Consensus of the 'Malasars' traditional aboriginal knowledge of medicinal plants in the Velliangiri holy hills, India

There are many vanishing cultures that possess a wealth of knowledge on the medicinal utility of plants. The Malasars of Dravidian Tamils are an indigenous society occupying the forests of the Western Ghats, South India. They are known to be exceptional healers and keepers of traditional aboriginal knowledge (TAK) of the flora in the Velliangiri holy hills. In fact, their expertise is well known throughout India as evidenced by the thousands of pilgrims that go to the Velliangiri holy hills for healing every year. Our research is the first detailed study of medicinal plants in India that considers variation in TAK among informants using a quantitative consensus analysis. A total of 95 species belonging to 50 families were identified for medicinal and general health purposes. For each species the botanical name, family, local name, parts used, summary of mode of preparation, administration and curing are provided. The consensus analysis revealed a high level of agreement among the informants usage of a particular plant at a local scale. The average consensus index value of an informant was FIC > 0.71, and over 0.80 for some ailments such as respiratory and jaundice. Some of the more common problems faced by the Malasars were gastrointestinal disorders, respiratory illness, dermatological problems and simple illness such as fever, cough, cold, wounds and bites from poisonous animals. We also discovered several new ethnotaxa that have considerable medicinal utility. This study supports claims that the Malasars possess a rich TAK of medicinal plants and that many aboriginals and mainstream people (pilgrims) utilize medicinal plants of the Velliangiri holy hills. Unfortunately, the younger generation of Malasars are not embracing TAK as they tend to migrate towards lucrative jobs in more developed urban areas. Our research sheds some light on a traditional culture that believes that a healthy lifestyle is founded on a healthy environment and we suggest that TAK such as that of the Malasars may serve toward a global lifestyle of health and environmental sustainability

The power of DNA based methods in probiotic authentication

IntroductionThe global probiotic market is growing rapidly, and strict quality control measures are required to ensure probiotic product efficacy and safety. Quality assurance of probiotic products involve confirming the presence of specific probiotic strains, determining the viable cell counts, and confirming the absence of contaminant strains. Third-party evaluation of probiotic quality and label accuracy is recommended for probiotic manufacturers. Following this recommendation, multiple batches of a top selling multi-strain probiotic product were evaluated for label accuracy.MethodsA total of 55 samples (five multi-strain finished products and 50 single-strain raw ingredients) containing a total of 100 probiotic strains were evaluated using a combination of molecular methods including targeted PCR, non-targeted amplicon-based High Throughput Sequencing (HTS), and non-targeted Shotgun Metagenomic Sequencing (SMS).ResultsTargeted testing using species-specific or strain-specific PCR methods confirmed the identity of all strains/species. While 40 strains were identified to strain level, 60 strains were identified to species level only due to lack of strain-specific identification methods. In amplicon based HTS, two variable regions of 16S rRNA gene were targeted. Based on V5–V8 region data, ~99% of total reads per sample corresponded to target species, and no undeclared species were detected. Based on V3–V4 region data, ~95%–97% of total reads per sample corresponded to target species, while ~2%–3% of reads matched undeclared species (Proteus species), however, attempts to culture Proteus confirmed that all batches were free from viable Proteus species. Reads from SMS assembled to the genomes of all 10 target strains in all five batches of the finished product.DiscussionWhile targeted methods enable quick and accurate identification of target taxa in probiotic products, non-targeted methods enable the identification of all species in a product including undeclared species, with the caveats of complexity, high cost, and long time to result

Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2

IntroductionReliable and accurate methods for probiotic identification and enumeration, at the strain level plays a major role in confirming product efficacy since probiotic health benefits are strain-specific and dose-dependent. In this study, real-time PCR methods were developed for strain specific identification and enumeration of L. paracasei 8700:2, a probiotic strain that plays a role in fighting the common cold.MethodsThe assay was designed to target a unique region in L. paracasei 8700:2 genome sequence to achieve strain level specificity. The identification assay was evaluated for specificity and sensitivity. The enumeration viability real-time PCR (v-qPCR) method was first optimized for the viability treatment, then the method was evaluated for efficiency, limit of quantification, precision, and its performance was compared to plate count (PC) and viability droplet digital PCR (v-ddPCR) methods.ResultsThe identification method proved to be strain specific and highly sensitive with a limit of detection of 0.5 pg of DNA. The optimal viability dye (PMAxx) concentration was 50 μM. The method was efficient (> 90% with R2 values > 0.99), with a linear dynamic range between 6*102 and 6*105 copies. The method was highly precise with a relative standard deviation below 5%. The Pearson correlation coefficient (r) was 0.707 for PC and v-qPCR methods, and 0.922 for v-qPCR and v-ddPCR. Bland-Altman method comparison showed that v-qPCR always gave higher values compared to PC method (relative difference ranging from 119% to 184%) and showed no consistent trend (relative difference ranging from −20% to 22%) when comparing v-qPCR and v-ddPCR methods.DiscussionThe difference between PC and v-PCR methods can potentially be attributed to the proportion of cells that exist in a viable but non culturable (VBNC) state, which can be count by v-PCR but not with PC. The developed v-qPCR method was confirmed to be strain specific, sensitive, efficient, with low variance, able to count VBNC cells, and has shorter time to results compared to plate count methods. Thus, the identification and enumeration methods developed for L. paracasei 8700:2 will be of great importance to achieve high quality and efficacious probiotic products

Ethnobotany genomics - discovery and innovation in a new era of exploratory research

We present here the first use of DNA barcoding in a new approach to ethnobotany we coined "ethnobotany genomics". This new approach is founded on the concept of 'assemblage' of biodiversity knowledge, which includes a coming together of different ways of knowing and valorizing species variation in a novel approach seeking to add value to both traditional knowledge (TK) and scientific knowledge (SK). We employed contemporary genomic technology, DNA barcoding, as an important tool for identifying cryptic species, which were already recognized ethnotaxa using the TK classification systems of local cultures in the Velliangiri Hills of India. This research is based on several case studies in our lab, which define an approach to that is poised to evolve quickly with the advent of new ideas and technology. Our results show that DNA barcoding validated several new cryptic plant species to science that were previously recognized by TK classifications of the Irulas and Malasars, and were lumped using SK classification. The contribution of the local aboriginal knowledge concerning plant diversity and utility in India is considerable; our study presents new ethnomedicine to science. Ethnobotany genomics can also be used to determine the distribution of rare species and their ecological requirements, including traditional ecological knowledge so that conservation strategies can be implemented. This is aligned with the Convention on Biological Diversity that was signed by over 150 nations, and thus the world's complex array of human-natural-technological relationships has effectively been re-organized

Valorizing the 'Irulas' traditional knowledge of medicinal plants in the Kodiakkarai Reserve Forest, India

A mounting body of critical research is raising the credibility of Traditional Knowledge (TK) in scientific studies. These studies have gained credibility because their claims are supported by methods that are repeatable and provide data for quantitative analyses that can be used to assess confidence in the results. The theoretical importance of our study is to test consensus (reliability/replicable) of TK within one ancient culture; the Irulas of the Kodiakkarai Reserve Forest (KRF), India. We calculated relative frequency (RF) and consensus factor (Fic) of TK from 120 Irulas informants knowledgeable of medicinal plants. Our research indicates a high consensus of the Irulas TK concerning medicinal plants. The Irulas revealed a diversity of plants that have medicinal and nutritional utility in their culture and specific ethnotaxa used to treat a variety of illnesses and promote general good health in their communities. Throughout history aboriginal people have been the custodians of bio-diversity and have sustained healthy life-styles in an environmentally sustainable manner. However this knowledge has not been transferred to modern society. We suggest this may be due to the asymmetry between scientific and TK, which demands a new approach that considers the assemblage of TK and scientific knowledge. A greater understanding of TK is beginning to emerge based on our research with both the Irulas and Malasars; they believe that a healthy lifestyle is founded on a healthy environment. These aboriginal groups chose to share this knowledge with society-at-large in order to promote a global lifestyle of health and environmental sustainability

Detection of Cistanches Herba (Rou Cong Rong) Medicinal Products Using Species-Specific Nucleotide Signatures

Cistanches Herba is a medicinal plant that has tonification properties and is commonly used in Asia. Owing to the imbalance between supply and demand, adulterants are frequently added for profit. However, there is no regulatory oversight because quality control tools are not sufficient for identifying heavily processed products. Thus, a novel molecular tool based on nucleotide signatures and species-specific primers was developed. The ITS2 regions from 251 Cistanches Herba and adulterant samples were sequenced. On the basis of SNP sites, four nucleotide signatures within 30~37 bp and six species-specific primers were developed, and they were validated by artificial experimental mixtures consisting of six different species and different ratios. This method was also applied to detect 66 Cistanches Herba products on the market, including extracts and Chinese patent medicines. The results demonstrated the utility of nucleotide signatures in identifying adulterants in mixtures. The market study revealed 36.4% adulteration: 19.7% involved adulteration with Cynomorium songaricum or Cistanche sinensis, and 16.7% involved substitution with Cy. songaricum, Ci. sinensis, or Boschniakia rossica. The results also revealed that Cy. songaricum was the most common adulterant in the market. Thus, we recommend the use of species-specific nucleotide signatures for regulating adulteration and verifying the quality assurance of medicinal product supply chains, especially for processed products whose DNA is degraded

Exploring DNA quantity and quality from raw materials to botanical extracts

Objectives: The aim of this study was to explore the variability in DNA quality and quantity along a gradient of industrial processing of botanical ingredients from raw materials to extracts. Methods: A data matrix was assembled for 1242 botanical ingredient samples along a gradient of industrial processing commonly used in the Natural Health Product (NHP) industry. Multivariate statistics was used to explore dependant variables for quality and quantity. The success of attaining a positive DNA test result along a gradient of industrial processing was compared among four biotechnologies: DNA barcoding, NGS, Sanger sequencing and qPCR. Results: There was considerable variance in DNA quality and quantity among the samples, which could be interpreted along a gradient from raw materials with greater quantities (50–120 ng/μL) of DNA and longer DNA (400-500bp) sequences to extracts, which were characterized by lower quantities (0.1–10.0 ng/μL) and short fragments (50-150bp). Conclusions: Targeted molecular diagnostic tests for species identity can be used in the NHP industry for raw and processed samples. Non-targeted tests or the use of NGS for any identity test needs considerable research and development and must be validated before it can be used in commercial operations as these methods are subject to considerable risk of false negative and positive results. Proper use of these tools can be used to ensure ingredient authenticity, and to avert adulteration, and contamination with plants that are a health concern. Lastly these tools can be used to prevent the exploitation of rare herbal species and the harvesting of native biodiversity for commercial purposes

Anthropogenic Disturbance and Population Viability of Woodland Caribou in Ontario

One of the most challenging tasks in wildlife conservation and management is to clarify how spatial variation in land cover due to anthropogenic disturbance influences wildlife demography and long‐term viability. To evaluate this, we compared rates of survival and population growth by woodland caribou (Rangifer tarandus caribou) from 2 study sites in northern Ontario, Canada that differed in the degree of anthropogenic disturbance because of commercial logging and road development, resulting in differences in predation risk due to gray wolves (Canis lupus). We used an individual‐based model for population viability analysis (PVA) that incorporated adaptive patterns of caribou movement in relation to predation risk and food availability to predict stochastic variation in rates of caribou survival. Field estimates of annual survival rates for adult female caribou in the unlogged ( x̄ = 0.90) and logged ( x̄ = 0.76) study sites recorded during 2010–2014 did not differ significantly (P \u3e 0.05) from values predicted by the individual‐based PVA model (unlogged:  x̄ = 0.87; logged:  x̄ = 0.79). Outcomes from the individual‐based PVA model and a simpler stage‐structured matrix model suggest that substantial differences in adult survival largely due to wolf predation are likely to lead to long‐term decline of woodland caribou in the commercially logged landscape, whereas the unlogged landscape should be considerably more capable of sustaining caribou. Estimates of population growth rates (λ) for the 2010–2014 period differed little between the matrix model and the individual‐based PVA model for the unlogged (matrix model  x̄ = 1.01; individual‐based model x̄ = 0.98) and logged landscape (matrix model x̄ = 0.88; individual‐based model x̄ = 0.89). We applied the spatially explicit PVA model to assess the viability of woodland caribou across 14 woodland caribou ranges in Ontario. Outcomes of these simulations suggest that woodland caribou ranges that have experienced significant levels of commercial forestry activities in the past had annual growth rates 0.96. These differences were strongly related to regional variation in wolf densities. Our results suggest that increased wolf predation risk due to anthropogenic disturbance is of sufficient magnitude to cause appreciable risk of population decline in woodland caribou in Ontario. © 2020 The Authors. The Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society

Multiple Multilocus DNA Barcodes from the Plastid Genome Discriminate Plant Species Equally Well

A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF–atpH, and psbK–psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85–100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK–psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69–71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems