Identification of Lens Cultivars in Market by Molecular Tools: DNA Barcoding and SSRs

Substitution of plant cultivars of high commercial value with a cheaper, lower-quality one is a common fraud committed against consumers and producers. Since it is one of the most widely grown legumes, lentil (Lens culinaris Medik.) are a suitable for such frauds. This study aimed to identify lentil cultivars which are registered and authorized in the market in Turkey by using current molecular methods. For this purpose, 26 lentil cultivars were analyzed for 15 SSR markers and 2 DNA barcode loci (trnH-psbA and matK). A high allele diversity was observed by 12 scorable SSR markers, and the average number of alleles was determined to be 16. One of the important findings was the presence of “cultivar-specific alleles” that can be used to identify each cultivar in the lentil market in Turkey. At least one “cultivar-specific allele” was obtained for each cultivar. The lentil cultivars were analyzed in terms of 2 DNA barcode regions as trnH-psbA and matK. Sequences that could identify 14 of the 26 cultivars were obtained. While the rate of the intra-species variation for the trnH-psbA region was observed to be low, a higher rate was found for matK. Nevertheless, it was observed that intra-species discrimination can be made more effective when both loci are used together. We expect that the results of this study, especially the cultivar-specific SSR alleles and DNA barcoding sequence data may be used routinely to identify on production and packaged products that are commercially available in markets.

Species discrimination of novel chloroplast DNA barcodes and their application for identification of Panax (Aralioideae, Araliaceae)

Certain species within the genus Panax L. (Araliaceae) contain pharmacological precious ginsenosides, also known as ginseng saponins. Species containing these compounds are of high commercial value and are thus of particular urgency for conservation. However, within this genus, identifying the particular species that contain these compounds by morphological means is challenging. DNA barcoding is one method that is considered promising for species level identification. However, in an evolutionarily complex genus such as Panax, commonly used DNA barcodes such as nrITS, matK, psbA-trnH, rbcL do not provide species-level resolution. A recent in silico study proposed a set of novel chloroplast markers, trnQ-rps16, trnS-trnG, petB, and trnE-trnT for species level identification within Panax. In the current study, the discriminatory efficiency of these molecular markers is assessed and validated using 91 reference barcoding sequences and 38 complete chloroplast genomes for seven species, one unidentified species and one sub-species of Panax, and two outgroup species of Aralia L. along with empirical data of Panax taxa present in Vietnam via both distance-based and tree-based methods. The obtained results show that trnQ-rps16 can classify with species level resolution every clade tested here, including the highly valuable Panax vietnamensis Ha et Grushv. We thus propose that this molecular marker to be used for identification of the species within Panax to support both its conservation and commercial trade.

Identification of CITES-Listed Euphorbia royleana through DNA Barcoding Technology: A New Facet in Wildlife Forensics

Disorganized and chaotic collection of the Euphorbia plant species from the wild is one of the major reasons for its endangered status. According to CITES, the trade in Euphorbia royleana species is prohibited under Appendix II. However, the trade continues unabated as current identification methods do not discriminate between closely related species. In the present study, a DNA barcoding method has been used to establish inter- and intra-specific divergences of both matK and rbcL regions by using pairwise genetic distance measurement methods for evaluating the maximum barcoding gap. The matk and rbcL yielded a 100% amplification and sequencing success rate to distinguish closely related species of Euphorbia royleana unambiguously. The matk and rbcL showed average interspecific genetic distance divergence values of 0.031and 0.015, respectively. The maximum number of species-specific SNPs was observed in matK sequences at seven consecutive sites, which could distinguish Euphorbia royleana from closely related species. The best candidate barcoding region to identify Euphorbia royleana was found to be matK with a single-locus barcoding approach. Furthermore, the species discrimination method was developed with the help of species-specific SNPs derived from the matK barcoding region to accurately authenticate Euphorbia royleana, and it provided 100% species resolution

The Feasibility of Two Handheld Spectrometers for Meat Speciation Combined with Chemometric Methods and Its Application for Halal Certification

Handheld visible-near-infrared (Vis-NIR) and near-infrared (NIR) spectroscopy can be cost-effective, rapid, non-destructive and transportable techniques for identifying meat species and may be valuable for enforcement authorities, retail and consumers. In this study, a handheld Vis-NIR (400–1000 nm) and a handheld NIR (900–1700 nm) spectrometer were applied to discriminate halal meat species from pork (halal certification), as well as speciation of intact and ground lamb, beef, chicken and pork (160 meat samples). Several types of class modeling multivariate approaches were applied. The presented one-class classification (OCC) approach, especially with the Vis-NIR sensor (95–100% correct classification rate), was found to be suitable for the application of halal from non-halal meat-species discrimination. In a discriminant approach, using the Vis-NIR data and support vector machine (SVM) classification, the four meat species tested could be classified with accuracies of 93.4% and 94.7% for ground and intact meat, respectively, while with partial least-squares discriminant analysis (PLS-DA), classification accuracies were 87.4% (ground) and 88.6% (intact). Using the NIR sensor, total accuracies of the SVM models were 88.2% and 81.5% for ground and intact meats, respectively, and PLS-DA classification accuracies were 88.3% (ground) and 80% (intact). We conclude that the Vis-NIR sensor was most successful in the halal certification (OCC approaches) and speciation (discriminant approaches) for both intact and ground meat using SVM.

Dead man’s fingers point towards new species: A taxonomic revision of Alcyonium aurantiacum with statistical discrimination methods and a survey of integrative taxonomy in Octocorallia

<p><b>Octocorals are a diverse group of sessile, colonial, filter-feeding anthozoan cnidarians, which form significant components of benthic marine communities worldwide. Globally, the most critical hurdle to the effective management of octocorals in the face of increasing anthropogenic pressure is the poor state of their species-level taxonomy, which hinders understanding of their biodiversity. New Zealand’s octocoral assemblage is among the most diverse of any country and is characterised by high levels of endemism, yet over half of its octocoral species remain undescribed. While progress is being made, this has focussed almost exclusively on protected deep-sea gorgonian octocorals.</b></p> <p>Unprotected coastal soft corals are less studied in New Zealand. This includes the endemic Alcyonium aurantiacum Quoy and Gaimard, 1833. Multiple, morphologically diverse forms have been attributed to this species. Here, the taxonomic status of A. aurantiacum is reviewed, and its phylogenetic relationships are examined using molecular data (nuclear 28S and mitochondrial MutS genes), which is compared to morphology in an integrative approach. As a result, evidence for two new, endemic genera and ten new species is presented. Alcyonium aurantiacum is referred to Kotatea gen. n. (as K. aurantiaca comb. n.), which contains seven additional new species. A second genus, Ushanaia gen. n., contains three new species.</p> <p>Of the new taxa described herein, K. aurantiaca and K. lobata sp. n. are the most commonly encountered and widespread, yet little is known regarding their biology. Both species co-occur in their natural habitat, could not be differentiated genetically with the tools used here, and can be difficult to distinguish without microscopic sclerite examinations. To facilitate the identification of these two similar species by non-taxonomists, a statistical model was developed that can discriminate them with up to 90% accuracy using easily obtainable measurements of gross colony morphology. Relationships between colony morphology and depth are also examined.</p> <p>Considering the difficulties associated with species discrimination among octocorals, a literature survey was conducted to review the use of integrative taxonomy in this group since the start of the 21st century, focusing particularly on morpho-molecular data comparisons. This revealed that, while description rates at family, genus, and species levels over the last twenty-one years rank among the highest ever, integrative techniques have been applied unevenly across taxonomic groups and geographic regions and overall remain a minority compared to taxonomic research based solely on morphology. Implementation of the integrative approach is increasing, however, as are the per-annum number of taxonomic publications and the total pool of authors associated with these publications.</p> <p>It is hoped that the research presented herein can contribute to ongoing global efforts of revising octocoral systematics and that the examination of integrative practices in octocoral taxonomy will serve as a baseline against which future taxonomic progress can be compared and promoted. For New Zealand specifically, elucidating the taxonomy and variability of these endemic taxa will enable aspects such as their contribution to ecosystem functioning and management needs to be examined accurately for the first time, which in turn may lead to their recognition as organisms worthy of legal protection.</p>

Dead man’s fingers point towards new species: A taxonomic revision of Alcyonium aurantiacum with statistical discrimination methods and a survey of integrative taxonomy in Octocorallia

<p><b>Octocorals are a diverse group of sessile, colonial, filter-feeding anthozoan cnidarians, which form significant components of benthic marine communities worldwide. Globally, the most critical hurdle to the effective management of octocorals in the face of increasing anthropogenic pressure is the poor state of their species-level taxonomy, which hinders understanding of their biodiversity. New Zealand’s octocoral assemblage is among the most diverse of any country and is characterised by high levels of endemism, yet over half of its octocoral species remain undescribed. While progress is being made, this has focussed almost exclusively on protected deep-sea gorgonian octocorals.</b></p> <p>Unprotected coastal soft corals are less studied in New Zealand. This includes the endemic Alcyonium aurantiacum Quoy and Gaimard, 1833. Multiple, morphologically diverse forms have been attributed to this species. Here, the taxonomic status of A. aurantiacum is reviewed, and its phylogenetic relationships are examined using molecular data (nuclear 28S and mitochondrial MutS genes), which is compared to morphology in an integrative approach. As a result, evidence for two new, endemic genera and ten new species is presented. Alcyonium aurantiacum is referred to Kotatea gen. n. (as K. aurantiaca comb. n.), which contains seven additional new species. A second genus, Ushanaia gen. n., contains three new species.</p> <p>Of the new taxa described herein, K. aurantiaca and K. lobata sp. n. are the most commonly encountered and widespread, yet little is known regarding their biology. Both species co-occur in their natural habitat, could not be differentiated genetically with the tools used here, and can be difficult to distinguish without microscopic sclerite examinations. To facilitate the identification of these two similar species by non-taxonomists, a statistical model was developed that can discriminate them with up to 90% accuracy using easily obtainable measurements of gross colony morphology. Relationships between colony morphology and depth are also examined.</p> <p>Considering the difficulties associated with species discrimination among octocorals, a literature survey was conducted to review the use of integrative taxonomy in this group since the start of the 21st century, focusing particularly on morpho-molecular data comparisons. This revealed that, while description rates at family, genus, and species levels over the last twenty-one years rank among the highest ever, integrative techniques have been applied unevenly across taxonomic groups and geographic regions and overall remain a minority compared to taxonomic research based solely on morphology. Implementation of the integrative approach is increasing, however, as are the per-annum number of taxonomic publications and the total pool of authors associated with these publications.</p> <p>It is hoped that the research presented herein can contribute to ongoing global efforts of revising octocoral systematics and that the examination of integrative practices in octocoral taxonomy will serve as a baseline against which future taxonomic progress can be compared and promoted. For New Zealand specifically, elucidating the taxonomy and variability of these endemic taxa will enable aspects such as their contribution to ecosystem functioning and management needs to be examined accurately for the first time, which in turn may lead to their recognition as organisms worthy of legal protection.</p>

Four new species of troglomorphic Coecobrya Yosii, 1956 (Collembola, Entomobryidae) from Thailand based on morphological and molecular evidence, with an updated key of Thai troglomorphic species

Four new species of troglomorphic Coecobrya Yosii, 1956 are described from caves located in the central and northeastern regions of Thailand. Coecobrya whittenisp. nov. and C. troglobiasp. nov. are from Khon Kaen province, C. ellisisp. nov. is from Phetchabun province and C. phitsanulokensissp. nov. is from Phitsanulok province. They all exhibit remarkable troglobitic characters i.e. elongated antennae, legs and furca, slender claw complex and large body size. Coecobrya whittenisp. nov. is similar to C. troglobiasp. nov. and they were found a distance of only 3.4 km from each other. However, they are mainly different in the number of An mac on dorsal head and number of chaetae of Th. II. Likewise, C. ellisisp. nov. is similar to C. phitsanulokensissp. nov.. However, they differ in the number of An mac on the dorsal head, the number of central mac on Abd. II, central mac on Abd. IV, lateral mac on Abd. IV and the number of inner teeth of the claw. Moreover, C. ellisisp. nov. has orange pigment dots on the body, a unique character, considering that all other troglomorphic Coecobrya species in Thailand are devoid of pigmentation. The results of the molecular approach based on two partial mitochondrial markers (COI and 16S rDNA) and a nuclear gene fragment (28S rDNA) supported the results of morphological species discrimination in separating the four nominal populations as valid species. An updated dichotomous key of Thai troglomorphic Coecobrya species is also given.

Chryseobacterium lecithinasegens sp. nov., a siderophore-producing bacterium isolated from soil at the bottom of a pond

Bacterial strain PAGU 2197T, which was isolated from soil collected from the bottom of a pond in Japan, is characterized in this study. Cells of strain PAGU 2197T were aerobic, Gram-negative, short rod-shaped, non-motile, flexirubin-producing, oxidase-positive, catalase-positive and lecithinase-negative. A phylogenetic study based on 16S rRNA gene sequences and multilocus sequence analysis (gyrB, rpoB and rpoD) indicated that strain PAGU 2197T belongs to the genus Chryseobacterium and is a member of an independent lineage including Chryseobacterium tructae CCUG 60111T (sequence similarity, 95.9 %), Chryseobacterium lactis CCUG 60566T (93.4 %) and Chryseobacterium viscerum CCUG 60103T (91.6 %). The average nucleotide identity values were 80.83–85.04 %. Because average nucleotide identity values of 95–96 % exceed the 70 % DNA–DNA hybridization cutoff value for species discrimination, strain PAGU 2197T represents a novel species in the genus Chryseobacterium . The genome of strain PAGU 2197T was 4 967 738 bp with a G+C content of 35.5 mol%. The sole respiratory quinone of strain PAGU 2197T was MK-6; the major cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3OH, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl); and the major polar lipids were phosphoglycolipids and phosphatidylethanolamine. These results indicate that strain PAGU 2197T should be classified as representing a novel species in the genus Chryseobacterium , for which the name Chryseobacterium lecithinasegens sp. nov. is proposed, with strain PAGU 2197T (=NBRC 114264T=CCUG 75150T) as the type strain.

Acuticoccus mangrovi sp. nov., with an antibacterial property, isolated from mangrove sediment

A Gram-stain-negative, aerobic, milky white bacterium, designated B2012T, was isolated from mangrove sediment collected at Beibu Gulf, South China Sea. Antimicrobial activity assay revealed that the isolate possesses the capability of producing antibacterial compounds. Strain B2012T shared the highest 16S rRNA gene sequence relatedness (96.9–95.5 %) with members of the genus Acuticoccus . The isolate and all known Acuticoccus species contain Q-10 as the main respiratory quinone and have the same polar lipid components (phosphatidylcholine, unidentified glycolipid, unidentified lipid, unidentified amino lipid and phosphatidylglycerol). However, genomic relatedness referred by values of average nucleotide identity, digital DNA–DNA hybridization, average amino acid identity and the percentage of conserved proteins between strain B2012T and other type strains of the genus Acuticoccus were below the proposed thresholds for species discrimination. The genome of strain B2012T was assembled into 65 scaffolds with an N50 size of 244239 bp, resulting in a 5.5 Mb genome size. Eight secondary metabolite biosynthetic gene clusters were detected in this genome, including three non-ribosomal peptide biosynthetic loci encoding yet unknown natural products. Strain B2012T displayed moderately halophilic and alkaliphilic properties, growing optimally at 2–3 % (w/v) NaCl concentration and at pH 8–9. The major cellular fatty acids (>10 %) were anteiso-C15 : 0, C16 : 0 dimethyl aldehyde (DMA) and C16 : 0. Combined data from phenotypic, genotypic and chemotaxonomic analyses suggested that strain B2012T represents a novel species of the genus Acuticoccus , for which the name Acuticoccus mangrovi sp. nov. is proposed. The type strain of the type species is B2012T (=MCCC 1K04418T=KCTC 72962T).