Using low-coverage whole genome sequencing (genome skimming) to delineate three introgressed species of buffalofish (Ictiobus).

Consumption of buffalofish has been sporadically associated with Haff disease-like illnesses involving sudden onset muscle pain and weakness due to skeletal muscle rhabdomyolysis, but determination of precisely which species are associated with these illnesses has been impeded by a lack of species-specific DNA-based markers. Here, three closely related species of buffalofish native to the Mississippi River Basin (Ictiobus bubalus, Ictiobus cyprinellus and Ictiobus niger) that have previously proven genetically indistinguishable using both mitochondrial and nuclear single-locus sequencing were reliably discriminated using low-coverage whole genome sequencing ('genome skimming'). Using 44 specimens representing the three species collected from the mid/upper (Missouri) and lower (Louisiana) regions of the species' native ranges, the SISRS (Site Identification from Short Read Sequences) bioinformatics pipeline was adapted to (1) identify over 620Mbp of putatively homologous nuclear sequence data and (2) isolate over 140,000 single-nucleotide polymorphisms (SNPs) that supported accurate species delimitation, all without the use of a reference genome or annotation data. These sites were used to classify Ictiobus spp. samples with genome-skim data, along with a larger set (n = 67) where ultraconserved elements (UCEs) were sequenced. Analyses of whole mitochondrial data revealed more limited signal. Nearly all samples matched their purported species based on morphologic identification, but two Missouri samples morphologically identified as I. niger grouped with samples of I. bubalus, albeit with significant enrichment of I. niger SNPs. To our knowledge this is the first report of a DNA-based tool to reliably discriminate these three morphologically distinct species.

A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States.

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.

The Low Copy Nuclear Gene Region, Granule Bound Starch Synthase (GBSS1), as a Novel Mini-DNA Barcode for the Identification of Different Sage (Salvia) Species.

Morphological similarity within species makes the identification and authentication of Salvia species challenging, especially in dietary supplements that contain processed root or leaf powder of different sage species. In the present study, the species discriminatory power of 2 potential DNA barcode regions from the nuclear genome was evaluated in 7 medicinally important Salvia species from the family Lamiaceae. The nuclear internal transcribed spacer 2 and the exon 9 - 14 region of low copy nuclear gene WAXY coding for granule-bound starch synthase 1 were tested for their species discrimination ability using distance, phylogenetic, and BLAST-based methods. A novel 2-step PCR method with 2 different annealing temperatures was developed to achieve maximum amplification from genomic DNA. The granule-bound starch synthase 1 region showed higher amplification and sequencing success rates, higher interspecific distances, and a perfect barcode gap for the tested species compared to the nuclear internal transcribed spacer 2. Hence, these novel mini-barcodes generated from low copy nuclear gene regions (granule-bound starch synthase) that were proven to be effective barcodes for identifying 7 Salvia species have potential for identification and authentication of other Salvia species.

Development and Validation of a Species-specific PCR Method for the Identification of Ginseng Species Using Orthogonal Approaches.

When testing botanical ingredients of herbal medicines and dietary supplements, the complexity of botanical matrixes often requires the use of orthogonal methods to establish identification procedures suitable for quality control purposes. Genomic-based botanical identification methods are evolving and emerging as useful quality control tools to complement traditional morphological and chemical identification methods. Species-specific polymerase chain reaction methods are being evaluated for botanical quality control and as a cost-effective approach to identify and discriminate between closely related botanical species. This paper describes orthogonal identification of Panax ginseng, P. quinquefolius, and P. notoginseng materials in commerce as an example of the development and validation of a set of species-specific polymerase chain reaction methods to establish botanical identity in ginseng roots. This work also explored the possibility of extending the application of species-specific polymerase chain reaction methods to provide species identity information for processed materials, such as steamed roots and hydroalcoholic extracts, and showed success with this approach. Finally, the paper provides recommendations for an out-of-specification investigation of samples that may pass some of the orthogonal tests and fail others.

Large outbreak of Jimsonweed (Datura stramonium) poisoning due to consumption of contaminated humanitarian relief food: Uganda, March-April 2019.

BACKGROUND: Jimsonweed (Datura stramonium) contains toxic alkaloids that cause gastrointestinal and central nervous system symptoms when ingested. This can be lethal at high doses. The plant may grow together with leguminous crops, mixing with them during harvesting. On 13 March 2019, more than 200 case-patients were admitted to multiple health centres for acute gastrointestinal and neurologic symptoms. We investigated to determine the cause and magnitude of the outbreak and recommended evidence-based control and prevention measures. METHODS: We defined a suspected case as sudden onset of confusion, dizziness, convulsions, hallucinations, diarrhoea, or vomiting with no other medically plausible explanations in a resident of Napak or Amudat District from 1 March-30 April 2019. We reviewed medical records and canvassed all villages of the eight affected subcounties to identify cases. In a retrospective cohort study conducted in 17 villages that reported the earliest cases, we interviewed 211 residents about dietary history during 11-15 March. We used modified Poisson regression to assess suspected food exposures. Food samples underwent chemical (heavy metals, chemical contaminants, and toxins), proteomic, DNA, and microbiological testing in one national and three international laboratories. RESULTS: We identified 293 suspected cases; five (1.7%) died. Symptoms included confusion (62%), dizziness (38%), diarrhoea (22%), nausea/vomiting (18%), convulsions (12%), and hallucinations (8%). The outbreak started on 12 March, 2-12 h after Batch X of fortified corn-soy blend (CSB +) was distributed. In the retrospective cohort study, 66% of 134 persons who ate CSB + , compared with 2.2% of 75 who did not developed illness (RRadj = 22, 95% CI = 6.0-81). Samples of Batch X distributed 11-15 March contained 14 tropane alkaloids, including atropine (25-50 ppm) and scopolamine (1-10 ppm). Proteins of Solanaceae seeds and Jimsonweed DNA were identified. No other significant laboratory findings were observed. CONCLUSION: This was the largest documented outbreak caused by food contamination with tropane alkaloids. Implicated food was immediately withdrawn. Routine food safety and quality checks could prevent future outbreaks.

HPLC-UV, Metabarcoding and Genome Skims of Botanical Dietary Supplements: A Case Study in Echinacea.

The use of DNA-based methods to authenticate botanical dietary supplements has been vigorously debated for a variety of reasons. More comparisons of DNA-based and chemical methods are needed, and concordant evaluation of orthogonal approaches on the same products will provide data to better understand the strengths and weaknesses of both approaches. The overall application of DNA-based methods is already firmly integrated into a wide array of continually modernizing stand alone and complementary authentication protocols. Recently, the use of full-length chloroplast genome sequences provided enhanced discriminatory capacity for closely related species of Echinacea compared to traditional DNA barcoding approaches (matK and rbcL). Here, two next-generation sequencing approaches were used: (1) genome skimming and (2) PCR amplicon (metabarcoding). The two genetic approaches were then combined with HPLC-UV to evaluate 20 commercially available dietary supplements of Echinacea representing "finished" products. The trade-offs involved in different DNA approaches were discussed, with a focus on how DNA methods support existing, accepted chemical methods. In most of the products (19/20), HPLC-UV suggested the presence of Echinacea spp. While metabarcoding was not useful with this genus and instead only resolved 7 products to the family level, genome skimming was able to resolve to species (9) or genus (1) with the 10/20 products where it was successful. Additional ingredients that HPLC-UV was unable to identify were also found in four products along with the relative sequence proportion of the constituents. Additionally, genome skimming was able to identify one product that was a different Echinacea species entirely.

Using full chloroplast genomes of 'red' and 'yellow' Bixa orellana (achiote) for kmer based identification and phylogenetic inference.

BACKGROUND: Full chloroplast genomes provide high resolution taxonomic discrimination between closely related plant species and are quickly replacing single and multi-locus barcoding regions as reference materials of choice for DNA based taxonomic annotation of plants. Bixa orellana, commonly known as "achiote" and "annatto" is a plant used for both human and animal foods and was thus identified for full chloroplast sequencing for the Center for Veterinary Medicine (CVM) Complete Chloroplast Animal Feed database. This work was conducted in collaboration with the Instituto de Medicina Tradicional (IMET) in Iquitos, Peru. There is a wide range of color variation in pods of Bixa orellana for which genetic loci that distinguish phenotypes have not yet been identified. Here we apply whole chloroplast genome sequencing of "red" and "yellow" individuals of Bixa orellana to provide high quality reference genomes to support kmer database development for use identifying this plant from complex mixtures using shotgun data. Additionally, we describe chloroplast gene content, synteny and phylogeny, and identify an indel and snp that may be associated with seed pod color. RESULTS: Fully assembled chloroplast genomes were produced for both red and yellow Bixa orellana accessions (158,918 and 158,823 bp respectively). Synteny and gene content was identical to the only other previously reported full chloroplast genome of Bixa orellana (NC_041550). We observed a 17 base pair deletion at position 58,399-58,415 in both accessions, relative to NC_041550 and a 6 bp deletion at position 75,531-75,526 and a snp at position 86,493 in red Bixa orellana. CONCLUSIONS: Our data provide high quality reference genomes of individuals of red and yellow Bixa orellana to support kmer based identity markers for use with shotgun sequencing approaches for rapid, precise identification of Bixa orellana from complex mixtures. Kmer based phylogeny of full chloroplast genomes supports monophylly of Bixaceae consistent with alignment based approaches. A potentially discriminatory indel and snp were identified that may be correlated with the red phenotype.

Characterization of Dinophysis spp. (Dinophyceae, Dinophysiales) from the mid-Atlantic region of the United States1.

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a "small Dinophysis sp." that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.

Assessment of the Authenticity of Herbal Dietary Supplements: Comparison of Chemical and DNA Barcoding Methods.

About 7 % of the U. S. population reports using botanical dietary supplements. Increased use of such supplements has led to discussions related to their authenticity and quality. Reports of adulteration with substandard materials or pharmaceuticals are of concern because such substitutions, whether inadvertent or deliberate, may reduce the efficacy of specific botanicals or lead to adverse events. Methods for verifying the identity of botanicals include macroscopic and microscopic examinations, chemical analysis, and DNA-based methods including DNA barcoding. Macroscopic and microscopic examinations may fail when a supplement consists of botanicals that have been processed beyond the ability to provide morphological characterizations. Chemical analysis of specific marker compounds encounters problems when these compounds are not distinct to a given species or when purified reference standards are not available. Recent investigations describing DNA barcoding analysis of botanical dietary supplements have raised concerns about the authenticity of the supplements themselves as well as the appropriateness of using DNA barcoding techniques with finished botanical products. We collected 112 market samples of frequently consumed botanical dietary supplements of ginkgo, soy, valerian, yohimbe, and St. John's wort and analyzed each for specific chemical markers (i.e., flavonol glycosides, total isoflavones, total valerenic acids, yohimbine, and hypericins, respectively). We used traditional DNA barcoding techniques targeting the nuclear ITS2 gene and the chloroplast gene psbA-trnH on the same samples to determine the presence of DNA of the labelled ingredient. We compared the results obtained by both methods to assess the contribution of each in determining the identity of the samples.

Development of a Reference Standard Library of Chloroplast Genome Sequences, GenomeTrakrCP.

Precise, species-level identification of plants in foods and dietary supplements is difficult. While the use of DNA barcoding regions (short regions of DNA with diagnostic utility) has been effective for many inquiries, it is not always a robust approach for closely related species, especially in highly processed products. The use of fully sequenced chloroplast genomes, as an alternative to short diagnostic barcoding regions, has demonstrated utility for closely related species. The U. S. Food and Drug Administration (FDA) has also developed species-specific DNA-based assays targeting plant species of interest by utilizing chloroplast genome sequences. Here, we introduce a repository of complete chloroplast genome sequences called GenomeTrakrCP, which will be publicly available at the National Center for Biotechnology Information (NCBI). Target species for inclusion are plants found in foods and dietary supplements, toxin producers, common contaminants and adulterants, and their close relatives. Publicly available data will include annotated assemblies, raw sequencing data, and voucher information with each NCBI accession associated with an authenticated reference herbarium specimen. To date, 40 complete chloroplast genomes have been deposited in GenomeTrakrCP (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA325670/), and this will be expanded in the future.

Suspected Palytoxin Inhalation Exposures Associated with Zoanthid Corals in Aquarium Shops and Homes - Alaska, 2012-2014.

On August 12, 2014, an Anchorage hospital notified the Alaska Section of Epidemiology (SOE) that a middle-aged male resident of Anchorage (patient A) had arrived in the emergency department with possible palytoxin exposure. Patient A complained of a bitter metallic taste, fever, weakness, cough, and muscle pain 7-8 hours after introduction of live zoanthid coral into his home aquarium. Palytoxin, a potent toxin known to produce the reported effects, is contained in zoanthid marine corals.

Tetrodotoxin poisoning outbreak from imported dried puffer fish--Minneapolis, Minnesota, 2014.

On June 13, 2014, two patients went to the Hennepin County Medical Center Emergency Department in Minneapolis, Minnesota, with symptoms suggestive of tetrodotoxin poisoning (i.e., oral paresthesias, weakness, and dyspnea) after consuming dried puffer fish (also known as globefish) purchased during a recent visit to New York City. The patients said two friends who consumed the same fish had similar, although less pronounced, symptoms and had not sought care. The Minnesota Department of Health conducted an investigation to determine the source of the product and samples were sent to the Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition for chemical and genetic analysis. Genetic analysis identified the product as puffer fish (Lagocephalus lunaris) and chemical analysis determined it was contaminated with high levels of tetrodotoxin. A traceback investigation was unable to determine the original source of the product. Tetrodotoxin is a deadly, potent poison; the minimum lethal dose in an adult human is estimated to be 2-3 mg. Tetrodotoxin is a heat-stable and acid-stable, nonprotein, alkaloid toxin found in many species of the fish family Tetraodontidae (puffer fish) as well as in certain gobies, amphibians, invertebrates, and the blue-ringed octopus. Tetrodotoxin exerts its effects by blocking voltage-activated sodium channels, terminating nerve conduction and muscle action potentials, leading to progressive paralysis and, in extreme cases, to death from respiratory failure. Because these fish were reportedly purchased in the United States, they pose a substantial U.S. public health hazard given the potency of the toxin and the high levels of toxin found in the fish.

Protocol for building a reference standard sequence library for DNA-based seafood identification.

With the recent adoption of a DNA sequencing-based method for the species identification for seafood products by the U.S. Food and Drug Administration (FDA), a library of standard sequences derived from reference specimens with authoritative taxonomic authentication was required. Provided here are details of how the FDA and its collaborators are building this reference standard sequence library that will be used to confirm the accurate labeling of seafood products sold in interstate commerce in the United States. As an example data set from this library, information for 117 fish reference standards, representing 94 species from 43 families in 15 orders, collected over a 4-year period from the Gulf of Mexico, U.S., that are now stored at the Smithsonian Museum Support Center in Suitland, MD, are provided.

Dynamics of actin evolution in dinoflagellates.

Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5' UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop codons. Overall, variation in the actin gene family fits best with the "birth and death" model of evolution based on recent duplications, pseudogenes, and incomplete lineage sorting. Divergence between species was similar to variation within species, so that actin may be too conserved to be useful for phylogenetic estimation of closely related species.

Reference-free discovery of nuclear SNPs permits accurate, sensitive identification of Carya (hickory) species and hybrids.

PREMISE: DNA-based species identification is critical when morphological identification is restricted, but DNA-based identification pipelines typically rely on the ability to compare homologous sequence data across species. Because many clades lack robust genomic resources, we present here a bioinformatics pipeline capable of generating genome-wide single-nucleotide polymorphism (SNP) data while circumventing the need for any reference genome or annotation data. METHODS: Using the SISRS bioinformatics pipeline, we generated de novo ortholog data for the genus Carya, isolating sites where genetic variation was restricted to a single Carya species (i.e., species-informative SNPs). We leveraged these SNPs to identify both full-species and hybrid Carya specimens, even at very low sequencing depths. RESULTS: We identified between 46,000 and 476,000 species-identifying SNPs for each of eight diploid Carya species, and all species identifications were concordant with the species of record. For all putative F1 hybrid specimens, both parental species were correctly identified in all cases, and more punctate patterns of introgression were detectable in more cryptic crosses. DISCUSSION: Bioinformatics pipelines that use only short-read sequencing data provide vital new tools enabling rapid expansion of DNA identification assays for model and non-model clades alike.

Haff disease associated with consumption of buffalofish (Ictiobus spp.) in the United States, 2010-2020, with confirmation of the causative species.

BACKGROUND: In the United States, buffalofish (Ictiobus spp.) are sporadically associated with sudden onset muscle pain and weakness due to rhabdomyolysis within 24 h of fish consumption (Haff disease). Previous genetic analyses of case-associated samples were unable to distinguish the three species of buffalofish that occur in the US, Ictiobus cyprinellus (bigmouth buffalo), Ictiobus bubalus (smallmouth buffalo), and Ictiobus niger (black buffalo). METHODS: Ten events were investigated between 2010 and 2020 and demographic and clinical information was collected for 24 individuals. Meal remnants were collected from 5 of 10 events with additional associated samples (n = 24) collected from another five of 10 events. Low-coverage whole-genome sequencing (genome skimming) was used to identify meal remnants. RESULTS: Patients (26-75 years of age) ranged from 1-4 per event, with 90% involving ≥2 individuals. Reported symptoms included muscle tenderness and weakness, nausea/vomiting, and brown/tea-colored urine. Median incubation period was 8 h. Ninety-six percent of cases were hospitalized with a median duration of four days. The most commonly reported laboratory finding was elevated creatine phosphokinase and liver transaminases. Treatment was supportive including intravenous fluids to prevent renal failure. Events occurred in California (1), Illinois (2), Louisiana (1), New York (1), Mississippi (1), Missouri (2), New Jersey (1), and Texas (1) with location of harvest, when known, being Illinois, Louisiana, Mississippi, Missouri, Texas, and Wisconsin. Meal remnants were identified as I. bubalus (n = 4) and I. niger (n = 1). Associated samples were identified as I. bubalus (n = 16), I. cyprinellus (n = 5), and I. niger (n = 3). DISCUSSION: Time course, presentation of illness, and clinical findings were all consistent with previous domestic cases of buffalofish-associated Haff disease. In contrast to previous reports that I. cyprinellus is the causative species in US cases, data indicate that all three buffalofish species are harvested but I. bubalus is most often associated with illness.

Screening the PRISM Library against Staphylococcus aureus Reveals a Sesquiterpene Lactone from Liriodendron tulipifera with Inhibitory Activity.

Infections caused by the bacterium Staphylococcus aureus continue to pose threats to human health and put a financial burden on the healthcare system. The overuse of antibiotics has contributed to mutations leading to the emergence of methicillin-resistant S. aureus, and there is a critical need for the discovery and development of new antibiotics to evade drug-resistant bacteria. Medicinal plants have shown promise as sources of new small-molecule therapeutics with potential uses against pathogenic infections. The principal Rhode Island secondary metabolite (PRISM) library is a botanical extract library generated from specimens in the URI Youngken Medicinal Garden by upper-division undergraduate students. PRISM extracts were screened for activity against strains of methicillin-susceptible S. aureus (MSSA). An extract generated from the tulip tree (Liriodendron tulipifera) demonstrated growth inhibition against MSSA, and a bioassay-guided approach identified a sesquiterpene lactone, laurenobiolide, as the active constituent. Intriguingly, its isomers, tulipinolide and epi-tulipinolide, lacked potent activity against MSSA. Laurenobiolide also proved to be more potent against MSSA than the structurally similar sesquiterpene lactones, costunolide and dehydrocostus lactone. Laurenobiolide was the most abundant in the twig bark of the tulip tree, supporting the twig bark's historical and cultural usage in poultices and teas.

Alveolate phylogeny inferred using concatenated ribosomal proteins.

Dinoflagellates and apicomplexans are a strongly supported monophyletic group in rDNA phylogenies, although this phylogeny is not without controversy, particularly between the two groups. Here we use concatenated protein-coding genes from expressed sequence tags or genomic data to construct phylogenies including "typical" dinophycean dinoflagellates, a parasitic syndinian dinoflagellate, Amoebophrya sp., and two related species, Oxyrrhis marina, and Perkinsus marinus. Seventeen genes encoding proteins associated with the ribosome were selected for phylogenetic analysis. The dataset was limited for the most part by data availability from the dinoflagellates. Forty-five taxa from four major lineages were used: the heterokont outgroup, ciliates, dinoflagellates, and apicomplexans. Amoebophrya sp. was included in this phylogeny as a sole representative of the enigmatic marine alveolate or syndinian lineage. The atypical dinoflagellate O. marina, usually excluded from rDNA analyses due to long branches, was also included. The resulting phylogenies were well supported in concatenated analyses with only a few unstable or weakly supported branches; most features were consistent when different lineages were pruned from the tree or different genes were concatenated. The least stable branches involved the placement of Cryptosporidium spp. within the Apicomplexa and the relationships between P. marinus, Amoebophrya sp., and O. marina. Both bootstrap and approximately unbiased test results confirmed that P. marinus, Amoebophrya sp., O. marina, and the remaining dinoflagellates form a monophyletic lineage to the exclusion of Apicomplexa.

A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance.

The U.S. Food and Drug Administration is responsible for ensuring that the nation's food supply is safe and accurately labeled. This task is particularly challenging in the case of seafood where a large variety of species are marketed, most of this commodity is imported, and processed product is difficult to identify using traditional morphological methods. Reliable species identification is critical for both foodborne illness investigations and for prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value. New methods that allow accurate and rapid species identifications are needed, but any new methods to be used for regulatory compliance must be both standardized and adequately validated. "DNA barcoding" is a process by which species discriminations are achieved through the use of short, standardized gene fragments. For animals, a fragment (655 base pairs starting near the 5' end) of the cytochrome c oxidase subunit 1 mitochondrial gene has been shown to provide reliable species level discrimination in most cases. We provide here a protocol with single-laboratory validation for the generation of DNA barcodes suitable for the identification of seafood products, specifically fish, in a manner that is suitable for FDA regulatory use.

Utilizing Big Data to Identify Tiny Toxic Components: Digitalis.

The botanical genus Digitalis is equal parts colorful, toxic, and medicinal, and its bioactive compounds have a long history of therapeutic use. However, with an extremely narrow therapeutic range, even trace amounts of Digitalis can cause adverse effects. Using chemical methods, the United States Food and Drug Administration traced a 1997 case of Digitalis toxicity to a shipment of Plantago (a common ingredient in dietary supplements marketed to improve digestion) contaminated with Digitalis lanata. With increased accessibility to next generation sequencing technology, here we ask whether this case could have been cracked rapidly using shallow genome sequencing strategies (e.g., genome skims). Using a modified implementation of the Site Identification from Short Read Sequences (SISRS) bioinformatics pipeline with whole-genome sequence data, we generated over 2 M genus-level single nucleotide polymorphisms in addition to species-informative single nucleotide polymorphisms. We simulated dietary supplement contamination by spiking low quantities (0-10%) of Digitalis whole-genome sequence data into a background of commonly used ingredients in products marketed for "digestive cleansing" and reliably detected Digitalis at the genus level while also discriminating between Digitalis species. This work serves as a roadmap for the development of novel DNA-based assays to quickly and reliably detect the presence of toxic species such as Digitalis in food products or dietary supplements using genomic methods and highlights the power of harnessing the entire genome to identify botanical species.