Morphology-based Identification Research Articles

Implications of taxonomic and numerical resolution on DNA metabarcoding-based inference of benthic macroinvertebrate responses to river restoration

It is essential to explore and define the level of taxonomic identification and quantification approaches for diversity and biomonitoring studies, given their potential influence on assessing and interpreting ecological outcomes. This study assessed the response of benthic macroinvertebrate communities to restore and construct gravel bars in a dam-impacted river, with non-dam influenced tributaries serving as reference sites. Additionally, we evaluated the performance of different taxonomic and numerical (i.e., abundance vs. presence/absence data) resolutions of DNA metabarcoding with consequent comparison to morphology-based identification and how it affects assessment outcomes. DNA metabarcoding detected 93% of the morphologically identified individuals and provided finer taxonomic resolution. We also detected significant correlations between morphological sample abundance, biomass, and DNA metabarcoding-read abundance. Furthermore, we observed a relatively high and significant congruence in macroinvertebrate community structure and composition between different taxonomic and numerical resolutions of both methods. This indicates a satisfactory surrogacy between the two approaches and their varying identification levels and data transformation. Additionally, community–environmental associations were significant for all datasets but showed significant varying associations with physicochemical parameters. Our observations imply that coarser taxonomic resolution could be highly advantageous for DNA metabarcoding-based applications in situations where the lack of taxonomic information, e.g., poor reference database, might severely affect biological assessment quality.

Discrimination of Tilletia controversa from the T. caries/T. laevis complex by MALDI-TOF MS analysis of teliospores

The fungal genus Tilletia includes a large number of plant pathogens of Poaceae. Only a few of those cause bunt of wheat, but these species can lead to significant yield losses in crop production worldwide. Due to quarantine regulations and specific disease control using appropriate seed treatments for the different disease agents, it is of high importance to distinguish Tilletia caries and Tilletia laevis as causal agents of common bunt accurately from Tilletia controversa, the causal agent of the dwarf bunt. Several studies have shown that matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful tool to differentiate closely related fungal species. The aim of this study was to assess whether MALDI-TOF MS analysis is able to distinguish specimens of the three closely related pathogens T. caries, T. laevis, and T. controversa and whether it may constitute an alternative method to the morphology-based identification or germination tests. Spectral data are available via ProteomeXchange with identifier PXD030401. Spectra-based hierarchical cluster analysis (HCA) and discriminant analysis of principal components (DAPC) of the obtained mass spectra showed two main clusters. One cluster included specimens of T. controversa, whereas the second cluster comprised T. laevis and T. caries specimens. Even though main spectral profiles (MSPs) for species identification are missing, MALDI-TOF MS has proven to be a useful method for distinguishing between T. controversa and the two causal agents of common bunt, using direct analysis of teliospores, but was unable to separate T. caries and T. laevis species.Key points• MALDI-TOF MS was developed to classify Tilletia species causing bunt of wheat.• Best results were achieved when combining HCA and DAPC analysis.• The method resulted in an accuracy of 98.51% testing 67 Tilletia specimens.Graphical abstract

The accuracy and clinical impact of the morphological identification of Aspergillus species in the age of cryptic species: A single-centre study.

Aspergillus spp. is identified morphologically without antifungal susceptibility tests (ASTs) in most clinical laboratories. The aim of this study was to examine the clinical impact of the morphological identification of Aspergillus spp. to ensure the adequate clinical management of Aspergillus infections. Aspergillus isolates (n=126) from distinct antifungal treatment-naïve patients with aspergillosis were first identified morphologically, followed by species-level identification via DNA sequencing. An AST for itraconazole (ITC) and voriconazole (VRC) was performed on each Aspergillus isolate. Based on the genetic test results, morphology-based identification was accurate for >95% of the isolates at the species sensu lato level although the test concordance of Aspergillus spp. with low detection rates was low. The rates of cryptic species were found to be 1.2% among the isolates of A.fumigatus complex and 96.8% in the A.niger complex. Cryptic species with lower susceptibilities to antifungal drugs than sensu stricto species among the same Aspergillus section were as follows: The A.lentulus (n=1) isolates had low susceptibilities to azoles among the A.fumigatus complex species (n=86), and A.tubingensis isolates (n=18) exhibited lower susceptibility to azoles among the A.niger complex species (n=31). Diagnostic accuracy was high at the A.fumigatus and A.niger complex level. However, in the presence of cryptic species, a solely morphological identification was insufficient. Particularly, ITC and VRC might be inappropriate for aspergillosis treatment when the A.niger complex is identified morphologically because it is possible that the Aspergillus isolate is A.tubingensis.

Morphological Characterization and DNA Barcoding of Duckweed Species in Saudi Arabia.

Duckweeds, or Lemnaceae, are widespread aquatic plants. Morphology-based identification of duckweed species is difficult because of their structural complexity. Hence, molecular tools provide significant advantages for characterizing and selecting species or clones for sustainable commercial use. In this study, we collected and characterized ten duckweed isolates from nine different regions in Saudi Arabia (SA). Based on the morphological characterization and phylogenetic analysis of intergenic spacer sequences of chloroplast DNA using six barcoding markers, the clones were classified into three genera, represented by seven species: Lemna gibba L., Lemna minor L., Lemna japonica Landolt, Lemna aequinoctialis Welw., Lemna perpusilla Torr., Spirodela polyryiza (L.) Schleid., and Landoltia punctate G. Mey. Lemna gibba was revealed to be a distinct dominant duckweed species in many regions of SA. Five barcoding markers showed that L. gibba, L. minor, and L. punctata were the most widely distributed species in the country. However, L. punctata, L. perpusilla, and S. polyryiza were the dominant species in the Al-Qassim, Madinah-1, and Madinah-2 regions, respectively. Moreover, the morphological traits revealed variations for these clones, relative to other studied duckweed clones. According to the results obtained in this study, three out of six plastid markers (trnH-psbA, matK, and atpF-atpH) helped to identify the dominant duckweed species in Saudi Arabia. Further evaluation based on adaptability, molecular genetic studies, and functional genomics is needed for these species to be used at the commercial level in Saudi Arabia.

Importance of Molecular Data to Identify Fungal Plant Pathogens and Guidelines for Pathogenicity Testing Based on Koch's Postulates.

Fungi are an essential component of any ecosystem, but they can also cause mild and severe plant diseases. Plant diseases are caused by a wide array of fungal groups that affect a diverse range of hosts with different tissue specificities. Fungi were previously named based only on morphology and, in many cases, host association, which has led to superfluous species names and synonyms. Morphology-based identification represents an important method for genus level identification and molecular data are important to accurately identify species. Accurate identification of fungal pathogens is vital as the scientific name links the knowledge concerning a species including the biology, host range, distribution, and potential risk of the pathogen, which are vital for effective control measures. Thus, in the modern era, a polyphasic approach is recommended when identifying fungal pathogens. It is also important to determine if the organism is capable of causing host damage, which usually relies on the application of Koch’s postulates for fungal plant pathogens. The importance and the challenges of applying Koch’s postulates are discussed. Bradford Hill criteria, which are generally used in establishing the cause of human disease, are briefly introduced. We provide guidelines for pathogenicity testing based on the implementation of modified Koch’s postulates incorporating biological gradient, consistency, and plausibility criteria from Bradford Hill. We provide a set of protocols for fungal pathogenicity testing along with a severity score guide, which takes into consideration the depth of lesions. The application of a standard protocol for fungal pathogenicity testing and disease assessment in plants will enable inter-studies comparison, thus improving accuracy. When introducing novel plant pathogenic fungal species without proving the taxon is the causal agent using Koch’s postulates, we advise the use of the term associated with the “disease symptoms” of “the host plant”. Where possible, details of disease symptoms should be clearly articulated.

Applicability of diatom metabarcoding in the ecological status assessment of Hungarian lotic and soda pan habitats

Diatoms are widely applied in the ecological status assessment of aquatic ecosystems using indices calculated from pollution sensitivity and indicator values of species. Traditional, morphology-based identification of species requires in-depth taxonomic knowledge and expertise. Identifying taxa according to their barcode sequences obtained with high-throughput sequencing (metabarcoding) would be a promising alternative. In this pilot study, we tested the applicability of metabarcoding of benthic diatom assemblages for the ecological status assessment of Hungarian water bodies, comparing its performance to that of morphology-based identification of species. The barcode region of the rbcL gene was investigated in samples from running waters with various trophic states and unique lentic habitats, namely soda pans. For running waters, the Specific Pollution Sensitivity Index (IPS) and for soda pans the H index and the Indice Biologique Diatomées (IBD) were calculated from the morphology- and sequence-based abundances. The ecological quality ratio was determined for running waters. Overall, more infrageneric taxa were found with microscopy than with metabarcoding in the lotic and lentic samples. The correspondence between taxon lists detected by the two methods was relatively low but increased considerably in the case of morphologically dominant taxa. Community composition based on microscopy and DNA sequence analysis showed a significant correlation and was determined by the same main environmental drivers. Morphology-based indices strongly correlated with sequence-based indices. Both aspects indicated the same ecological status class for more than half of the lotic samples. In other cases, a status shift from good to moderate was frequent, a phenomenon that could prove problematic because the Water Framework Directive prescribes intervention for waters with a moderate or worse status. Considering discrepancies between the results obtained with microscopy and metabarcoding, using both methods in parallel could be proposed until the reference database has been suitably updated.

Congruence between morphology-based species and Barcode Index Numbers (BINs) in Neotropical Eumaeini (Lycaenidae).

BackgroundWith about 1,000 species in the Neotropics, the Eumaeini (Theclinae) are one of the most diverse butterfly tribes. Correct morphology-based identifications are challenging in many genera due to relatively little interspecific differences in wing patterns. Geographic infraspecific variation is sometimes more substantial than variation between species. In this paper we present a large DNA barcode dataset of South American Lycaenidae. We analyze how well DNA barcode BINs match morphologically delimited species.MethodsWe compare morphology-based species identifications with the clustering of molecular operational taxonomic units (MOTUs) delimitated by the RESL algorithm in BOLD, which assigns Barcode Index Numbers (BINs). We examine intra- and interspecific divergences for genera represented by at least four morphospecies. We discuss the existence of local barcode gaps in a genus by genus analysis. We also note differences in the percentage of species with barcode gaps in groups of lowland and high mountain genera.ResultsWe identified 2,213 specimens and obtained 1,839 sequences of 512 species in 90 genera. Overall, the mean intraspecific divergence value of CO1 sequences was 1.20%, while the mean interspecific divergence between nearest congeneric neighbors was 4.89%, demonstrating the presence of a barcode gap. However, the gap seemed to disappear from the entire set when comparing the maximum intraspecific distance (8.40%) with the minimum interspecific distance (0.40%). Clear barcode gaps are present in many genera but absent in others. From the set of specimens that yielded COI fragment lengths of at least 650 bp, 75% of the a priori morphology-based identifications were unambiguously assigned to a single Barcode Index Number (BIN). However, after a taxonomic a posteriori review, the percentage of matched identifications rose to 85%. BIN splitting was observed for 17% of the species and BIN sharing for 9%. We found that genera that contain primarily lowland species show higher percentages of local barcode gaps and congruence between BINs and morphology than genera that contain exclusively high montane species. The divergence values to the nearest neighbors were significantly lower in high Andean species while the intra-specific divergence values were significantly lower in the lowland species. These results raise questions regarding the causes of observed low inter and high intraspecific genetic variation. We discuss incomplete lineage sorting and hybridization as most likely causes of this phenomenon, as the montane species concerned are relatively young and hybridization is probable. The release of our data set represents an essential baseline for a reference library for biological assessment studies of butterflies in mega diverse countries using modern high-throughput technologies an highlights the necessity of taxonomic revisions for various genera combining both molecular and morphological data.

New species and a new record of Phylloporia from Benin

Species of the wood-decay genus Phylloporia (Hymenochaetaceae, Hymenochaetales, Basidiomycota) are widely distributed in the tropics. Phylloporia species are, however, morphologically and ecologically diverse, which makes morphology-based species identification challenging. In this study, we re-examined species of Phylloporia reported from Benin (West Africa). Using an integrative approach combining morphology, ecology, and phylogenetic analyses, we describe Phylloporia beninensis sp. nov. and report Phylloporia littoralis for the first time outside of its type locality. Phylloporia beninensis sp. nov. is characterized by its annual and imbricate basidiomata, duplex context with a black zone separating the upper context from the lower one, dimitic hyphal system, presence of cystidioles, basidia of 9–12 × 4–5 μm, and subglobose to ellipsoid basidiospores measuring 3–4.6 × 2.1–3.6 μm. Detailed descriptions with illustrations for the new species are provided. With the addition of the new species, 15 Phylloporia species are now known to occur in tropical Africa. Our discovery of a new Phylloporia species in Benin should stimulate further mycological investigations in tropical African ecosystems to discover other new polypore species. To facilitate further taxonomy studies on tropical African Phylloporia taxa, a key to the known tropical African species is provided.

Identification of culturable microalgae diversity in the River Nile in Egypt using enrichment media

This study aimed to morphologically identify the highest possible microalgae biodiversity in a sample collected from the River Nile using the culture-based method. Water samples were cultured on the two commonly used media BG-11 and BBM media to obtain a broad spectrum of microalgae biodiversity. Likewise, the effects of nutrients concentration and vitamin supplements to BG-11 and BBM for supporting the maximum diversity of culturable microalgae were tested. Cell counts and morphology-based identification were conducted to evaluate the population diversity. A total of 35 species have been identified using the two media combined. The standard BG-11 and BBM media enriched 25 and 27 species, respectively, while the reduced nutrient concentration of BG-11 and BBM had a species richness of 19 and 24, respectively. The vitamin-enriched media each sustained BG-11+Vit (23) and BBM+Vit (20). We found that some algae species were uniquely identified on the reduced nutrient concentration or vitamin-enriched media. The results of this study report the current algae diversity in the Nile demonstrating that the usage of single-culture media for algal enrichment would result in underestimating the species richness. The diversity identified can be used as a reference for continuos monitoring of the River Nile microalgae diversity in Cairo.

Morphology-based identification and classification of Pediastrum through AlexNet Convolution Neural Network

In general solutions and outcomes to an issue in image processing, a ton of trail and examination with an enormous assortment of test photographs is incorporated. Pediastrum, which comprises of pigments like carotene, xanthophyll, chlorophylls, and other nourishing elements like complex sugars, major and minor minerals, and protein, enzymes, fiber, is a minute coccal shaped colony forming green algae. In this research, an attempt was made to solve the problem of identifying and classifying different species of Pediastrum with the aid of CNN based deep learning model. The informational collection comprising of 12,000 algal pictures was being utilized by, AlexNet CNN (Convolution Neural Network) model for the training and validating purposes. The features like the presence of sporopollenin in the cell wall, the structures and the functions of the cells, and the structural properties of coenobia make the basis for automated classification by AlexNet deep learning model. The efficacy of the proposed approach is demonstrated by an experimental outcome of 99.54 percent classification accuracy with precision and Fl-score more than 0.98.

<p><strong>True branching and phenotypic plasticity in the planktonic cyanobacterium <em>Dolichospermum brachiatum sp. nov.</em> (Nostocales, Aphanizomenonaceae), from south-eastern Australia</strong></p>

Phenotypic plasticity in the cyanobacteria provides adaptive strategies to cope with changes in prevailing environmental conditions. Plasticity may manifest in morphological, physiological, or behavioural responses. Determining if variable phenotypes are truly novel expressions evolved via adaptive genetic change, rather than intrinsic characteristics within the spectrum of intraspecific diversity has rarely been studied. Poorly characterised or unrecognised intraspecific variability is as problematic as cryptic diversity in terms of imposing limitations on the use of morphology-based identifications to recognise algal diversity. We isolated strains from a population of filamentous cyanobacteria from the plankton of a temperate lake in Western Victoria, Australia putatively identified as the cylindrospermopsin producer Chrysosporum ovalisporum which displayed aberrant vegetative and apical cells and akinete morphology, and T-type true branching. In culture, strains grown in phosphorus-free media showed morphology and true branching consistent with the wild types suggesting that these features are a phenotypic response to P-limitation. Results of the phylogenetic analyses of these strains using the 16S-23S rRNA, and the secondary structure of the ITS region of the 16S-23S rRNA operon confirmed that the isolated strains were a novel species, here designated Dolichospermum brachiatum. This is the first record of true branching in the genus Dolichospermum and only the second observation from the Aphanizomenonaceae. These findings support our current understanding that true branching in cyanobacteria is a polyphyletic trait, having arisen several times in the evolution of this Phylum.

Re-evaluating the presence of Carex microcarpa (Cyperaceae) in Italy based on herbarium material and DNA barcoding

Carex microcarpa (sect. Rhynchocystis) is a Central Mediterranean endemic known from Corsica, Sardinia and the Tuscan archipelago, that has been also reported from scattered localities in mainland Italy (Abruzzo, Lazio and Apulia). We tested by molecular means the identification of three specimens classified as C. microcarpa from continental Italy, as well as a voucher from the Montecristo island (Tuscany) and one from Sardinia, whose taxonomic identity could not be determined with certainty by morphological examination. We performed a DNA barcoding study based on four DNA regions (two nuclear, two plastid). Our results reveal that none of previous mainland Italian records corresponds to C. microcarpa, but to C. pendula, which also belong to section Rhynchocystis, and in other cases possibly to C. acutiformis, from a different species group (sect. Paludosae). The specimen from Montecristo island was confirmed to be C. microcarpa. We conclude that C. microcarpa is likely a Tyrrhenian endemic exclusively known with certainty in Corsica, Sardinia and the Tuscan Archipelago. The presence of C. microcarpa in the Italian peninsula remains highly doubtful and we recommend its exclusion from checklists. Our study highlights the utility of DNA barcoding approaches in Carex when a reliable morphology-based identification is not possible. Supplemental data for this article is available online at https://doi.org/10.1080/11263504.2021.1897703 .

New insights into Danube’s macroinvertebrate communities from DNA metabarcoding as part of the Joint Danube Survey 4 (JDS4)

The Joint Danube Survey (JDS) is a multinational effort in monitoring Danube’s water quality, including its major tributaries. The Danube river stretches over a distance of 2,800 km and flows through or borders 10 different countries to which it is of utter importance as a source of potable water and hydrodynamic power. The JDS is conducted every 6 years and provides a unique opportunity to collect comprehensive data on both abiotic parameters and organisms and to raise awareness of the importance of water as a natural resource. As part of JDS and as a biological quality element in many monitoring programs worldwide, macroinvertebrates are monitored as indicators for various environmental conditions. However, due to their diverse taxonomic composition, associated difficulties with their morphology-based identification as well as their sheer abundance, macroinvertebrates are often analysed with a low taxonomic resolution (i.e., above species level). As an alternative, DNA metabarcoding offers a promising approach to capture this species diversity more accurately. Here, we used DNA metabarcoding to investigate the macrozoobenthic diversity of 46 sites from the latest JDS sampling campaign in 2019. To analyse macroinvertebrate diversity, bulk samples were taken by kick-net sampling and analysed using two different approaches, analysing the bulk sample fixative and analysing homogenised organisms from complete bulk samples. DNA metabarcoding of the sample fixative revealed 1,146 Operational Taxonomic Units (OTUs) and 231 species compared to 833 OTUs and 333 species from homogenised sample analysis. While more dipterans, in particular Chironomidae, were detected in fixative (136 species) than homogenised bulk (90 species) analyses, the latter picked up more Trichoptera (19 vs. 2), Amphipoda (10 vs. 4) and Bivalvia species (13 vs. 5). Even though these results of a DNA-based assessment deliver new insights into species richness and composition of Danube’s macroinvertebrate communities from the Danube source to its delta already, it is evident that the majority of OTUs was not assigned to species. While filling this lack of reference sequences poses a major challenge, the JDS consortium also offers a unique opportunity to complement reference databases in a multinational effort towards a more comprehensive Danube assessment and monitoring.

Establishing (e)DNA-Methods for Governmental Biomonitoring Efforts and Ecological Assessment in German Federal Waterways

The German Federal Institute of Hydrology (BfG, for its acronym in German) is the nationwide advising departmental research institution regarding hydrology, water use and quality, ecology and water protection of the German Federal Waterways that comprise inland waterways as well as marine coastal waters. The Federal Waterways of Germany are important habitats and distribution corridors for aquatic biodiversity. However, the ecology of these large rivers is heavily impacted by shipping, hydromorphological alterations, chemical pollution and the distribution and establishment of invasive species. All these factors have led to a decline of native biodiversity and ecological status. Since the 1970s, tremendous efforts have been undertaken to restore water quality and biodiversity. Continuous monitoring and assessment of both chemical components and biological quality elements (BQEs) required by law should indicate the success or failure of restoration measures as well as ongoing and future ecological consequences of multiple stakeholders’ interests and demands. Standardized ecological assessment procedures are already in place for both rivers and streams. However, these widely used procedures have several shortcomings and require a high level of taxonomic competence, which is being lost at an increasing rate. DNA and environmental DNA (eDNA)-based methods, such as DNA barcoding and (e)DNA metabarcoding, are promising alternatives to overcome the impediments to the morphology-based identification of organisms. But, these new methods need to be tailored to the challenges of large river bioassessment. The implementation of such state-of-the-art methods for future biomonitoring and ecological assessment efforts in large rivers should therefore ultimately serve the legal requirements regarding the EU Water Framework Directive (WFD) and promote the application of (e)DNA methods from basic and applied research to water managers and regulatory authorities. Here we present the goals and approaches of our ongoing project that are 1) to create standard operation procedures (SOPs) for (e)DNA analyses for large rivers and 2) update or establish new methods for biodiversity assessment and biomonitoring (e.g. presence-absence analyses) of the federal waterways; and 3) to transfer the gained knowledge to water managers and practitioners. To accomplish these goals, the focus of our research relies on the BQEs fish and macrozoobenthos (led by Saskia Schmidt), as well as phytobenthos and phytoplankton (led by Demetrio Mora).

Applicability of diatom metabarcoding in the ecological status assessment of Hungarian lotic and soda pan habitats

Diatoms, eukaryotic algae with silica cell wall have been proved to be reliable bioindicators and are applied in ecological status assessment of aquatic ecosystems using indices calculated from pollution sensitivity and indicator values of species. Traditional method of identification of diatom species is based on morphological features of frustule that requires in-depth taxonomical knowledge and expertise. Metabarcoding that combines barcodes and high-throughput sequencing offers a promising alternative. In this pilot study we tested the applicability of metabarcoding of benthic diatom assemblages for ecological status assessment of water bodies in Hungary comparing its performance to that of morphology-based identification of species. Samples from 78 rivers and streams arranged along a trophic gradient and 14 soda pans representing unique types of habitats were investigated. Sequences of the barcode region of rbcL gene suggested by Vasselon et al. (2017) were acquired revealing 1135 diatom amplicon sequence variants (ASVs) of which 54% could be assigned at species level using Diat.barcode (Rimet et al. 2019) as reference database. Morphological investigation detected more species and intraspecific taxa in the lotic and lentic samples (413 and 78, respectively) than metabarcoding (190 and 75). Correspondence between taxa detected based on morphology and DNA sequences was relatively low (on average 24% in lotic and 26% in lentic samples) but considerably increased (on average 66% and 56%, respectively) when taking into account only the taxa reaching higher than 5% relative abundance in microscopy counting. The differences mainly derived from that a considerable portion (59% and 32%, respectively) of the morphologically identified taxa were not recorded in the reference database. Community structure of samples from both running and standing waters based on microscopy and DNA sequence analysis showed significant correlation as revealed using Mantel test. For rivers and streams diatom index IPS (Specific Pollution Sensitivity Index, (Coste in Cemagref, 1982) for soda pans indices H (Ziemann et al., 2001) and IBD (Indice Biologique Diatomées, Lenoir and Coste, 1996) were calculated from the results of the two aspects. Strong correlation was found between morphology- and sequence-based indices and in about half of the samples, the ecological status class obtained with the two methods coincided. Our results suggest that metabarcoding inheres a great opportunity and could be successfully applied in benthic diatom-based ecological status assessment of Hungarian waters after the reference database is supplemented with taxa frequently occurring in these habitats. References Cemagref, 1982. Etude des méthodes biologiques quantitativesd’appréciation de la qualité des eaux, Rapport Division Qualité des Eaux Lyon. Agence financière de Bassin Rhône-Méditerranée-Corse, Pierre-Bénite, France. Lenoir, A., Coste, M., 1996. Development of a practical diatom index of overall water quality applicable to the French National Water Board Network. Presented at the International symposium, Volksbildungsheim Grilhof Vill, AUT, 17-19 September 1995, Universität Innsbruck, pp. 29–43. Rimet, F., Gusev, E., Kahlert, M., Kelly, M.G., Kulikovskiy, M., Maltsev, Y., Mann, D.G., Pfannkuchen, M., Trobajo, R., Vasselon, V., Zimmermann, J., Bouchez, A., 2019. Diat.barcode, an open-access curated barcode library for diatoms. Sci. Rep. 9, 15116. Vasselon, V., Rimet, F., Tapolczai, K., Bouchez, A., 2017. Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island, France). Ecol. Indic. 82, 1–12. Ziemann, H., Kies, L., Schulz, C.-J., 2001. Desalinization of running waters: III. Changes in the structure of diatom assemblages caused by a decreasing salt load and changing ion spectra in the river Wipper (Thuringia, Germany). Limnologica 31, 257–280.

Searchlight trapping reveals seasonal cross-ocean migration of fall armyworm over the South China Sea

The fall armyworm (FAW), Spodoptera frugiperda, is a migratory noctuid pest that has recently invaded eastern Asia. From 2017 up till 2020, searchlight trapping was used to assess the extent of FAW migration across the South China Sea. Molecular and morphology-based identification confirmed that FAW made its first appearance on Yongxing Island on 11 April 2019, with most trapped individuals belonging to the S. frugiperda “corn-strain”. Carbon isotope analysis further showed that FAW moths originated from C4 host plants, while trajectory analyses revealed that migratory S. frugiperda adults are able to cross the South China Sea and enter mainland China. This long-distance migration process plausibly results in frequent genetic mixing between domestic FAW populations and those of neighboring Southeast Asian countries. Overall, this work provides unique insights into FAW migration across eastern Asia and ultimately can help advance pest forecasting, risk assessment and area-wide pest management.

Phylogenetic analysis of the distribution of deadly amatoxins among the little brown mushrooms of the genus Galerina.

Some but not all of the species of 'little brown mushrooms' in the genus Galerina contain deadly amatoxins at concentrations equaling those in the death cap, Amanita phalloides. However, Galerina's ~300 species are notoriously difficult to identify by morphology, and the identity of toxin-containing specimens has not been verified with DNA barcode sequencing. This left open the question of which Galerina species contain toxins and which do not. We selected specimens for toxin analysis using a preliminary phylogeny of the fungal DNA barcode region, the ribosomal internal transcribed spacer (ITS) region. Using liquid chromatography/mass spectrometry, we analyzed amatoxins from 70 samples of Galerina and close relatives, collected in western British Columbia, Canada. To put the presence of toxins into a phylogenetic context, we included the 70 samples in maximum likelihood analyses of 438 taxa, using ITS, RNA polymerase II second largest subunit gene (RPB2), and nuclear large subunit ribosomal RNA (LSU) gene sequences. We sequenced barcode DNA from types where possible to aid with applications of names. We detected amatoxins only in the 24 samples of the G. marginata s.l. complex in the Naucoriopsis clade. We delimited 56 putative Galerina species using Automatic Barcode Gap Detection software. Phylogenetic analysis showed moderate to strong support for Galerina infrageneric clades Naucoriopsis, Galerina, Tubariopsis, and Sideroides. Mycenopsis appeared paraphyletic and included Gymnopilus. Amatoxins were not detected in 46 samples from Galerina clades outside of Naucoriopsis or from outgroups. Our data show significant quantities of toxin in all mushrooms tested from the G. marginata s.l. complex. DNA barcoding revealed consistent accuracy in morphology-based identification of specimens to G. marginata s.l. complex. Prompt and careful morphological identification of ingested G. marginata s.l. has the potential to improve patient outcomes by leading to fast and appropriate treatment.

Comparison of Larval Fish Detections Using Morphology-Based Taxonomy versus High-Throughput Sequencing for Invasive Species Early Detection.

When first introduced, invasive species typically evade detection; DNA barcoding coupled with high-throughput sequencing (HTS) may be more sensitive and accurate than morphology-based taxonomy, and thereby improve invasive (or rare) species detection. We quantified the relative error of species detection between morphology-based and HTS-based taxonomic identification of ichthyoplankton collections from the Port of Duluth, Minnesota, an aquatic non-native species introduction 'hot-spot' in the Laurentian Great Lakes. We found HTS-based taxonomy identified 28 species and morphology-based taxonomy 30 species, of which 27 were common to both. Among samples, 76% of family-level taxonomic assignments agreed; however, only 42% of species assignments agreed. Most errors were attributed to morphology-based taxonomy, whereas HTS-based taxonomy error was low. For this study system, for most non-native fishes, the detection probability by randomized survey for larvae was similar to that by a survey that is optimized for non-native species early detection of juveniles and adults. We conclude that classifying taxonomic errors by comparing HTS results against morphology-based taxonomy is an important step toward incorporating HTS-based taxonomy into biodiversity surveys.

Adding DNA barcoding to stream monitoring protocols - What's the additional value and congruence between morphological and molecular identification approaches?

Although aquatic macroinvertebrates and freshwater fishes are important indicators for freshwater quality assessments, the morphological identification to species-level is often impossible and thus especially in many invertebrate taxa not mandatory during Water Framework Directive monitoring, a pragmatism that potentially leads to information loss. Here, we focus on the freshwater fauna of the River Sieg (Germany) to test congruence and additional value in taxa detection and taxonomic resolution of DNA barcoding vs. morphology-based identification in monitoring routines. Prior generated morphological identifications of juvenile fishes and aquatic macroinvertebrates were directly compared to species assignments using the identification engine of the Barcode of Life Data System. In 18% of the invertebrates morphology allowed only assignments to higher systematic entities, but DNA barcoding lead to species-level assignment. Dissimilarities between the two approaches occurred in 7% of the invertebrates and in 1% of the fishes. The 18 fish species were assigned to 20 molecular barcode index numbers, the 104 aquatic invertebrate taxa to 113 molecular entities. Although the cost-benefit analysis of both methods showed that DNA barcoding is still more expensive (5.30–8.60€ per sample) and time consuming (12.5h), the results emphasize the potential to increase taxonomic resolution and gain a more complete profile of biodiversity, especially in invertebrates. The provided reference DNA barcodes help building the foundation for metabarcoding approaches, which provide faster sample processing and more cost-efficient ecological status determination.

Wound Myiasis in Diabetic Foot Ulcer: Calliphoridae and Sarcophagidae Family.

Background:Myiasis is a parasitic infestation of tissues or body cavities of mammals with dipterous larvae. The patients with diabetic foot ulcers are more vulnerable to acquiring infestation; however, the infestation may be neglected and mistreated in some cases.Methods:Data were collected of twelve myiasis cases with diabetic foot ulcers in Nazli-Selim Eren Chronic Wound and Infections Care Unit, Aydin, Turkey between 2017 and 2019. Demographic, clinical characteristics of the patients and clinical examination of the wound were recorded. To morphology-based identification method of the agents, the developmental stages of the maggots were examined.Results:The cases aged between 46 and 81 years (10 males, two females). Eight of the larvae collected from wounds had Calliphoridae and four had Sarcophagidae family. The larvae were infested right/left foot sole, thumb, ankle, and mostly left toes. The number of larvae collected from the cases ranged from 2 to 48. Third-stage larvae (L3) were mostly detected. Mixed (L1-L2, L2-L3) larvae were detected in a patient. The infestations were more common in July and August. According to the score of Infectious Diseases Society of America (IDSA), ten (83%) cases had moderate and two (17%) cases were mild diabetic foot infections (DFIs).Conclusion:Diabetic foot ulcers should be evaluated in terms of myiasis. This was the first study in our province indicating that myiasis should not be neglected and different species of flies were responsible for myiasis cases.