Authentication of garlic (Allium sativum L.) supplements using a trnLUAA mini-barcode.

Garlic (Allium sativum), a widely distributed plant with great cultural and medicinal significance, is one of the most popular herbal dietary supplements in Europe and North America. Garlic supplements are consumed for a variety of reasons, including for their purported antihypertensive, antibacterial, and anticarcinogenic effects. The steady increase in the global herbal dietary supplement market paired with a global patchwork of regulatory frameworks makes the development of assays for authentication of these products increasingly important. A DNA mini-barcode assay was developed using the P6 loop of the plastid trnLUAA intron to positively identify A. sativum products. Analysis of 43 commercially available garlic herbal dietary supplements produced mini-barcode sequences for 33 supplements, all of which contained detectable amounts of A. sativum. The trnLUAA P6 mini-barcode can be highly useful for specimen identification, particularly for samples that may contain degraded DNA.

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.

Authentication of Ginkgo biloba herbal dietary supplements using DNA barcoding.

Ginkgo biloba L. (known as ginkgo or maidenhair tree) is a phylogenetically isolated, charismatic, gymnosperm tree. Herbal dietary supplements, prepared from G. biloba leaves, are consumed to boost cognitive capacity via improved blood perfusion and mitochondrial function. A novel DNA mini-barcode assay was designed and validated for the authentication of G. biloba in herbal dietary supplements (n = 22; sensitivity = 1.00, 95% CI = 0.59-1.00; specificity = 1.00, 95% CI = 0.64-1.00). This assay was further used to estimate the frequency of mislabeled ginkgo herbal dietary supplements on the market in the United States of America: DNA amenable to PCR could not be extracted from three (7.5%) of the 40 supplements sampled, 31 of 37 (83.8%) assayable supplements contained identifiable G. biloba DNA, and six supplements (16.2%) contained fillers without any detectable G. biloba DNA. It is hoped that this assay will be used by supplement manufacturers to ensure that their supplements contain G. biloba.

A functional phylogenomic view of the seed plants.

A novel result of the current research is the development and implementation of a unique functional phylogenomic approach that explores the genomic origins of seed plant diversification. We first use 22,833 sets of orthologs from the nuclear genomes of 101 genera across land plants to reconstruct their phylogenetic relationships. One of the more salient results is the resolution of some enigmatic relationships in seed plant phylogeny, such as the placement of Gnetales as sister to the rest of the gymnosperms. In using this novel phylogenomic approach, we were also able to identify overrepresented functional gene ontology categories in genes that provide positive branch support for major nodes prompting new hypotheses for genes associated with the diversification of angiosperms. For example, RNA interference (RNAi) has played a significant role in the divergence of monocots from other angiosperms, which has experimental support in Arabidopsis and rice. This analysis also implied that the second largest subunit of RNA polymerase IV and V (NRPD2) played a prominent role in the divergence of gymnosperms. This hypothesis is supported by the lack of 24nt siRNA in conifers, the maternal control of small RNA in the seeds of flowering plants, and the emergence of double fertilization in angiosperms. Our approach takes advantage of genomic data to define orthologs, reconstruct relationships, and narrow down candidate genes involved in plant evolution within a phylogenomic view of species' diversification.

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.

The genome of the Wollemi pine, a critically endangered "living fossil" unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

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.

Relationships within Podocarpaceae based on DNA sequence, anatomical, morphological, and biogeographical data.

Despite considerable recent progress in understanding intergeneric relationships, a comprehensive analysis of Podocarpaceae at the species level using molecular data, biogeography, anatomy, and morphology has not been previously attempted. Here we present sequence analyses of rbcL, nrITS1 and NEEDLY intron 2 for two-thirds (183 accessions of 145 taxa) of all Podocarpaceae species representing all genera except Parasitaxus. These analyses include many more species and accessions than previous studies and result in a more resolved phylogeny. The comprehensive anatomical and morphological study ensures that the identification of taxa is correct and also provides clade support. Bayesian and parsimony analyses were used to resolve 20 well-supported monophyletic groups including 11 groups of the formerly poorly resolved subgenera Podocarpus and Foliolatus. The well-resolved topology is supported by anatomical and morphological features and is highly congruent with geographical distribution. © The Willi Hennig Society 2011.

DNA barcode identification of black cohosh herbal dietary supplements.

Black cohosh (Actaea racemosa) herbal dietary supplements are commonly consumed to treat menopausal symptoms, but there are reports of adverse events and toxicities associated with their use. Accidental misidentification and/or deliberate adulteration results in harvesting other related species that are then marketed as black cohosh. Some of these species are known to be toxic to humans. We have identified two matK nucleotides that consistently distinguish black cohosh from related species. Using these nucleotides, an assay was able to correctly identify all of the black cohosh samples in the validation set. None of the other Actaea species in the validation set were falsely identified as black cohosh. Of 36 dietary supplements sequenced, 27 (75%) had a sequence that exactly matched black cohosh. The remaining nine samples (25%) had a sequence identical to that of three Asian Actaea species (A. cimicifuga, A. dahurica, and A. simplex). Manufacturers should routinely test plant material using a reliable assay to ensure accurate labeling.

A unified index of sequence quality and contig overlap for DNA barcoding.

SUMMARY: Barcode quality index (B) is a novel, unified measure of sequence quality and contig overlap tailored to the needs of DNA barcoding. Re-analysis of published data demonstrates the utility of B. AVAILABILITY AND IMPLEMENTATION: A GPL PERL script is available for download (http://www.nybg.org/files/scientists/dlittle/B.html).

DNA barcoding: a new tool for palm taxonomists?

BACKGROUND AND AIMS: In the last decade, a new tool - DNA barcoding - was proposed to identify species. The technique of DNA barcoding is still being developed. The Consortium for the Barcode of Life's Plant Working Group (CBOL-PWG) selected two core markers (matK and rbcL) that now must be tested in as many taxa as possible. Although the taxonomy of palms (Arecaceae/Palmae) has been greatly improved in the past decades, taxonomic problems remain. Species complexes, for example, could significantly benefit from DNA barcoding. Palms have never before been subjected to a DNA barcoding test. METHODS: For this study, 40 out of the 48 species of the southeast Asian tribe Caryoteae (subfamily Coryphoideae) were included. In total, four DNA markers - three plastid encoded (matK, rbcL and psbA-trnH) and one nuclear encoded (nrITS2) - were analysed to determine if adequate variation exists to discriminate among species. KEY RESULTS: The combination of three markers - matK, rbcL and nrITS2 - results in 92 % species discrimination. This rate is high for a barcoding experiment. The two core markers suggested by the CBOL-PWG, rbcL and matK, have a low species discrimination rate and need to be supplemented by another marker. In Caryoteae, nrITS2 should be chosen over psbA-trnH to supplement the two 'core' markers. CONCLUSIONS: For the first time a test of DNA barcoding was conducted in Arecaceae. Considering that palms have highly variable mutation rates compared with other angiosperms, the results presented here are encouraging for developing DNA barcoding as a useful tool to identify species within this ecologically important tropical plant family.

Expression analyses in Ginkgo biloba provide new insights into the evolution and development of the seed.

Although the seed is a key morphological innovation, its origin remains unknown and molecular data outside angiosperms is still limited. Ginkgo biloba, with a unique place in plant evolution, being one of the first extant gymnosperms where seeds evolved, can testify to the evolution and development of the seed. Initially, to better understand the development of the ovules in Ginkgo biloba ovules, we performed spatio-temporal expression analyses in seeds at early developing stages, of six candidate gene homologues known in angiosperms: WUSCHEL, AINTEGUMENTA, BELL1, KANADI, UNICORN, and C3HDZip. Surprisingly, the expression patterns of most these ovule homologues indicate that they are not wholly conserved between angiosperms and Ginkgo biloba. Consistent with previous studies on early diverging seedless plant lineages, ferns, lycophytes, and bryophytes, many of these candidate genes are mainly expressed in mega- and micro-sporangia. Through in-depth comparative transcriptome analyses of Ginkgo biloba developing ovules, pollen cones, and megagametophytes we have been able to identify novel genes, likely involved in ovule development. Finally, our expression analyses support the synangial or neo-synangial hypotheses for the origin of the seed, where the sporangium developmental network was likely co-opted and restricted during integument evolution.

DNA Barcode Authentication of Devil's Claw Herbal Dietary Supplements.

Devil's claw is the vernacular name for a genus of medicinal plants that occur in the Kalahari Desert and Namibia Steppes. The genus comprises two distinct species: Harpagophytum procumbens and H. zeyheri. Although the European pharmacopeia considers the species interchangeable, recent studies have demonstrated that H. procumbens and H. zeyheri are chemically distinct and should not be treated as the same species. Further, the sale of H. zeyheri as an herbal supplement is not legal in the United States. Four markers were tested for their ability to distinguish H. procumbens from H. zeyheri: rbcL, matK, nrITS2, and psbA-trnH. Of these, only psbA-trnH was successful. A novel DNA mini-barcode assay that produces a 178-base amplicon in Harpagophytum (specificity = 1.00 [95% confidence interval = 0.80-1.00]; sensitivity = 1.00 [95% confidence interval = 0.75-1.00]) was used to estimate mislabeling frequency in a sample of 23 devil's claw supplements purchased in the United States. PCR amplification failed in 13% of cases. Among the 20 fully-analyzable supplements: H. procumbens was not detected in 75%; 25% contained both H. procumbens and H. zeyheri; none contained only H. procumbens. We recommend this novel mini-barcode region as a standard method of quality control in the manufacture of devil's claw supplements.

The Herbarium 2021 Half-Earth Challenge Dataset and Machine Learning Competition.

Herbarium sheets present a unique view of the world's botanical history, evolution, and biodiversity. This makes them an all-important data source for botanical research. With the increased digitization of herbaria worldwide and advances in the domain of fine-grained visual classification which can facilitate automatic identification of herbarium specimen images, there are many opportunities for supporting and expanding research in this field. However, existing datasets are either too small, or not diverse enough, in terms of represented taxa, geographic distribution, and imaging protocols. Furthermore, aggregating datasets is difficult as taxa are recognized under a multitude of names and must be aligned to a common reference. We introduce the Herbarium 2021 Half-Earth dataset: the largest and most diverse dataset of herbarium specimen images, to date, for automatic taxon recognition. We also present the results of the Herbarium 2021 Half-Earth challenge, a competition that was part of the Eighth Workshop on Fine-Grained Visual Categorization (FGVC8) and hosted by Kaggle to encourage the development of models to automatically identify taxa from herbarium sheet images.

Recognition of Latin scientific names using artificial neural networks.

PREMISE: The automated recognition of Latin scientific names within vernacular text has many applications, including text mining, search indexing, and automated specimen-label processing. Most published solutions are computationally inefficient, incapable of running within a web browser, and focus on texts in English, thus omitting a substantial portion of biodiversity literature. METHODS AND RESULTS: An open-source browser-executable solution, Quaesitor, is presented here. It uses pattern matching (regular expressions) in combination with an ensembled classifier composed of an inclusion dictionary search (Bloom filter), a trio of complementary neural networks that differ in their approach to encoding text, and word length to automatically identify Latin scientific names in the 16 most common languages for biodiversity articles. CONCLUSIONS: In combination, the classifiers can recognize Latin scientific names in isolation or embedded within the languages used for >96% of biodiversity literature titles. For three different data sets, they resulted in a 0.80-0.97 recall and a 0.69-0.84 precision at a rate of 8.6 ms/word.

An algorithm competition for automatic species identification from herbarium specimens.

PREMISE: Plant biodiversity is threatened, yet many species remain undescribed. It is estimated that >50% of undescribed species have already been collected and are awaiting discovery in herbaria. Robust automatic species identification algorithms using machine learning could accelerate species discovery. METHODS: To encourage the development of an automatic species identification algorithm, we submitted our Herbarium 2019 data set to the Fine-Grained Visual Categorization sub-competition (FGVC6) hosted on the Kaggle platform. We chose to focus on the flowering plant family Melastomataceae because we have a large collection of imaged herbarium specimens (46,469 specimens representing 683 species) and taxonomic expertise in the family. As is common for herbarium collections, some species in this data set are represented by few specimens and others by many. RESULTS: In less than three months, the FGVC6 Herbarium 2019 Challenge drew 22 teams who entered 254 models for Melastomataceae species identification. The four best algorithms identified species with >88% accuracy. DISCUSSION: The FGVC competitions provide a unique opportunity for computer vision and machine learning experts to address difficult species-recognition problems. The Herbarium 2019 Challenge brought together a novel combination of collections resources, taxonomic expertise, and collaboration between botanists and computer scientists.

Gymnosperms on the EDGE.

Driven by limited resources and a sense of urgency, the prioritization of species for conservation has been a persistent concern in conservation science. Gymnosperms (comprising ginkgo, conifers, cycads, and gnetophytes) are one of the most threatened groups of living organisms, with 40% of the species at high risk of extinction, about twice as many as the most recent estimates for all plants (i.e. 21.4%). This high proportion of species facing extinction highlights the urgent action required to secure their future through an objective prioritization approach. The Evolutionary Distinct and Globally Endangered (EDGE) method rapidly ranks species based on their evolutionary distinctiveness and the extinction risks they face. EDGE is applied to gymnosperms using a phylogenetic tree comprising DNA sequence data for 85% of gymnosperm species (923 out of 1090 species), to which the 167 missing species were added, and IUCN Red List assessments available for 92% of species. The effect of different extinction probability transformations and the handling of IUCN data deficient species on the resulting rankings is investigated. Although top entries in our ranking comprise species that were expected to score well (e.g. Wollemia nobilis, Ginkgo biloba), many were unexpected (e.g. Araucaria araucana). These results highlight the necessity of using approaches that integrate evolutionary information in conservation science.

Relative quantitation of virus population size in mixed genotype infections using sequencing chromatograms.

In order to quantitatively distinguish between highly similar RNA sequences, specific primers or probes must be designed. Unfortunately, consistent and reliable results are not always obtained with conventional techniques. This study uses reverse transcription-PCR coupled with direct terminator sequencing to economically and efficiently distinguish between sequence types in pooled samples while providing accurate relative quantification. As an example, the method is applied to measure template concentration of two Barley yellow dwarf virus (BYDV; family Luteoviridae) species in doubly infected wheat plants. A PERL script (polySNP) was developed that uses PHRED to automatically extract relative peak areas and heights from sequencing chromatograms at polymorphic sites. Peak measurements from experimental samples were compared to a standard curve generated by mixing in vitro transcribed RNA from BYDV-PAV and PAS templates in several ratios (ranging from 1:9 to 9:1 PAV:PAS) prior to RT-PCR amplification and sequencing. The relative amount of RNA template added to a sample was regressed onto the proportion of the chromatogram peak height or area corresponding to one virus species. The function of the best fit line was used to calculate template frequency in the experimental samples.

A comparison of algorithms for the identification of specimens using DNA barcodes: examples from gymnosperms.

In order to use DNA sequences for specimen identification (e.g., barcoding, fingerprinting) an algorithm to compare query sequences with a reference database is needed. Precision and accuracy of query sequence identification was estimated for hierarchical clustering (parsimony and neighbor joining), similarity methods (BLAST, BLAT and megaBLAST), combined clustering/similarity methods (BLAST/parsimony and BLAST/neighbor joining), diagnostic methods (DNA-BAR and DOME ID), and a new method (ATIM). We offer two novel alignment-free algorithmic solutions (DOME ID and ATIM) to identify query sequences for the purposes of DNA barcoding. Publicly available gymnosperm nrITS 2 and plastid matK sequences were used as test data sets. On the test data sets, almost all of the methods were able to accurately identify sequences to genus; however, no method was able to accurately identify query sequences to species at a frequency that would be considered useful for routine specimen identification (42-71% unambiguously correct). Clustering methods performed the worst (perhaps due to alignment issues). Similarity methods, ATIM, DNA-BAR, and DOME ID all performed at approximately the same level. Given the relative precision of the algorithms (median = 67% unambiguous), the low accuracy of species-level identification observed could be ascribed to the lack of correspondence between patterns of allelic similarity and species delimitations. Application of DNA barcoding to sequences of CITES listed cycads (Cycadopsida) provides an example of the potential application of DNA barcoding to enforcement of conservation laws.

Parallel reductions in phenolic constituents resulting from the domestication of eggplant.

Crop domestication is often accompanied by changes in metabolite compositions that alter traits such as flavor, color, or other beneficial properties. Fruits of eggplants (Solanum melongena L.) and related species are abundant and diverse in pharmacologically interesting phenolic compounds, particularly hydroxycinnamic acid (HCA) conjugates such as the antioxidant caffeoylquinic acids (CQA) and HCA-polyamine amides (HCAA). To understand metabolite variability through the lens of natural and artificial selection, HPLC-DAD was used to generate phenolic profiles for 32 compounds in fruits from 93 accessions representing 9 Solanum species. Profiles were used for identification of species-level and infraspecific chemical patterns across both genetic distance and landscape. Sampling of plant lines included the undomesticated progenitor of eggplant and Asian landraces with a genetic background associated with three Asian regions near proposed separate centers of domestication to test whether chemical changes were convergent despite different origins. Results showed ten compounds were unique to species, and ten other compounds varied significantly in abundance among species. Five CQAs and three HCA-polyamine conjugates were more abundant in wild (undomesticated) versus domesticated eggplant, indicating that artificial selection may have led to reduced phenolic levels. No chemical abundance patterns were associated with site-origin. However, one genetically distinct lineage of geographically-restricted SE Asian eggplants (S. melongena subsp. ovigerum) had a higher HCAA content and diversity than other lineages, which is suggested to be related to artificial selection for small, firm fruit. Overall, patterns show that fruit size, palatability and texture were preferentially selected over health-beneficial phytochemical content during domestication of several nightshade crops.