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The focus of this study was on obtaining DNA sequence information from selected rare plant species of Tennessee and their close relatives for the nuclear ribosomal ITS region, which is widely used for molecular barcoding. New sequence data was obtained for 71 species from 18 genera. The ITS region provided a good molecular barcode for nine of the genera. For the other genera, the results suggested that the ITS region would be uninformative, problematic, or require detailed analysis for application. For the orchid genus Platanthera, data from the ITS marker allowed unambiguous identification of vegetative plants of the federally listed P. integrilabia, which will help make efficient use of resources for conservation of the species. For the aquatic genus Potamogeton, the ITS data suggested the presence of unsuspected interspecific hybridization involving the rare species P. tennesseensis which showed the need for further investigation. The sequencing results provided validation of the separation of Lobelia gattingeri as distinct from L. appendiculata. The results of the study expanded the database of DNA sequences for rare plants of Tennessee and also highlighted the need for further study of the flora of the state and of the southeastern United States.

Like all obligately ectomycorrhizal plants, pines require ectomycorrhizal fungal symbionts to complete their life cycle. Pines introduced into regions far from their native range are typically incompatible with local ectomycorrhizal fungi, and, when they invade, coinvade with fungi from their native range. While the identities and distributions of coinvasive fungal symbionts of pine invasions are poorly known, communities that have been studied are notably depauperate. However, it is not yet clear whether any number of fungal coinvaders is able to support a Pinaceae invasion, or whether very depauperate communities are unable to invade. Here, we ask whether there is evidence for a minimum species richness of fungal symbionts necessary to support a pine/ectomycorrhizal fungus coinvasion. We sampled a Pinus contorta invasion front near Coyhaique, Chile, using molecular barcoding to identify ectomycorrhizal fungi. We report that the site has a total richness of four species, and that many invasive trees appear to be supported by only a single ectomycorrhizal fungus, Suillus luteus. We conclude that a single ectomycorrhizal (ECM) fungus can suffice to enable a pine invasion.

Background The ixodid tick genera Rhipicephalus and Haemaphysalis contain several species of medical and/or veterinary importance, but their diversity in some regions of the world remains under-explored. For instance, very few modern studies have been performed on the taxonomy of these genera on the Arabian Peninsula. Methods In this study, we trapped small mammals in the 'Asir Mountains of south-western Saudi Arabia and collected tick specimens for morphological examination and molecular barcoding, targeting three mitochondrial loci: cox1 , 16S rRNA and 12S rRNA. Results We obtained a total of 733 ticks (608 Haemaphysalis spp. and 125 Rhipicephalus spp.) from 75 small mammal hosts belonging to six species. All tick specimens were immature except for nine adults recovered from a hedgehog ( Paraechinus aethiopicus ). Morphologically, the Rhipicephalus ticks resembled R. camicasi , but the Haemaphysalis ticks showed differences in palp morphology compared with species previously described from Saudi Arabia. Phylogenetic analysis and automatic barcode gap discovery identified a novel clade of Rhipicephalus sp. representing most of the nymphs. This was most closely related to R. leporis , R. guilhoni and R. linnaei . The adult ticks and a small proportion of nymphs clustered with R. camicasi sequences from a previous study. Finally, the Haemaphysalis nymphs formed two distinct clades that were clearly separated from all reference sequences but closest to some African species. Conclusions This apparent high level of tick diversity observed in a single study site of only ~ 170 km 2 , on a relatively small number of hosts, highlights the potential for the discovery of new tick species on the Arabian Peninsula. Graphical Abstract

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides , revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.

Myxomycetes are a unique branch of life, recognisable by sporophores showing a fungus-like dispersal biology. These structures bear nearly all diagnostic characters for species identification and develop by rapid transformation of plasmodia. During this short period of time, external factors can significantly influence the formation of morphological characters. Therefore, the description of a new species must be carried out with utmost care. Over the last 50 years, approximately 10–15 new species of myxomycetes have been described per year and only some of the latest publications underpin this with molecular data. In this paper, we discuss a set of recommendations for the description of myxomycete species new to science, striving for the following goals: (i) to minimise the number of erroneous descriptions of the species, whose names later have to be put into synonymy; (ii) to make all respective data easily accessible for the scientific community; and (iii) to comply with existing rules of nomenclature. We recommend (1) whenever possible not to describe a new taxon from a single specimen; however, an exception could be made only if supported by molecular data and by unique morphological characters which are unlikely to fall in the range of infraspecific variation of related species; (2) preparing detailed descriptions, including data on developmental stages, microhabitats, ecology, phenology and associated species; (3) providing at least two independent diagnostic characters that tell the new species apart from all others; (4) obtaining a molecular barcode and, whenever possible, providing proof for reproductive isolation of the new species from related taxa; and (5) depositing type specimens in public herbaria. To comply with nomenclatural rules, (6) the new name must be registered in a recognised repository, (7) all published names should be checked for usability before proposing a new name and (8) a unique name should be chosen, preferably highlighting a distinct character of the new species.

We present a simple molecular indexing method for quantitative targeted RNA sequencing, in which mRNAs of interest are selectively captured from complex cDNA libraries and sequenced to determine their absolute concentrations. cDNA fragments are individually labeled so that each molecule can be tracked from the original sample through the library preparation and sequencing process. Multiple copies of cDNA fragments of identical sequence become distinct through labeling, and replicate clones created during PCR amplification steps can be identified and assigned to their distinct parent molecules. Selective capture enables efficient use of sequencing for deep sampling and for the absolute quantitation of rare or transient transcripts that would otherwise escape detection by standard sequencing methods. We have also constructed a set of synthetic barcoded RNA molecules, which can be introduced as controls into the sample preparation mix and used to monitor the efficiency of library construction. The quantitative targeted sequencing revealed extremely low efficiency in standard library preparations, which were further confirmed by using synthetic barcoded RNA molecules. This finding shows that standard library preparation methods result in the loss of rare transcripts and highlights the need for monitoring library efficiency and for developing more efficient sample preparation methods.

Understanding vertebrate–vector interactions is vitally important for understanding the transmission dynamics of arthropod-vectored pathogens and depends on the ability to accurately identify the vertebrate source of blood-engorged arthropods in field collections using molecular methods. A decade ago, molecular techniques being applied to arthropod blood meal identification were thoroughly reviewed, but there have been significant advancements in the techniques and technologies available since that time. This review highlights the available diagnostic markers in mitochondrial and nuclear DNA and discusses their benefits and shortcomings for use in molecular identification assays. Advances in real-time PCR, high resolution melting analysis, digital PCR, next generation sequencing, microsphere assays, mass spectrometry, and stable isotope analysis each offer novel approaches and advantages to bloodmeal analysis that have gained traction in the field. New, field-forward technologies and platforms have also come into use that offer promising solutions for point-of-care and remote field deployment for rapid bloodmeal source identification. Some of the lessons learned over the last decade, particularly in the fields of DNA barcoding and sequence analysis, are discussed. Though many advancements have been made, technical challenges remain concerning the prevention of sample degradation both by the arthropod before the sample has been obtained and during storage. This review provides a roadmap and guide for those considering modern techniques for arthropod bloodmeal identification and reviews how advances in molecular technology over the past decade have been applied in this unique biomedical context.

Background Hippobosca longipennis (Diptera: Hippoboscidae) is an obligate hematophagous ectoparasite that infests a wide range of vertebrate hosts across Africa, Southern Europe, the Middle East, and Asia. It is a potential vector of Acanthocheilonema dracunculoides (Filarioidea: Onchocercidae) and serves as a phoretic host for Cheyletiella yasguri (Acari: Cheyletiellidae), a known causative agent of dermatitis in both dogs and humans. Due to the lack of data on hippoboscids in Morocco, this study aimed to investigate the louse fly fauna of sheltered dogs in the country as well as the filarial nematodes they may harbor. Methods Between April and November 2022, 230 sheltered dogs from four cities in Central Morocco were randomly examined as part of an entomological and epidemiological study on arthropod vectors and canine vector-borne pathogens. All visible louse flies on the domestic dogs were randomly collected and then morphologically and molecularly identified. DNA was subsequently extracted for screening of filarial nematodes. Results A total of 30 dogs (13.1%) were infested with 35 H. longipennis louse flies, consisting of 33 adults (10 males, 19 non-gravid females, and four gravid females) and two larvae. Two representative specimens were confirmed through DNA barcoding of the cytochrome oxidase subunit I gene. All fly pools (gravid females, non-gravid females, males, and larvae) tested negative for filarial nematodes in the 12S rRNA PCR. Conclusions This study represents the first morphological and molecular characterization of H. longipennis flies in Morocco. Further national-scale investigations are needed to address gaps in the knowledge of unrecorded hippoboscid species and the pathogens of medical and veterinary importance that they may carry. Graphical Abstract

Two new species of lichens, Oset, Flakus & Guzow-Krzem. and Oset, Flakus & Guzow-Krzem., are described based on material collected in Bolivia. The study is based on morphological and anatomical examinations, molecular phylogenetic analysis and haplotype network analysis of the nuITS rDNA sequences. Phenotypically, is characterised by the long, concentrically arranged setae on the surface of the thallus and densely pilose margins, whereas in the diagnostic features are the brown upper surface thallus when fresh, pale yellow to orange-yellow when dry, with slightly visible concentric colour zonation when dry and also with involute, creamy white margins. In addition, three species, i.e., B. Moncada, C. Rodr. & Lücking, Holgado, Rivas Plata & Perlmutter, and L.Y. Vargas, B. Moncada & Lücking, are reported as new to Bolivia. New records of Wilk, Lücking & E. Morales from Bolivia are reported.

The recent increase in international fish trade leads to the need for improving the traceability of fishery products. In relation to this, consistent monitoring of the production chain focusing on technological developments, handling, processing and distribution via global networks is necessary. Molecular barcoding has therefore been suggested as the gold standard in seafood species traceability and labelling. This review describes the DNA barcoding methodology for preventing food fraud and adulteration in fish. In particular, attention has been focused on the application of molecular techniques to determine the identity and authenticity of fish products, to discriminate the presence of different species in processed seafood and to characterize raw materials undergoing food industry processes. In this regard, we herein present a large number of studies performed in different countries, showing the most reliable DNA barcodes for species identification based on both mitochondrial (COI, cytb, 16S rDNA and 12S rDNA) and nuclear genes. Results are discussed considering the advantages and disadvantages of the different techniques in relation to different scientific issues. Special regard has been dedicated to a dual approach referring to both the consumer’s health and the conservation of threatened species, with a special focus on the feasibility of the different genetic and genomic approaches in relation to both scientific objectives and permissible costs to obtain reliable traceability.