidate genes involved in the core chlorogenic acid synthesis pathway in eggplant (PAL, C4H, 4CL, HCT, C3'H, HQT) as well as five polyphenol oxidase (PPO1, PPO2, PPO3, PPO4, PPO5) were mapped. Except for 4CL and HCT chlorogenic acid genes were not linked. On the contrary, all PPO genes clustered together. Candidate genes important in domestication such as fruit shape (OVATE, SISUN1) and prickliness were also located. CONCLUSIONS: The achievements in location of candidate genes will allow the search of favorable alleles employing marker-assisted selection in order to develop new varieties with higher chlorogenic content alongside a lower polyphenol oxidase activity. This will result into an enhanced product showing a lower fruit flesh browning with improved human health properties.

The reference genome of an endangered Asteraceae, Deinandra increscens subsp. villosa, endemic to the Central Coast of California.

We present a reference genome for the federally endangered Gaviota tarplant, Deinandra increscens subsp. villosa (Madiinae, Asteraceae), an annual herb endemic to the Central California coast. Generating PacBio Hifi, Oxford Nanopore Technologies, and Dovetail Omni-C data, we assembled a haploid consensus genome of 1.67 Gbp as 28.7 K scaffolds with a scaffold N50 of 74.9 Mb. We annotated repeat content in 74.8% of the genome. Long terminal repeats (LTR) covered 44.0% of the genome with Copia families predominant at 22.9% followed by Gypsy at 14.2%. Both Gypsy and Copia elements were common in ancestral peaks of LTR, and the most abundant element was a Gypsy element containing nested Copia/Angela sequence similarity, reflecting a complex evolutionary history of repeat activity. Gene annotation produced 33,257 genes and 68,942 transcripts, of which 99% were functionally annotated. BUSCO scores for the annotated proteins were 96.0% complete of which 77.6% was single copy and 18.4% duplicates. Whole genome duplication (WGD) synonymous mutation rates of Gaviota tarplant and sunflower (Helianthus annuus) shared peaks that correspond to the last Asteraceae polyploidization event and subsequent divergence from a common ancestor at ∼27 mya. Regions of high-density tandem genes were identified, pointing to potentially important loci of environmental adaptation in this species.

Critical considerations for communicating environmental DNA science.

The economic and methodological efficiencies of environmental DNA (eDNA) based survey approaches provide an unprecedented opportunity to assess and monitor aquatic environments. However, instances of inadequate communication from the scientific community about confidence levels, knowledge gaps, reliability, and appropriate parameters of eDNA-based methods have hindered their uptake in environmental monitoring programs and, in some cases, has created misperceptions or doubts in the management community. To help remedy this situation, scientists convened a session at the Second National Marine eDNA Workshop to discuss strategies for improving communications with managers. These include articulating the readiness of different eDNA applications, highlighting the strengths and limitations of eDNA tools for various applications or use cases, communicating uncertainties associated with specified uses transparently, and avoiding the exaggeration of exploratory and preliminary findings. Several key messages regarding implementation, limitations, and relationship to existing methods were prioritized. To be inclusive of the diverse managers, practitioners, and researchers, we and the other workshop participants propose the development of communication workflow plans, using RACI (Responsible, Accountable, Consulted, Informed) charts to clarify the roles of all pertinent individuals and parties and to minimize the chance for miscommunications. We also propose developing decision support tools such as Structured Decision-Making (SDM) to help balance the benefits of eDNA sampling with the inherent uncertainty, and developing an eDNA readiness scale to articulate the technological readiness of eDNA approaches for specific applications. These strategies will increase clarity and consistency regarding our understanding of the utility of eDNA-based methods, improve transparency, foster a common vision for confidently applying eDNA approaches, and enhance their benefit to the monitoring and assessment community.

Reply to J. Samuels: Taxonomic notes on several wild relatives of Solanum melongena L.

The purpose of the study "Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication" by Meyer et al. (2012) was to use new and expanded accession sets coupled with molecular data to evaluate possible scenarios of eggplant domestication with as little influence as possible from any previously published nomenclatural scheme, and taking into consideration multiple sources of evidence regarding the history of eggplant in Asia. Samuels (2013) disfavored this system and in his Letter to the Editor attempted to re-evaluate the results according to his system. However, Samuels appears to have misread Meyer et al. and also makes several claims without the support of evidence. We stand by the results of Meyer et al., which are in agreement with the recent and much needed new taxonomic treatment for wild relatives of eggplant.

Evolutionary meta-analysis of solanaceous resistance gene and solanum resistance gene analog sequences and a practical framework for cross-species comparisons.

Cross-species comparative genomics approaches have been employed to map and clone many important disease resistance (R) genes from Solanum species-especially wild relatives of potato and tomato. These efforts will increase with the recent release of potato genome sequence and the impending release of tomato genome sequence. Most R genes belong to the prominent nucleotide binding site-leucine rich repeat (NBS-LRR) class and conserved NBS-LRR protein motifs enable survey of the R gene space of a plant genome by generation of resistance gene analogs (RGA), polymerase chain reaction fragments derived from R genes. We generated a collection of 97 RGA from the disease-resistant wild potato S. bulbocastanum, complementing smaller collections from other Solanum species. To further comparative genomics approaches, we combined all known Solanum RGA and cloned solanaceous NBS-LRR gene sequences, nearly 800 sequences in total, into a single meta-analysis. We defined R gene diversity bins that reflect both evolutionary relationships and DNA cross-hybridization results. The resulting framework is amendable and expandable, providing the research community with a common vocabulary for present and future study of R gene lineages. Through a series of sequence and hybridization experiments, we demonstrate that all tested R gene lineages are of ancient origin, are shared between Solanum species, and can be successfully accessed via comparative genomics approaches.

Patterns and processes in crop domestication: an historical review and quantitative analysis of 203 global food crops.

Domesticated food crops are derived from a phylogenetically diverse assemblage of wild ancestors through artificial selection for different traits. Our understanding of domestication, however, is based upon a subset of well-studied 'model' crops, many of them from the Poaceae family. Here, we investigate domestication traits and theories using a broader range of crops. We reviewed domestication information (e.g. center of domestication, plant traits, wild ancestors, domestication dates, domestication traits, early and current uses) for 203 major and minor food crops. Compiled data were used to test classic and contemporary theories in crop domestication. Many typical features of domestication associated with model crops, including changes in ploidy level, loss of shattering, multiple origins, and domestication outside the native range, are less common within this broader dataset. In addition, there are strong spatial and temporal trends in our dataset. The overall time required to domesticate a species has decreased since the earliest domestication events. The frequencies of some domestication syndrome traits (e.g. nonshattering) have decreased over time, while others (e.g. changes to secondary metabolites) have increased. We discuss the influences of the ecological, evolutionary, cultural and technological factors that make domestication a dynamic and ongoing process.

Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication.

The domestication history of eggplant (Solanum melongena L.) has long been debated, with studies unable to narrow down where domestication occurred within a broad region of tropical Asia. The most commonly hypothesized region is India, however China has an equally old written record of eggplant use dating ca. 2000 years before present. Both regions have a high diversity of landraces and populations of putatively wild eggplant: Solanum incanum L. in India and Solanum undatum Lam. in SE Asia. An additional complication is that there is taxonomic confusion regarding the two candidate progenitors. Here, we synthesize historic, morphologic, and molecular data (nrITS sequence and AFLP) to interpret the phylogeographic relationships among candidate progenitors and Asian eggplant landraces in order to test theories of domestication. A minimum of two domestication events is supported: one in India and one in southern China/SE Asia. Results also support separate domestication of S. melongena subsp. ovigerum, a group of morphologically distinct eggplants found in SE Asia, and suggest Asian S. incanum and S. undatum may not be genetically distinct. Routes of the spread of eggplant cultivation throughout Asia are proposed, and evolutionary relationships among allied species are discussed.

Antioxidant glucosylated caffeoylquinic acid derivatives in the invasive tropical soda apple, Solanum viarum.

Eggplant and related Solanum species contain abundant caffeoylquinic acid (CQA) derivatives. Fruit of the invasive species Solanum viarum Dunal contain numerous complex CQA derivatives, but only a few have been identified. The structures of two new compounds isolated from methanolic extracts of S. viarum fruit by C(18)-HPLC-DAD were determined using 2D NMR and MS data. Both include two 5-CQA molecules joined by glucose via ester and glycosidic linkages. The structures of compounds 1 and 2 (viarumacids A and B) are, respectively, 5-caffeoyl- and 3-malonyl-5-caffeoyl-[4-(1ß-[6-(5-caffeoyl)quinate]glucopyranosyl)]quinic acid. The antioxidant activities determined by ABTS(•+) and DPPH(•) assays were in the order 1 > 2 > 5-CQA.

A manager's guide to using eDNA metabarcoding in marine ecosystems.

Environmental DNA (eDNA) metabarcoding is a powerful tool that can enhance marine ecosystem/biodiversity monitoring programs. Here we outline five important steps managers and researchers should consider when developing eDNA monitoring program: (1) select genes and primers to target taxa; (2) assemble or develop comprehensive barcode reference databases; (3) apply rigorous site occupancy based decontamination pipelines; (4) conduct pilot studies to define spatial and temporal variance of eDNA; and (5) archive samples, extracts, and raw sequence data. We demonstrate the importance of each of these considerations using a case study of eDNA metabarcoding in the Ports of Los Angeles and Long Beach. eDNA metabarcoding approaches detected 94.1% (16/17) of species observed in paired trawl surveys while identifying an additional 55 native fishes, providing more comprehensive biodiversity inventories. Rigorous benchmarking of eDNA metabarcoding results improved ecological interpretation and confidence in species detections while providing archived genetic resources for future analyses. Well designed and validated eDNA metabarcoding approaches are ideally suited for biomonitoring applications that rely on the detection of species, including mapping invasive species fronts and endangered species habitats as well as tracking range shifts in response to climate change. Incorporating these considerations will enhance the utility and efficacy of eDNA metabarcoding for routine biomonitoring applications.

Revisiting the effect of PCR replication and sequencing depth on biodiversity metrics in environmental DNA metabarcoding.

Environmental DNA (eDNA) metabarcoding is an increasingly popular tool for measuring and cataloguing biodiversity. Because the environments and substrates in which DNA is preserved differ considerably, eDNA research often requires bespoke approaches to generating eDNA data. Here, we explore how two experimental choices in eDNA study design-the number of PCR replicates and the depth of sequencing of PCR replicates-influence the composition and consistency of taxa recovered from eDNA extracts. We perform 24 PCR replicates from each of six soil samples using two of the most common metabarcodes for Fungi and Viridiplantae (ITS1 and ITS2), and sequence each replicate to an average depth of ~84,000 reads. We find that PCR replicates are broadly consistent in composition and relative abundance of dominant taxa, but that low abundance taxa are often unique to one or a few PCR replicates. Taxa observed in one out of 24 PCR replicates make up 21-29% of the total taxa detected. We also observe that sequencing depth or rarefaction influences alpha diversity and beta diversity estimates. Read sampling depth influences local contribution to beta diversity, placement in ordinations, and beta dispersion in ordinations. Our results suggest that, because common taxa drive some alpha diversity estimates, few PCR replicates and low read sampling depths may be sufficient for many biological applications of eDNA metabarcoding. However, because rare taxa are recovered stochastically, eDNA metabarcoding may never fully recover the true amplifiable alpha diversity in an eDNA extract. Rare taxa drive PCR replicate outliers of alpha and beta diversity and lead to dispersion differences at different read sampling depths. We conclude that researchers should consider the complexity and unevenness of a community when choosing analytical approaches, read sampling depths, and filtering thresholds to arrive at stable estimates.

ranacapa: An R package and Shiny web app to explore environmental DNA data with exploratory statistics and interactive visualizations.

Environmental DNA (eDNA) metabarcoding is becoming a core tool in ecology and conservation biology, and is being used in a growing number of education, biodiversity monitoring, and public outreach programs in which professional research scientists engage community partners in primary research. Results from eDNA analyses can engage and educate natural resource managers, students, community scientists, and naturalists, but without significant training in bioinformatics, it can be difficult for this diverse audience to interact with eDNA results. Here we present the R package ranacapa, at the core of which is a Shiny web app that helps perform exploratory biodiversity analyses and visualizations of eDNA results. The app requires a taxonomy-by-sample matrix and a simple metadata file with descriptive information about each sample. The app enables users to explore the data with interactive figures and presents results from simple community ecology analyses. We demonstrate the value of ranacapa to two groups of community partners engaging with eDNA metabarcoding results.