Traditional Morphological Identification Research Articles

Development of recombinase polymerase amplification-based colorimetric detection assay for rapid identification of invasive cassava mealybug, Phenacoccus manihoti Matile-Ferrero

Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for P. manihoti based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of P. manihoti, and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of P. manihoti only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in P. manihoti samples. The assay successfully amplified P. manihoti DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring P. manihoti in quarantine stations and facilitating in development of a portable diagnostic kit.

Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria).

Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Cuticular chemical compounds of puparial cases of three forensically important blow flies from Egypt: potential for accurate identification and forensic investigations

AbstractThe distinct and species-specific chemical compounds found on the insect cuticle have demonstrated effectiveness in various applications, including species identification. Accurate identification of fly species becomes challenging when only damaged empty puparial cases are available, making it difficult to use traditional morphological and molecular identification methods. This study aimed to analyze the chemical compositions of puparial exuviae from three forensically and medically important fly species; Lucilia sericata, Chrysomya albiceps, and Chrysomya marginalis. Gas chromatography/mass spectrometry (GC–MS) was employed to assess the chemical profiles of these exuviae and evaluate their accuracy in identifying Dipteran insects. The study revealed the presence of twelve classes of chemical compounds across the three species, with retention times ranging from 18.78 to 35.03. A total of forty-two compounds with chain lengths ranging from C12 to C45 were identified. The profiles of Ch. albiceps and L. sericata displayed similarities, with alcohol being the most abundant compound (28.6%) in L. sericata. However, alkanes, including n-alkanes, branched alkanes, and cycloalkanes, constituted the main components of the cuticles in the three species, with Ch. marginalis displaying the highest percentage. These findings represent an initial step towards utilizing hydrocarbon composition as a practical tool for distinguishing between forensic species in Egypt.

Artificial neuronal networks are revolutionizing entomological research

AbstractThe application of artificial intelligence (AI) in entomological research has gained significant attention in recent years. This review summarizes the current state of research on the potential of AI methods in various subfields of entomology, such as behavioural biology, biodiversity research, climate change research, pest management, and disease vector control. In some cases, AI‐based species identification methods based on deep learning neuronal network models have been shown to outperform traditional morphological identification methods in terms of accuracy and speed. Behavioural biology research has been enhanced through the use of AI‐based tracking systems that can classify insect behaviour and movement patterns. Habitat modelling has also been improved with the use of AI, allowing for the creation of more accurate models that can predict insect distribution and abundance. Climate change and biodiversity research have benefited from AI‐driven tools that can analyse large datasets and predict the impact of environmental changes on insect populations. In agriculture, pest management has been revolutionized by AI methods, with the development of smart traps and monitoring systems that can detect and identify pest species in real‐time, enabling targeted control measures. Disease vector control has also been improved through the use of AI‐based predictive models, which can identify areas at risk of disease transmission and aid in the development of effective control strategies. In conclusion, AI methods have the potential to revolutionize many aspects of entomological research. Future research should focus on developing more sophisticated AI tools and integrating them into entomological research to address even more complex ecological questions.

Assessing arthropod biodiversity with DNA barcoding in Jinnah Garden, Lahore, Pakistan.

Previous difficulties in arthropod taxonomy (such as limitations in conventional morphological approaches, the possibility of cryptic species and a shortage of knowledgeable taxonomists) has been overcome by the powerful tool of DNA barcoding. This study presents a thorough analysis of DNA barcoding in regards to Pakistani arthropods, which were collected from Lahore's Jinnah Garden. The 88 % (9,451) of the 10,792 specimens that were examined were able to generate DNA barcodes and 83% (8,974) of specimens were assigned 1,361 barcode index numbers (BINs). However, the success rate differed significantly between the orders of arthropods, from 77% for Thysanoptera to an astounding 93% for Diptera. Through morphological exams, DNA barcoding, and cross-referencing with the Barcode of Life Data system (BOLD), the Barcode Index Numbers (BINs) were assigned with a high degree of accuracy, both at the order (100%) and family (98%) levels. Though, identifications at the genus (37%) and species (15%) levels showed room for improvement. This underscores the ongoing need for enhancing and expanding the DNA barcode reference library. This study identified 324 genera and 191 species, underscoring the advantages of DNA barcoding over traditional morphological identification methods. Among the 17 arthropod orders identified, Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera from the class Insecta dominated, collectively constituting 94% of BINs. Expected malaise trap Arthropod fauna in Jinnah Garden could contain approximately 2,785 BINs according to Preston log-normal species distribution, yet the Chao-1 Index predicts 2,389.74 BINs. The Simpson Index of Diversity (1-D) is 0.989, signaling high species diversity, while the Shannon Index is 5.77, indicating significant species richness and evenness. These results demonstrated that in Pakistani arthropods, DNA barcoding and BOLD are an invaluable tool for improving taxonomic understanding and biodiversity assessment, opening the door for further eDNA and metabarcoding research.

Capturing drifting species and molecules—Lessons learned from integrated approaches to assess marine metazoan diversity in highly dynamic waters

AbstractMarine community diversity surveys require a reliable assessment to estimate ecosystem functions and their dynamics. For these, non‐invasive environmental DNA (eDNA) metabarcoding is increasingly applied in zoological studies to complement or even replace traditional morphological identification methods. However, uncertainties remain about the accuracy of the diversity detected with eDNA to capture the actual diversity in the field. Here, we validate the reliability of eDNA metabarcoding in identifying metazoan biodiversity in highly dynamic marine waters of the North Sea. We analyzed biodiversity from water (eDNA) and zooplankton samples with cytochrome c oxidase subunit 1 (COI) and 18S rRNA (18S) metabarcoding at Helgoland Roads and validated the optimal molecular resolution by morphological and molecular zooplankton identification (metabarcoding) with the result of merely a few false‐negative detections. eDNA and zooplankton metabarcoding resolved 354 species from all major and in total 16 metazoan phyla. This molecular genetic species inventory overlapped by 95.9% (COI) and 81.9% (18S) with published inventories of local, morphologically identified species, among them neozoa and rediscovered species. Even though half of all species were detected by both eDNA and zooplankton metabarcoding, the methods differed significantly in their detected diversity. eDNA metabarcoding performed very well in cnidarians and annelids, whereas zooplankton metabarcoding identified higher numbers of fish and malacostraca. Species assemblages significantly differed between the individual sampling events and the cumulative number of identified species increased steadily over the sampling period and did not reach saturation. About a third of the species were detected only once while a core community of 22 species was identified continuously. Our study confirms eDNA metabarcoding to be a powerful tool to identify and analyze North Sea fauna in highly dynamic waters and we recommend investing in high sampling efforts by repetitive sampling and replication using at least 0.45 μm filters to increase filtration volume.

Benefits of combined environmental DNA and microscopy for diversity monitoring in rotifer community: A mesocosm experiment

Rotifers are crucial for the monitoring of aquatic ecosystems because of the sensitive response of rotifer community structure to environmental changes. Comparisons and combinations of meta-barcoding (sequence analysis) and traditional morphological identification for rotifer community responses are rare. To better understand the differences between the two approaches, we conducted a study using 48 mesocosms (2500 L) and a combination of warming, nutrient loading, and pesticide to evaluate the applicability of both methods to rotifer communities in order to better understand how rotifer communities respond to multiple environmental stressors. The findings demonstrated that the degree of matching between both techniques was limited, despite the fact that there was a substantial positive association between the morphology abundance determined by morphological identification and the sequence abundances determined by eDNA metabarcoding. Although the two methods had similar responses to various environmental stresses, sequence analysis can identify rotifers that are difficult to identify with traditional methods such as Bdelloidea, while classical morphological methods were more suitable for assessing the effects of various environmental changes on rotifer communities. This study provided valuable implications for monitoring and managing aquatic ecosystems since it enables us to spot changes in the rotifer community structure early on and take the necessary precautions to safeguard these sensitive ecosystems.

Ultra-deep sequencing of 45S rDNA to discern intragenomic diversity in three Chrysodeixis species for molecular identification

Species identification is necessary to prevent introductions of exotic plant pests through global trade. Many of these pests are understudied and lack publicly available DNA sequence data on which rapid molecular identification methods can be based. One such lineage is the genus Chrysodeixis, which includes three species of potential concern for United States trade initiatives: C. includens, C. chalcites, and C. eriosoma. Here we describe a method to generate robust 45S rDNA profiles using long read sequencing in order to clarify evolutionary relationships and develop a real-time PCR identification technique. Such an identification tool will be useful in rapidly differentiating between Chrysodeixis species of quarantine concern where traditional morphological identification methods are insufficient. Molecular methods such as this greatly reduce the time spent identifying each specimen, allow for detection of eDNA, vastly increase throughput, and increase the probability of detection. The methods presented here will be generally adaptable to any understudied lepidopteran taxa that necessitates a molecular diagnostic assay and, with adjustment or testing of the primers, could be applied to any group for which development of rDNA profiles in a benchtop setting would prove useful.

A New, Non-Invasive Methodology for the Molecular Identification of Adult Sarcophagidae from Collections.

Correct species identification is the cornerstone of all scientific studies that involve insects. Alongside traditional morphological identification techniques, molecular identification based on the characterization and analysis of specific mitochondrial or nuclear gene regions is becoming commonplace. Despite the good results that can be achieved, DNA extraction usually involves invasive techniques that lead to the partial or total destruction of specimens. In this work, a non-invasive DNA extraction technique is described. The technique was tested on the abdomens of dry-preserved Sarcophagidae (Diptera) specimens collected between 1889 and 2015. This allowed for the correct identification of species without impairing diagnostic morphological structures useful for further studies.

Morphological and Molecular Identification of Aporrectodea trapezoides (Duges, 1828) in Baghdad

In this study, Aporrectodea trapezoides were collected from three locations in the north, central and south of Baghdad (Rashdiyah, Jadriyah and Zafaraniyah). The species was initially identified using morphological characteristics, and then genetic links among the researched species in the three locations were analyzed using molecular techniques (DNA barcoding). In order to validate the species of earthworms examined, their nuclear 18S rDNA gene sequences were analyzed and a phylogenetic tree was constructed. The nucleotide sequences were compared to those in GenBank by using the online Basic Local Alignment Search Tool (Blast). The DNA barcoding of the nuclear 18S rDNA has been shown to be an effective tool for species identification of earthworms, and it provides a powerful complement to traditional morphological identification as shown by a Blast analysis showing 99-100% similarity with sequence annotated in GenBank at 18S region. DNA sequence from one of the species under study has been submitted in GenBank with an accession number.

A new species of Megastigmus (Hymenoptera, Megastigmidae) from China.

Most species of Megastigmus are considered important economic pests that grow in seeds, especially of conifers. Accurate identification of species is a crucial step for the biological research of parasitic pests and the further application of biological control. However, their large variety, small size, similar morphology and different growth and development stages have brought great challenges to taxonomic research. Traditional morphological identification often takes a long time and this requires us to seek a new method for rapid and accurate identification. Therefore, the better identification of Megastigmus urgently needs to be combined with molecular methods to help taxonomic development. Here, Megastigmusdaduheensis sp. n. (Chalcidoidea: Megastigmidae) was identified, based on morphology and molecular markers, such as COI and Cytb. M.daduheensis sp. n. is distinct from other known species of the same genus in the morphology. The results of the molecular phylogenetic tree, similarity alignment and genetic distance indicate that the COI and Cytb sequences of M.daduheensis sp. n. are highly similar to M.sobinae and M.duclouxiana, but there are some genetic differences. The genetic distances of M.daduheensis sp. nov. with M.duclouxiana and M.sabinae were 0.34 and 0.33 and the percentages of shared base pairs were 76.3% and 76.8%, respectively. Both morphological and molecular data classified M.daduheensis sp. n. as a new species. The obtained COI and Cytb sequences of M.daduheensis sp. n. can be used as DNA barcodes, providing molecular data for rapid and accurate identification of this species in the future.

Replicate DNA metabarcoding can discriminate seasonal and spatial abundance shifts in river macroinvertebrate assemblages.

The delivery of consistent and accurate fine-resolution data on biodiversity using metabarcoding promises to improve environmental assessment and research. Whilst this approach is a substantial improvement upon traditional techniques, critics note that metabarcoding data are suitable for establishing taxon occurrence, but not abundance. We propose a novel hierarchical approach to recovering abundance information from metabarcoding, and demonstrate this technique using benthic macroinvertebrates. To sample a range of abundance structures without introducing additional changes in composition, we combined seasonal surveys with fish-exclusion experiments at Catamaran Brook in northern New Brunswick, Canada. Five monthly surveys collected 31 benthic samples for DNA metabarcoding divided between caged and control treatments. A further six samples per survey were processed using traditional morphological identification for comparison. By estimating the probability of detecting a single individual, multispecies abundance models infer changes in abundance based on changes in detection frequency. Using replicate detections of 184 genera (and 318 species) from metabarcoding samples, our analysis identified changes in abundance arising from both seasonal dynamics and the exclusion of fish predators. Counts obtained from morphological samples were highly variable, a feature that limited the opportunity for more robust comparison, and emphasizing the difficulty standard methods also face to detect changes in abundance. Our approach is the first to demonstrate how quantitative estimates of abundance can be made using metabarcoding, both among species within sites as well as within species among sites. Many samples are required to capture true abundance patterns, particularly in streams where counts are highly variable, but few studies can afford to process entire samples. Our approach allows study of responses across whole communities, and at fine taxonomic resolution. We discuss how ecological studies can use additional sampling to capture changes in abundance at fine resolution, and how this can complement broad-scale biomonitoring using DNA metabarcoding.

Descriptive DNA barcoding of Argas (Persicargas) arboreus and Argas (Persicargas) persicus ticks (Ixodida: Argasidae) infesting birds in Egypt

Argas ticks are primary parasites of birds with veterinary importance. Nevertheless, these ticks have received little attention regarding molecular identification studies. DNA barcoding is a powerful technique for identifying tick species besides traditional morphological identification. The present work is a first effort to divulge DNA sequences of Argas (Persicargas) arboreus from Egypt and worldwide. We used cytochrome c oxidase subunit I (COI) from A. arboreus infesting herons, and from the fowl tick Argas (Persicargas) persicus. Our results pointed out another success for the Folmer primers that are widely used in DNA barcoding, permitting the discrimination of morphologically similar A. arboreus and A. persicus.

Study on Analysis of Several Molecular Identification Methods for Ciliates of Colpodea (Protista, Ciliophora)

The application of molecular techniques to accurately identify protozoan species can correct previous misidentifications based on traditional morphological identification. Colpodea ciliates have many toxicological and cytological applications, but their subtle morphological differences and small body size hinder species delineation. Herein, we used Cox I and β-tubulin genes, alongside fluorescence in situ hybridization (FISH), to evaluate each method in delineating Colpodea species. For this analysis, Colpoda harbinensis n. sp., C. reniformis, two populations of C. inflata, Colpoda compare grandis, and five populations of Paracolpoda steinii, from the soil in northeastern China, were used. We determined that (1) the Cox I gene was more suitable than the β-tubulin gene as a molecular marker for defining intra- and interspecific level relationships of Colpoda. (2) FISH probes designed for Colpoda sp., C. inflata, Colpoda compare grandis, and Paracolpoda steinii, provided rapid interspecific differentiation of Colpodea species. (3) Colpoda harbinensis n. sp. was established and mainly characterized by its size in vivo (approximately 80 × 60 μ m ), a reniform body in outline, one macronucleus, its spherical shape, a sometimes nonexistent micronucleus, 11–15 somatic kineties, and five or six postoral kineties. In conclusion, combining oligonucleotide probes, DNA barcoding, and morphology for the first time, we have greatly improved the delineation of Colpodea and confirmed that Cox I gene was a promising DNA barcoding marker for species of Colpodea, and FISH could provide useful morphological information as complementing traditional techniques such as silver carbonate.

DNA Metabarcoding of Preservative Ethanol Reveals Changes in Invertebrate Community Composition Following Rotenone Treatment

The botanical compound rotenone is extensively used to eradicate populations and reduce the negative impacts of freshwater invasive animals. The method is controversial as non-target organisms often are negatively affected, but these effects are highly variable among taxa and may be difficult to monitor on species-level as many invertebrates are challenging or costly to identify using morphology. We investigated the effect of rotenone treatment on freshwater invertebrates in two Nordic lakes using both traditional morphological identification and DNA metabarcoding of the preservative ethanol DNA (peDNA). We used a before-after-control-impact design in assessing the effect of the rotenone treatment, with two treated and two control lakes, all sampled immediately before the rotenone treatment, immediately after and 1 year after the treatment. The two methods of identification gave different results: The peDNA based method detected 333 taxa, while 90 taxa were recorded by morphological identification. Twenty-eight taxa were in common for both methods. Both traditional morphological identification and identification using metabarcoding of peDNA showed a significant effect of the rotenone treatment on invertebrate community composition expressed as Bray-Curtis dissimilarities. Using the results from peDNA, the number of taxa decreased just after the treatment and increased again 1 year after the treatment in the treated lakes, indicating a short-term effect of the treatment and a subsequent recovery of the invertebrate community. We conclude that DNA metabarcoding of preservative ethanol has a promising potential to record effects of anthropogenic stressors such as rotenone treatment. However, as peDNA based metabarcoding missed several taxa, and detected DNA from species not living in the actual sampling sites, the method needs further improvement and should perhaps be used in combination with morphological-based identification until sufficiently refined.

Disentangling bias for non-destructive insect metabarcoding.

A fast and reliable method for obtaining a species-level identification is a fundamental requirement for a wide range of activities, from plant protection and invasive species management to biodiversity assessments and ecological studies. For insects, novel molecular techniques such as DNA metabarcoding have emerged as a rapid alternative to traditional morphological identification, reducing the dependence on limited taxonomic experts. Until recently, molecular techniques have required a destructive DNA extraction, precluding the possibility of preserving voucher specimens for future studies, or species descriptions. Here we paired insect metabarcoding with two recent non-destructive DNA extraction protocols, to obtain a rapid and high-throughput taxonomic identification of diverse insect taxa while retaining a physical voucher specimen. The aim of this work was to explore how non-destructive extraction protocols impact the semi-quantitative nature of metabarcoding, which alongside species presence/absence also provides a quantitative, but biased, representation of their relative abundances. By using a series of mock communities representing each stage of a typical metabarcoding workflow we were able to determine how different morphological (i.e., insect biomass and exoskeleton hardness) and molecular traits (i.e., primer mismatch and amplicon GC%), interact with different protocol steps to introduce quantitative bias into non-destructive metabarcoding results. We discuss the relevance of taxonomic bias to metabarcoding identification of insects and potential approaches to account for it.

Discrimination of 15 Amazonian Anopheline Mosquito Species by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism.

Precise identification of anopheline species is paramount for incrimination of malaria vectors and implementation of a sustainable control program. Anopheline mosquitoes are routinely identified morphologically, a technique that is time-consuming, needs high level of expertise, and prone to misidentifications especially when considering Amazonian species. The aim of this study was therefore to develop a DNA-based identification technique to supplement traditional morphological identification methods for the discrimination of anopheline mosquitoes collected in French Guiana. The internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA) for anopheline species was amplified by polymerase chain reaction (PCR), and digested with AluI/MspI restriction enzymes. PCR-restriction fragments length polymorphism (RFLP) assay was compared to sequencing of the ITS2 region for validation. Fifteen Anopheles species have shown distinct PCR-RFLP profiles. A concordance of 100% was obtained when identification by PCR-RFLP was compared to sequencing of ITS2. A high throughput, fast, and cost-effective PCR-RFLP assay has been developed for unambiguous discrimination of fifteen anopheline mosquito species from French Guiana including primary and suspected secondary malaria vectors.

Towards harmonization of DNA metabarcoding for monitoring marine macrobenthos: the effect of technical replicates and pooled DNA extractions on species detection

DNA-based monitoring methods are potentially faster and cheaper compared to traditional morphological benthic identification. DNA metabarcoding involves various methodological choices which can introduce bias leading to a different outcome in biodiversity patterns. Therefore, it is important to harmonize DNA metabarcoding protocols to allow comparison across studies and this requires a good understanding of the effect of methodological choices on diversity estimates. This study investigated the impact of DNA and PCR replicates on the detection of macrobenthos species in locations with high, medium and low diversity. Our results show that two to three DNA replicates were needed in locations with a high and medium diversity to detect at least 80% of the species found in the six DNA replicates, while three to four replicates were needed in the location with low diversity. In contrast to general belief, larger body size or higher abundance of the species in a sample did not increase its detection prevalence among DNA replicates. However, rare species were less consistently detected across all DNA replicates of the location with high diversity compared to locations with less diversity. Our results further show that pooling of DNA replicates did not significantly alter diversity patterns, although a small number of rare species was lost. Finally, our results confirm high variation in species detection between PCR replicates, especially for the detection of rare species. These results contribute to create reliable, time and cost efficient metabarcoding protocols for the characterization of macrobenthos.

Geographical range expansion of Nitzschia volvendirostrata Ashworth, D\u0105bek & Witkowski, 2016 (Bacillariophyta: Bacillariaceae) based on morphological and molecular analysis

In diatoms the use of molecular tools to corroborate traditional (morphological) identification offers a new perspective in the field of biogeography. This manuscript reports the first record of the raphid pennate diatom Nitzschia volvendirostrata along the coast of Mexico, which in turn represents an expansion of the geographic range along the American continent. The cells were isolated from benthic samples taken from Balandra lagoon, La Paz, Baja California Sur, Mexico and cultured as a monoclonal culture. Morphology and morphometry of the diatom obtained from light and electron microscopy reveal that they correspond to the original description of N. volvendirostrata and also its chloroplast sequences, rbcL and psbC, showed 98.7 to 100 % similarity and a close phylogenetic relationship with N. volvendirostrata. The reported places for this taxon show that it has a tropical-temperate biogeographical affinity.

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.