Reliable Species Identification Research Articles

Taxonomic shortcuts lead to long delays in species discovery, delineation, and identification

Invasive species threaten biodiversity, ecosystem functions, and human health around the world. Accurate and reliable species delineation and identification are prerequisites for any effort to control the introduction of new species and the spread of those already introduced. The advent of DNA assisted taxonomic tools has fueled the development of diverse and innovative approaches often touted as simple, inexpensive, and universally applicable for addressing impediments to rapid identification of non-native species. Ampullariidae (apple snails) are a family of freshwater snails with at least five species that have been introduced outside their native range and a prime example of how taxonomic uncertainty may hamper our understanding of invasion biology. Matsukura et al. (Appl Entomol Zool 43(4):535–540, 2008) and Cooke et al. (Conserv Genet Resour 4(3):591–593, 2012) developed species-specific PCR primers as rapid, simple, and inexpensive approaches to distinguishing among and identifying the most notorious and widespread invasive apple snails, Pomacea canaliculata and Pomacea maculata, and a third species in the aquarium trade, Pomacea diffusa. Neither approach was evaluated within the context of the overall genetic diversity of the target species, or with consideration for the species diversity within the family. To evaluate both shortcut approaches, the primers were tested for their utility in delineating and identifying the target species and other closely related taxa, including 24 apple snails representing 21 species. The results indicate that the primers are of limited taxonomic utility, and fail to consistently identify the target taxa, or congenerics. More importantly, use of these primers without additional data leads to misidentifications, potentially endangering native biodiversity and allowing novel introduced species to go undetected.

Intraspecies variability of the 16S rRNA gene of the soil bacteria Acinetobacter lwoffii and Paenibacillus taichungensis

The functioning of well-studied key groups of soil microorganisms depends on the microbial ecosystem in which they function in interaction with species belonging to other ecological groups. An analysis of the composition of the soil microbiota, and the possibility of analyzing the number of representatives of specific species, remains an urgent task of soil microbiology. Such analysis could be performed using modern and expensive methods such as metagenomics and mass-spectrometry, but some questions could also be answered using real-time PCR. This well-known approach is rather cheap for massive analysis and is ready to present reproducible results for practical agricultural applications. Understanding the variability of the primary structure of 16S rRNA is key to the reliable identification of bacterial species and provides an opportunity to choose the optimal pathways for their detection by PCR. In this work, analysis of the sequences of 16S rRNA of two species of soil bacteria, Acinetobacter lwoffii and Paenibacillus taichungensis is carried out. The most variable and most conservative areas on the level of species are detected. It was proved that conventional variable and conservative areas of the gene have on average nearly the same level of variability on the intraspecies level.

Association of oral candidal species with human immunodeficiency virus patients of West Godavari district, Andhra Pradesh - An in vitro study.

Introduction:Human immunodeficiency virus (HIV) infection is a major worldwide health problem characterized by progressive immunosuppression. The morbidity of HIV patients is due to its association with opportunistic infections among which oral candidiasis is common. Regardless of HIV status, candidiasis can prevail when their immune system is depressed. Oral candidiasis can thus serve as a useful marker for both restoration of immune functions and HIV disease progression. Routine identification of Candida species is laborious and time-consuming. HICHROM agar stains different species into different colors facilitating rapid reliable identification of candida species as they differ in their virulence and sensitivity to antifungal drugs.Materials and Methods:This cross-sectional study includes a total of 200 HIV seropositive patients from Anti-Retroviral Therapy Centre, West Godavari District. Unstimulated saliva samples were collected in a screw-capped universal container. Five microliters of each sample using a sterile inoculating loop is streaked on the chromogenic agar culture media. The colonies formed are counted using a magnifying glass and LAPIZ colony counter. Candidal colony-forming units per milliliter were analyzed, compared and correlated among different study groups. Different candida species were also identified in the study.Results:Nonalbicans Candida was the most common species isolated in our study accounting for 53% and Candida albicans accounting for 47%. Considering initial and final CD4 counts, there is improvement in patients on retroviral therapy.Conclusion:Identification of the species is important for epidemiological reasons and for treatment purposes to ensure a better prognosis since some species present reduced susceptibility to azoles.

Advances in the Detection of Toxic Algae Using Electrochemical Biosensors.

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using different redox mediators and signal probes with different labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells L−1 for some species by using a fast (~2 h), simple (PCR-free) and cheap methodology (~2 EUR/determination) that will allow this methodology to be integrated into easy-to-use portable systems.

Taxonomic revision of the genus Xenopholis Peters, 1869 (Serpentes: Dipsadidae): Integrating morphology with ecological niche.

A reliable identification and delimitation of species is an essential pre-requisite for many fields of science and conservation. The Neotropical herpetofauna is the world’s most diverse, including many taxa of uncertain or debated taxonomy. Here we tackle one such species complex, by evaluating the taxonomic status of species currently allocated in the snake genus Xenopholis (X. scalaris, X. undulatus, and X. werdingorum). We base our conclusions on concordance between quantitative (meristic and morphometric) and qualitative (color pattern, hemipenes and skull features) analyses of morphological characters, in combination with ecological niche modeling. We recognize all three taxa as valid species and improve their respective diagnosis, including new data on color in life, pholidosis, bony morphology, and male genitalia. We find low overlap among the niches of each species, corroborating the independent source of phenotypic evidence. Even though all three species occur in the leaf litter of distinct forested habitats, Xenopholis undulatus is found in the elevated areas of the Brazilian Shield (Caatinga, Cerrado and Chaco), whereas X. scalaris occurs in the Amazon and Atlantic rainforests, and X. werdingorum in the Chiquitanos forest and Pantanal wetlands. We discuss the disjunct distribution between Amazonian and Atlantic Forest snake species in the light of available natural history and ecological aspects. This study shows the advantages of combining multiple data sources for reliable identification and circumscription of ecologically similar species.

Avoiding common numts to provide reliable species identification for tiger parts

Tigers are killed to supply a demand for many wildlife products despite a ban on commercial international trade. As populations decrease, products from substitute species (i.e. lions and leopards) have been fraudulently sold as tiger. DNA forensic techniques are needed to definitively identify tiger in order to secure prosecutions although this is complicated by the presence of numts. Therefore, we have developed and validated a CO1 genetic marker that preferentially amplifies the mtDNA CO1 region and excludes the nuclear CO1 pseudogene, which we expect to be of use in tiger forensic casework.

Geometric morphometric wing analysis represents a robust tool to identify female mosquitoes (Diptera: Culicidae) in Germany

Accurate species identification is the prerequisite to assess the relevance of mosquito specimens, but is often hindered by missing or damaged morphological features. The present study analyses the applicability of wing geometric morphometrics as a low-cost and practical alternative to identify native mosquitoes in Germany. Wing pictures were collected for 502 female mosquitoes of five genera and 19 species from 80 sampling sites. The reliable species identification based on interspecific wing geometry of 18 landmarks per specimen was tested. Leave-one-out cross validation revealed an overall accuracy of 99% for the genus and 90% for the species identification. Misidentifications were mainly due to three pairings of Aedes species: Aedes annulipes vs. Aedes cantans, Aedes cinereus vs. Aedes rossicus and Aedes communis vs. Aedes punctor. Cytochrome oxidase subunit I (COI) gene region was sequenced to validate the morphological and morphometric identification. Similar to the results of the morphometric analysis, the same problematic three Aedes-pairs clustered, but most other species could be well separated. Overall, our study underpins that morphometric wing analysis is a robust tool for reliable mosquito identification, which reach the accuracy of COI barcoding.

Technical Limitations Associated With Molecular Barcoding of Arthropod Bloodmeals Taken From North American Deer Species.

Accurate species-level identification of the source of arthropod bloodmeals is important for deciphering blood feeding patterns of field-collected specimens. Cytochrome c oxidase I (COI) mitochondrial gene sequencing has been used for this purpose; however, species resolution can be difficult to obtain from certain vertebrate genera, including Odocoileus. Sanger sequencing of mitochondrial genes was employed to identify the bloodmeal source of wild-caught mosquitoes trapped in Greeley, Colorado. Initial sequencing of the COI gene of mitochondrial DNA in bloodmeals was inadequate for species-level resolution of bloodmeals from deer in the genus Odocoileus, with current databases returning low fidelity matches to multiple genera. The use of the hypervariable D loop of the control region provided species-level identification of white-tailed deer (Order: Artiodactyla, Family: Cervidae, Odocoileus virginianus); however, taxonomic identification was successful only to genus for mule (O. hemionus hemionus) and black-tailed deer (O. hemionus columbianus). We advocate the use of multiple loci for bloodmeal analysis and the buildout of available databases to include multiple mitochondrial reference genes for reliable host species identification.

Problems, Limitations, and Challenges in Species Identification of Ascomycota Members on the Basis of ITS Regions

The internal transcribed spacer (ITS) region is regarded as a formal fungal primary barcode with a high probability of the correct identification for a broad group of fungi. ITS sequences have been widely used to determine many fungal species and analysis of rDNA ITS is still one of the most popular tools used in mycology. However, this region is not equally variable in all groups of fungi; therefore, identification may be problematic and result in ambiguous data, especially in some species-rich genera of Ascomycota. For these reasons, identification based on rDNA ITS is usually complemented by morphological observations and analysis of additional genes. Reliable species identification of Ascomycota members is essential in diagnosing plant diseases, verifying air quality and the effectiveness of agronomic practices, or analyzing relationships between microorganisms. Therefore, the present study aimed to verify, using specific examples, the extent to which ITS sequence analysis is useful in species identification of pathogens and saprobionts from Ascomycota and demonstrate problems related to such identification in practice. We analyzed 105 ITS sequences of isolates originating from air and plant material. Basic local alignment search tool (BLASTn) significantly contributed to the reliable species identification of nearly 80% of isolates such as <em>Arthrinium arundinis</em>, <em>Beauveria bassiana</em>, <em>Boeremia exigua</em>, <em>Cladosporium cladosporioides</em>, <em>Epicoccum nigrum</em>, <em>Nigrospora oryzae</em>, <em>Sclerotinia sclerotiorum</em>, or <em>Sordaria fimicola </em>and members of the genera <em>Alternaria </em>and <em>Trichoderma</em>. However, for most isolates, additional morphological observations, information regarding the isolate origin and, where possible, a PCR with species-specific primers were helpful and complementary. Using our practical approach, we determined that ITS-based species identification and comparative analysis with GenBank sequences significantly helps identifying Ascomycota members. However, in many cases, this should be regarded as suggestive of a taxon because the data usually require the use of additional tools to verify the results of such analysis.

COMPARATIVE WOOD ANATOMICAL PROPERTIES OF GENUS SYZYGIUM (FAMILY MYRTACEAE) FROM MANIPUR, INDIA

Syzygium belongs to family Myrtaceae and comprises of mostly trees and a few shrubs. The main objectives were to study anatomical and physical characteristics of five Syzygium species namely Syzygium cumini, Syzygium fruticosum, Syzygium jambos, Syzygium nervosum and Syzygium praecox and to see intra and inter species variation among them. The wood samples were taken at breast-height from straight bole and uniform crowned trees. The present study showed that all the selected species shared common features like simple perforation plate, vestured inter-vessel pits, vessel-ray pits, disjunctive ray parenchyma cells, diffuse, diffuse-in-aggregate, vasicentric, aliform and confluent types of axial parenchyma Diffuse porous and indistinct fruticosum were observed. The vessels were mostly barrel shaped with small or long tails at one or both ends in all species except tube shaped in S. jambos and drum shaped in S. fruticosum. Spiral thickenings were present in the tails of S. nervosum and S. fruticosum. Fibres were thin walled and non-septate. Occasional septate fibres and vasicentric tracheids were present in S. nervosum and S. jambos. Crystals in ray of S. nervosum and S. fruticosum and silica bodies in axial parenchyma of S. jambos were observed. The fibre percentage and wood density were maximum in S. jambos whereas moisture content was minimum in S. jambos. Principal Component Analysis revealed a close relationship among all species. Therefore, the qualitative characteristics and all quantitative anatomical characteristics can be used for reliable identification of Syzygium species.

Identification of polymer species in a complex mixture by pyrolysis-gas chromatography-atmospheric pressure chemical ionization-high resolution time-of-flight mass spectrometry as a basis for environmental microplastic analysis

Pyrolysis-gas chromatography coupled with atmospheric pressure chemical ionization-high resolution time-of-flight mass spectrometry (Py-GC-APCI-TOFMS) was applied for the reliable identification of polymer species in a complex mixture to establish this system as a platform for environmental microplastic analysis. The method was evaluated on a model sample of mixed microplastics comprised of five major polymers: polyethylene (PE), polypropylene (PP), poly(vinyl chloride) (PVC), polystyrene (PSt), and poly(ethylene terephthalate) (PET). The representative ions in the mass spectra of the pyrolyzates were identified by Py-GC-APCI-TOFMS measurements of the individual polymer species. The unique APCI ionization/fragmentation process and the high mass resolution of TOFMS were exploited to confirm the presence of the respective polymer species in the extracted ion chromatograms (EICs) of the mixed sample for the selected key ions. PE and PP were successfully and uniquely identified in the mixed sample by focusing on specific fragment ions of the pyrolyzates formed during APCI (m/z 114.093 ± 0.020 and m/z 156.139 ± 0.020, respectively). The resultant EICs allowed for the specific analysis of these species, even in the presence of interfering species produced from the other polymers. Similar processes were applied to the detection of PVC, PSt, and PET in the mixed sample. These results demonstrated that Py-GC-APCI-TOFMS is a promising tool to characterize a complex mixture of environmental microplastics.

Fish Species Identification Using the Rhombic Squamation Pattern

The shape of fish scales is to a considerable degree species-specific, making it possible to identify species using only one scale per specimen. However, the shape of the rhombic lamination pattern has not been considered to identify species. This study used geometric morphometric statistical approaches to address two specific questions: (1) whether the rhombic lamination pattern of fish scales along the longitudinal axis varies within species and sex and (2) how many fish scales of the rhombic squamation pattern should be considered to obtain an adequate identification. These questions were assessed with Mancova and cross-validated quadratic discriminant analysis (DA) using the rhombus of one, three and six scales, and six, 14 and 26 landmarks, respectively, in order to discriminate between two co-generic species, Mugil cephalus and M. curema. The proportions of the total shape variance explained by the total length and the centroid size were 2.3, 11.8 and 10.5% and 4.2, 5.1 and 5.4% for 1, 3 and 6 scales, respectively. Analyses were carried out on the shape and the form (shape plus size). The Mancova and DA analyses were found to be effective in detecting differences in scale pattern shape between species (except in the case of 3 scales; p=0.079 for the shape and p=0.065 for the form), whereas no differences were recorded between size and sex in all cases. The findings indicate that a good identification of species is possible, with no significant differences when using shape or form. The discriminant analyses provided values of 75.8, 75.0 and 73.4% based on the shape, and of 72.7, 75.8 and 75.0% based on the form, for 1, 3 and 6 scales respectively. Thus, it is possible to obtain a rapid and reliable identification of species using the rhombus of one scale only without considering the size. This is a useful finding in practical terms since scaling requires data on length. In addition, the finding of a suitable discrimination using only the rhombus of one scale raises the possibility of using an ocular adaptor on a camera or mobile, allowing many individuals to be easily screened without having to collect scales.

Forensic identification of CITES Appendix I Cupressaceae using anatomy and mass spectrometry

Abstract Alerce (Fitzroya cupressoides (Mol.) Johnst.) and Guaitecas cypress (Pilgerodendron uviferum (Don) Florin) are two of the three closely-related species of conifers in the Cupressaceae that are endemic to southern Chile and Argentina. Both are listed in Appendix I of the Convention on International Trade in Endangered Species of Fauna and Flora (CITES). The presence or absence of nodular (conspicuously pitted) end walls in the parenchyma cells provide good diagnostic characters to separate the two species wood anatomically, but the latter is sometimes difficult to distinguish. Therefore, a collaborative project was designed to study the chemical-molecular expression of these species by analyzing the heartwood using DART TOFMS (Direct Analysis in Real-Time (DART) Time-of-Flight Mass Spectrometry (TOFMS). This study compares the anatomical features of heartwood for both species and demonstrates that anatomy in conjunction with chemistry can separate them. DART TOFMS analysis combined with PCA was able to unequivocally determine taxonomic source with a statistical certainty of 99%. The mass spectra results obtained from heartwood demonstrated that identification is feasible after a few seconds, using a very small sample. DART TOFMS is a robust tool for reliable species identification and is useful to identify the taxonomic source of finished products or timber that are suspected of being illegally harvested.

Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance

Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical for the prosecution of illegally-traded wildlife products, conservation-based biodiversity research, and identification of blood-meal hosts of hematophagous invertebrates. However, forensic identification of vertebrate tissue relies on sequencing of the mitochondrial cytochrome oxidase I (COI) ‘barcode’ gene, which remains costly for purposes of screening large numbers of unknown samples during routine surveillance. Here, we adapted a rapid, low-cost approach to differentiate 10 domestic and 24 wildlife species that are common in the East African illegal wildlife products trade based on their unique high-resolution melting profiles from COI, cytochrome b, and 16S ribosomal RNA gene PCR products. Using the approach, we identified (i) giraffe among covertly sampled meat from Kenyan butcheries, and (ii) forest elephant mitochondrial sequences among savannah elephant reference samples. This approach is being adopted for high-throughput pre-screening of potential bushmeat samples in East African forensic science pipelines.

Wing morphometrics for the identification of Nearctic and Palaearctic Piophilidae (Diptera) of forensic relevance

The Piophilidae (Diptera) are a family comprising about 80 species, several of them of high economic and forensic relevance. An unequivocal species identification is crucial for designing effective control measures or to provide reliable estimations of the minimum post mortem interval. However, the identification may sometimes not be possible, either because the diagnostic morphological characters are not easily observable or very fragile, or because of a poor DNA quality and/or unavailability of reference molecular signatures. In the recent years, the use of wing morphometric techniques has emerged as a powerful tool for the identification of different families of forensically important Diptera. The present study applies, for the first time, this technique to the identification of 11 Piophilidae species of forensic relevance in the Nearctic and Palaearctic regions, as well as to the discrimination of sexes, distinct geographical populations and, in the case of the dimorphic species Prochyliza nigrimana (Meigen), seasonal morphs. Wing shape variation was analysed using 14 landmarks located at wing vein junctions and a cross-variation analysis was used to test the reliability of identifications. The present results demonstrate that wing shape can be used to identify most species with relatively high success, whereas cross-validation analyses indicated that discrimination between sexes, populations or morphs was less effective, even if significant differences were observed in every comparison. We conclude that wing morphometrics can be a powerful identification tool that might be used in combination with other methods in order to achieve accurate and reliable species identifications—independently of the sex, geographic origin or colour variation of the samples—even with those piophilid species that have been object of frequent misidentifications.

The need to employ reliable and reproducible species identifications in coralline algal research

Coralline algae perform important ecological roles in nearshore marine ecosystems globally by promoting the settlement of invertebrate larvae and enhancing biodiversity by creating habitat. However, these roles are severely threatened by global environmental changes. Most coralline algae are extremely difficult to identify, and DNA sequencing has revealed rampant inaccuracy of morpho-anatomical approaches to distinguish species, and even genera. If appropriate identification methods are not reported, or even used, we will be left with an uninterpretable body of literature where the species-specific biology of coralline algae cannot be validated. This will make it difficult to determine the impact a changing ocean may have on these ecologically important species. We reveal the magnitude of the issue in coralline algal research—both the identification methods used and the reporting of identification protocols. An analysis of 341 articles over the past decade revealed that only 7.6% used molecular methods, with over 70% not reporting any details of how species were identified. While many coralline algal taxonomists understand that the majority of species cannot be identified morphologically, this message has not disseminated to the ecological and physiological community. We provide a series of guidelines for conducting DNA-based identifications and strongly recommend the use of these methods over less informative morpho-anatomical techniques. Most importantly, the methods of identification should be adequately reported. Without following these guidelines, research on coralline algae runs the risk of collecting uninterpretable data, and conducting irreproducible science, slowing our ability to determine how these important species will respond to future ocean conditions.

Dolphin whistle repertoires around São Miguel (Azores): Are you common or spotted?

Short-beaked common dolphins (Delphinus delphis) and Atlantic spotted dolphins (Stenella frontalis) are both common in the Azores archipelago during summer. Because both species are sympatric, at least in part of their range, they may use acoustic features to recognize conspecifics and maintain school cohesion throughout their different activities. Delphinid whistles were recorded with a 96-kHz sampling rate using towed hydrophone system during surveys held in summer of 2013 and 2014 around São Miguel Island (Azores, Portugal). A total of 256 whistles attributed to either short-beaked common dolphin (n = 133) or Atlantic spotted dolphin (n = 123) were selected and processed with a contour extraction software. Elementary statistical analysis showed that duration, frequency and slope variables were significantly different for both species, although in most cases their range overlapped. We performed a discriminant analysis to test species classification: the dataset was randomly split into one calibration subset (186 whistles) and one validation subset (70 whistles). The discriminant analysis retained four variables (global slope, duration, minimal and final frequencies) as useful for classification. The discriminant function resulted in correct classification rates of 78.5% (calibration subset) and 81.4% (validation subset). Common dolphin whistles were better classified than Atlantic spotted dolphin whistles (83.4% and 74.8%) respectively. This study shows that reliable species identification can be achieved for common and spotted dolphins using their whistle repertoire characteristics.

Open Set Recognition of Timber Species Using Deep Learning for Embedded Systems

Reliable and rapid identification of timber species is a very relevant issue for many countries in South America and especially for Peru, which is the second country with the largest extent of tropical forest, and that is because this issue is a necessity in order to develop an effective management of the forest resources, such as inspection and control of the timber commerce. Since current methods of identification are based on a closed set recognition approach, they are not reliable enough to be used in a practical application because scenarios of identification of timber species are by nature an open set recognition problem. For that reason, in this work we propose a convolutional neural network that has two main characteristics, being able to run in a real-time embedded system and being able to handle the open set recognition problem, that is, this model can discriminate between known and unknown species. In order to evaluate it, tests are performed in two timber species datasets and some experiments are developed in the embedded system Raspberry Pi3B+ to measure energy consumption. The results present high metrics, which means that it manages to discriminate the unknown species with accuracy and F1 score above 91% for two sets of images used. In addition to this, our proposed model obtain lower maximum power value (10-12%) and computational resource usage (5-13%) than a classical convolutional model and MobileNetsV2 measured on the Raspberry Pi3B+.

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S\u201323S rRNA region

BackgroundMany members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S–23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S–23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S–23S rRNA region.MethodsThirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S–23S rRNA region.ResultsThis study allowed obtainment and deposition of reference sequences of the 16S–23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S–23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level.ConclusionsBased on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S–23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S–23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

Standard reference material (SRM) DNA barcode library approach for authenticating Antidesma bunius (L.) Spreng. (bignay) derived herbal medicinal products

ABSTRACTIn the Philippines, the herbal medicinal product (HMP) market is flourishing due to the abundance of pharmacologically important species, and the high level of ethnomedicinal knowledge still widely accepted by the public. As such, herbal products from Antidesma bunius (L.) Spreng., locally known as bignay, are popular as medicine for various ailments of the circulatory and digestive systems. Though efficacy is guaranteed, the authenticity of the marketed products is still in question as several other herbal plants can provide the said benefits. Similar morphology between wild species also hinders species identification and contributes confusion especially to the general consumer. The authenticity of the marketed HMPs was established by means of DNA barcoding techniques which offers quick and reliable species identification by means of (1) the Basic Local Alignment Search Tool (BLASTn) and (2) the establishment of the first Standard Reference Material (SRM) Herbal barcode library for Antidesma spp. A total of 56 gene accessions from matK-psbA-trnH-rbcL sequences of 9 wild Antidesma spp. comprised the SRM which was then used to confirm the identity of 11 randomly sampled bignay-derived HMPs. Following the BLASTn and the SRM (maximum likelihood tree reconstruction) criterion, the subjected sequences revealed that only three of the 11 HMPs were authentic A. bunius-derived products. The other eight HMPs contained substitutes that were either fillers or different herbal medicinal plant not indicated in the product labels. These results indicate that product safety should be reinforced with complete HMP authentication using traditional methods supported by molecular data.