Spacers ITS1 Research Articles

Identification of pathogenic fungi in garlic bulbs during storage and in the root zone during plant growth

Relevance and methods. Losses of agricultural crops are associated not only with the development of diseases during the growing season, but also during post-harvest storage. Garlic is a popular vegetable and aromatic crop in world. Significant losses in garlic yield during cultivation and storage are associated with fungal pathogens, the most harmful of which are representatives of the genus Fusarium. In the Moscow region, the defeat of garlic by Fusarium occurs annually, but with varying intensity. At the Federal Scientific Vegetable Center (FSVC), it was shown that rot and wilting of garlic plants is caused by a complex of pathogenic fungi, including mainly different species of Fusarium. At the same time, the ratio of Fusarium species in the pathogenic complex changes from year to year, new Fusarium species and their races are registered. The aim of this study was to identify fungal phytopathogens causing dry rot of garlic cloves during post-harvest storage. To carry out the work, garlic bulbs of cultivars Dubkovsky and Strelets were taken from the FSVC storage.Results. As a result of visual examination, cloves with symptoms of dry rot were identified. The diseased cloves tissues were plated on potato dextrose agar to obtain fungal colonies. Analysis of the morphological and cultural characteristics of fungal isolates, as well as the nucleotide sequences of four DNA regions (ITS spacers, genes EF1α, RPB1, and RPB2) showed that the causative agent of dry rot of garlic cloves is the pathogenic fungus Fusarium proliferatum. In addition, in the field, identification was carried out based on the analysis of the sequences of spacers ITS and the EF1αgene of phytopathogenic fungi inhabiting the root zone of garlic plants. As a result, two species of fungi of the genus Fusarium (F. proliferatum and F. oxysporum f. sp. cepae), as well as the species Rhizoctonia solani, Volutella rosea, and Ceratobasidium sp. were found in the root zone of garlic cultivars.

A Novel Model for the RNase MRP-Induced Switch between the Formation of Different Forms of 5.8S rRNA.

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3′ end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5′ end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5′ end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5′ end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.

Microbial profiles of Greek PDO cheeses assessed with amplicon metabarcoding

Greece is a country possessing many cheese products granted with a PDO (Protected Designation of Origin) certificate, with high exporting activities. In this study, we analyzed six popular cheese PDO products purchased from different industries to assess their microbial communities using amplicon metabarcoding analysis. To this end, using Next Generation Sequencing technology, we sequenced the 16S rRNA gene and the ITS spacer for prokaryotes and fungi, respectively. Alpha diversity indices revealed higher bacterial species richness for some cheeses (Kopanisti, Batzos) and poor for others (Feta, Galotiri). Kopanisti, together with Kalathaki and Anevato, also presented increased species diversity concerning fungal populations. Results showed that lactic acid bacteria (LAB) prevailed the bacterial populations in all samples (Lactococcus, Lactobacillus, Streptococcus, Leuconostoc), whereas for fungi, members of the Saccharomycetaceae, Dipodascaceae and Debaryomycetaceae families prevailed the fungal populations. Several other genera were identified that make up each product's microbiome leading to the creation of the unique organoleptic attributes of Greek PDO cheeses. However, the identified species could not be directly linked to certain cheese types, assuming that starter and adjunct cultures, combined with the raw material used during production greatly impact the microbial communities in cheeses. Our data, produced for the first time for six Greek PDO cheeses, can be exploited in the process of creating a core microbial signature within each cheese type, supporting the Greek brand name and valorizing cheese products.

Phylogenetic analyses of ribosomal transcription units from Haplorchis taichui and H. pumilio species of Heterophyidae (Platyhelminthes: Opisthorchiata)

Heterophyidiasis caused by minute intestinal flukes becomes of public concern in many countries worldwide. Haplorchis taichui and H. pumilio, belonging to the family Heterophyidae (Platyhelminthes: Trematoda) are two of many infecting humans and commonly found in Vietnam. Sequence study of these two small intestinal flukes is still very limited, hence we need more prospective markers for taxonomic identification and classification. This study provides complete coding sequence of the ribosomal transcription units (rTU) from H. taichui and H. pumilio (Vietnamese samples) and demonstrates the use of complete 28S rDNA sequences for phylogenetic analysis.The complete coding sequence of the rTU (from 5' 18S to 3' 28S), consisting of complete 18S, ITS-1, 5.8S, ITS2 and complete 28S rRNA genes and spacers, from H. taichui (7,268 bp) and H. pumilio (7,416 bp) from human hosts in Vietnam, were determined and annotated. The 18S and 5.8S genes of both species were of the same length (1,992 bp/18S, 160 bp/5.8S), but 28S genes differed (3,875 bp/H. taichui and 3,870 bp/H. pumilio). ITS-1 in H. taichui (797 bp) and ITS-2 in H. pumilio (280 bp) do not contain tandem repeat units (TRUs), while ITS-1 in H. pumilio (1,106 bp) contains 3 TRUs of 136 bp/each and 2 TRUs of 116 bp/each and ITS-2 in H. taichui (444 bp) contain 3 TRUs (83–85 bp/each). A phylogenetic tree inferred from the alignment of complete 28S rDNA sequences of 32 trematode strains/species, including 2 Vietnamese Haplorchis spp. and 24 species of 8 families in the suborders Xiphidiata (families Nanophyetidae, Paragonimidae, Collyriclidae), Opisthorchiata (Heterophyidae, Opisthorchiidae), and Echinostomata (Echinostomatidae, Fasciolidae). and Schistosoma japonicum of the family Schistosomatidae is used as an outgroup. The topology of the phylogenetic tree clearly confirmed the status of the Vietnamese H. taichui and H. pumilio species. These species gathered in a group (in the family Heterophyidae) clearly identified in the position of "sister” group to those in the family Opisthorchiidae (suborder Opisthorchiata, superfamily Opisthorchioidea).

Insights on a rare species, 'Geum atlanticum', new data to differentiate from 'Geum sylvaticum'

Geum atlanticum is a species described from North Africa and later found in Southern Iberian Peninsula. However, according to Flora iberica taxonomic treatment, it has been considered synonymous with G. sylvaticum, a species mainly distributed by Southeast of France, Iberian Peninsula, and North of Africa, sharing part of the distribution range and habitat. In this work, herbarium specimens of both G. atlanticum and G. sylvaticum were revised to evaluate morphological differences. Furthermore, the nuclear ribosomal ITS spacer was sequenced to study phylogenetic relationships among G. atlanticum and Iberian Geum species. The morphological study indicated clear differences with G. sylvaticum, and the phylogenetic analysis suggests that G. atlanticum was sister to G. sylvaticum. However, it shared several mutations with other Iberian Geum species. The number of populations is relatively low in the Iberian Peninsula, so its conservation status and its further inclusion in lists of threatened plants have been evaluated in this work.

Differentiation of Two Pleurotus Species Based on the Restrictive Digestion Profile of the Internal Transcribed Spacer Region

Two oyster mushrooms (Pleurotus eous P-31 and P. ostreatus EM-1) are under either cottage industry or semi-commercial cultivation in Ghana. The latter (P. ostreatus) is already well known to the public and on the shelf of some leading supermarkets. There is morphological resemblance between the two species making it difficult for the untrained eye to distinguish between them except for the colour difference. In this study, molecular methods were em­ployed to differentiate among the two species. The Internal Transcribed Spacer ITS 1 and ITS 4 regions of the rDNA of the two oyster species were amplified by the conventional PCR using the universal primer pair, ITS 1 and ITS 4 followed by restrictive digestion with enzymes, (Hh I, Hinf I, Rsa I and Hae III). The two species could not be separated based on the ampli­fied bands only, as both produced a characteristic band size of 650 bp. Gel profiling showing restrictive patterns generated by the four enzymes indicated that only the Hae III restrictive enzyme was effective in separating P. eous P-31 and P. ostreatus EM-1. This is the first record of the separation of the Ghanaian Pleurotus species by molecular methods indicating their genetic differences.

The Effect of a High-Grain Diet on the Rumen Microbiome of Goats with a Special Focus on Anaerobic Fungi.

This work investigated the changes of the rumen microbiome of goats switched from a forage to a concentrate diet with special attention to anaerobic fungi (AF). Female goats were fed an alfalfa hay (AH) diet (0% grain; n = 4) for 20 days and were then abruptly shifted to a high-grain (HG) diet (40% corn grain, 60% AH; n = 4) and treated for another 10 days. Rumen content samples were collected from the cannulated animals at the end of each diet period (day 20 and 30). The microbiome structure was studied using high-throughput sequencing for bacteria, archaea (16S rRNA gene) and fungi (ITS2), accompanied by qPCR for each group. To further elucidate unclassified AF, clone library analyses were performed on the ITS1 spacer region. Rumen pH was significantly lower in HG diet fed goats, but did not induce subacute ruminal acidosis. HG diet altered prokaryotic communities, with a significant increase of Bacteroidetes and a decrease of Firmicutes. On the genus level Prevotella 1 was significantly boosted. Methanobrevibacter and Methanosphaera were the most abundant archaea regardless of the diet and HG induced a significant augmentation of unclassified Thermoplasmatales. For anaerobic fungi, HG triggered a considerable rise in Feramyces observed with both ITS markers, while a decline of Tahromyces was detected by ITS2 and decrease of Joblinomyces by ITS1 only. The uncultured BlackRhino group revealed by ITS1 and further elucidated in one sample by LSU analysis, formed a considerable part of the AF community of goats fed both diets. Results strongly indicate that the rumen ecosystem still acts as a source for novel microorganisms and unexplored microbial interactions and that initial rumen microbiota of the host animal considerably influences the reaction pattern upon diet change.

Occurrence, Molecular Characterization and Phylogenetic Relationship of <i>Aspergillus </i>Species Isolated from Garri Sold in Benue State, North Central, Nigeria

Characterization and identification of molds based on cultural and morphological characteristics are often not reliable and frail with limitations. The occurrence of Aspergillus species in garri on sale in markets in Benue State, Nigeria, was studied by molecular techniques. Aspergillus species were isolated and purified on Potato Dextrose Agar. DNA from the purified isolates was extracted using the ZR fungal DNA miniprep and amplified by PCR mix made up of 12.5 μL of Taq 2X Master Mix. Primer sequences for the fungi characterization were internal transcribed spacers ITS 4 and ITS 5. The phylogenetic tree was plotted between the isolated organisms and reference sequences and evolutionary analysis was conducted in MEGA X. Result revealed that one thousand, six hundred and forty-six Aspergilli were isolated comprising of 980 and 666 isolates from the white and yellow garri respectively. Aspergillus flavus, A. fumigatus, A. niger, A. aculeatinus, and A. aculeatus were identified. Twenty percent (20%) of the strains had aflatoxin D structural gene, 50% amplified AFLP and 70% of the strains expressed AFLQ genes needed for the biosynthesis of aflatoxin B1. Majority of the strains that showed the expression of these structural genes were consistent with Aspergillus flavus. Phylogenetic tree showed a close relationship among the isolates and their most identical sequence in the NCBI database.

Analyses of molecular markers and leaf morphology of two rare birches, Betula obscura and B. pendula var. carelica

Trees and shrubs belonging to the Betula L. genus seem to represent one of the most confused interspecific relationships, and hybridization is considered to be one of the main factors responsible for the substantial genetic and morphological variation of birches. In the present investigation, the internal transcribed spacer ITS1 and ITS2 regions of nuclear ribosomal DNA, nuclear alcohol dehydrogenase (ADH) gene sequences and amplified fragment length polymorphisms (AFLPs) as well as the Atkinson discriminant function (ADF) of leaf morphology were used to verify hypotheses concerning the origin of the dark barked Betula obscura and B. pendula var. carelica with the patterned wood. Both plants were considered by different authors to be distinct species, intraspecific forms of common B. pendula or B. pubescens, or hybrid taxa between B. pendula and B. pubescens. In the phylogenetic trees, the ITS and ADH gene sequences of both B. obscura and B. pendula var. carelica clustered with those of B. pendula, whereas B. pubescens trees were somewhat distinct. In turn, the AFLPs revealed genetic similarity of B. pendula var. carelica to both frequent species, whereas B. obscura was clearly distinct from other birches. Values of the ADF indices of the rare birches were typical for B. pendula. In the light of the results obtained, we imply that B. obscura and B. pendula var. carelica represent an intraspecific variation of B. pendula, they are neither intraspecific taxa of B. pubescens nor hybrid species between the common birches. Different grouping of B. obscura in the AFLPs and DNA sequences analyses is likely a result of an unreliable phylogenetic signal of the former molecular markers.

Summer bloom of Vulcanodinium rugosum in Cienfuegos Bay (Cuba) associated to dermatitis in swimmers

The marine dinoflagellate Vulcanodinium rugosum produces powerful paralyzing and cytotoxic compounds named pinnatoxins (PnTX) and portimines. Even though, no related human intoxication episodes following direct exposure in seawater or the ingestion of contaminated seafood have been documented so far. This study aimed at investigating a dinoflagellate bloom linked to acute dermatitis cases in two recreational beaches in Cienfuegos Bay, Cuba. We used epidemiological and clinical data from 60 dermatitis cases consisting of individuals in close contact with the bloom. Seawater physical-chemical properties were described, and the microorganism causing the bloom was identified by means of light and scanning electron microscopy. Morphological identification was confirmed genetically by sequencing the internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA region. Toxic compounds were identified from a bloom extract using liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS), and their concentrations were estimated based on low-resolution tandem mass spectrometry (LC-MS/MS). Sixty people who had prolonged contact with the dinoflagellate bloom suffered acute dermal irritation. Most patients (79.2%) were children and had to be treated with antibiotics; some required >5-day hospitalization. Combined morphological and genetic characters indicated V. rugosum as the causative agent of the bloom. rDNA sequences of the V. rugosum genotype found in the bloom aligned with others from Asia, including material found in the ballast tank of a ship in Florida. The predominant toxins in the bloom were portimine, PnTX-F and PnTX-E, similar to strains originating from the Pacific Ocean. This bloom was associated with unusual weather conditions such as frequent and prolonged droughts. Our findings indicate a close link between the V. rugosum bloom and a dermatitis outbreak among swimmers in Cienfuegos Bay. Phylogenetic evidence suggests a recent introduction of V. rugosum from the Pacific Ocean into Caribbean waters, possibly via ballast water.

Phyllodistomum kupermani n. sp. from the European perch, Perca fluviatilis L. (Perciformes: Percidae), and redescription of Phyllodistomum macrocotyle (L\xfche, 1909) with notes on the species diversity and host specificity in the European Phyllodistomum spp. (Trematoda: Gorgoderidae)

BackgroundEuropean species of the large genus Phyllodistomum Braun, 1899 had historically been erected based solely on morphological characters. Unfortunately, many of them are still poorly known and inadequately described. Molecular approaches are critical to delineate species which were impossible to differentiate based on morphology alone.MethodsNew samples of adult Phyllodistomum spp. were collected from the urinary bladder and/or ureters of European freshwater fishes and fixed to conduct a light and scanning electron microscopy study, and to obtain sequences of nuclear (ITS2 spacer and 28S rRNA gene), to be analysed in the context of a molecular phylogeny.ResultsBased on new findings, a new species of Phyllodistomum from the urinary bladder of the European perch, Perca fluviatilis, in Volga River basin, Russia, is described. Additionally, new data on the morphology and tegumental surface topography of P. macrocotyle (Lühe, 1909) Odhner, 1911 from ureters of the common rudd, Scardinius erythrophthalmus, is presented. The host range of P. folium, confirmed by DNA analysis, is extended to other cyprinid fish species.ConclusionsThe present study has again shown that species of the genus Phyllodistomum are in dire need of revision based on both molecular analysis and detailed morphological redescriptions of the forms attributed to the genus. Morphologically, P. kupermani n. sp. most closely resembles P. pseudofolium, a highly host-specific parasite of Gymnocephalus cernuus (L.), but molecular phylogenetic analyses based on ITS2 and 28S rDNA sequences showed that these species are distantly related. Phyllodistomum kupermani n. sp. was found to be phylogenetically most closely related to the type-species of Phyllodistomum, P. folium. Phylogenetic analyses revealed that Phyllodistomum kupermani n. sp. and P. folium formed a clade with other freshwater species for which cystocercous cercariae develop in bivalves of the family Sphaeriidae. The micromorphology and tegumental surface topography of P. macrocotyle revealed in the present study provide a valuable taxonomic criterion for congeneric species differentiation.

Identification Problems and Cryptic Diversity of Chlorella-Clade Microalgae (Chlorophyta)

The article considers the system of the Chlorella-clade based on morphological, ecological, and molecular genetic data. Diagnostic characteristics of some genera and species are discussed, as well as the systematic position of some taxa. Molecular phylogenetic analysis of members of the Chlorella-clade showed that the use of only the 18S rRNA gene, which is considered the main phylogenetic marker for green microalgae, does not reliably distinguish species and genera within the clade. Application of more variable spacers ITS1 and ITS2 resolved the tree topology and strengthened the phylogenetic signal. The polyphyly of the genus Chlorella was confirmed. Some of the species of the genus Chlorella formed a true clade of Chlorella species, including the type species C. vulgaris, C. heliozoae, C. pituita, C. chlorelloides, C. variabilis, and Lobosphaeropsis lobophora. Other species (C. pulchelloides, C. colonials, C. rotunda, C. singularis, C. elongata, C. sorokiniana, C. lewinii, C. volutis, and C. thermophila) were found to belong to other groups, and their taxonomic affiliation requires clarification. The CBC approach and the search for compensatory mutations in conservative ITS2 regions as a tool for distinguishing species was not effective for most Chlorella-clade representatives. Efficiency of using the intron as a criterion for separating closely related species was confirmed. Analysis of genetic distances made it possible to clearly identify the strains as species of the genera Didymogenes, Hindakia, and Heynigia. Members of the Chlorella-clade are characterized by a high cryptic diversity: none of the phenotypic characteristics considered separately was sufficient for unequivocal determination of the taxonomic position of members of the clade. Some morphological features were variable, often changed or became lost after instrumental manipulations during sample preparation for the water bodies monitoring. Using a combination of morphological, biochemical, ultrastructural, physiological, ecological, and molecular genetic features (a polyphasic approach), we were able to characterize eight groups within the Chlorella-clade and to make assumptions about the division of the genera and species within these groups.

Genetic Diversity of Aquatic Ranunculus (Batrachium, Ranunculaceae) in One River Basin Caused by Hybridization.

Aquatic Ranunculus (sect. Batrachium) include homophyllous and heterophyllous plants. The development of floating leaves may be induced by genetic mechanisms or/and environmental conditions and this fact complicates the morphologically based identification of species. DNA-based studies provide the opportunity to expand the knowledge of this complicated group. We studied heterophyllous Ranunculus with well-developed capillary and intermediate leaves and visually homophyllous plants with capillary leaves from a single river basin, with the aim to evaluate their genetic polymorphism and taxonomic status—whether the plants with well-developed and weakly expressed intermediate leaves belong to different forms (taxa) or if they just express morphological variation of one or two taxa in a specific, very variable river environment. The studied heterophyllous and homophyllous plants from different rivers showed high genetic differentiation and a low level of genetic diversity within these groups. The molecular analysis did not reveal any inter simple sequence repeat (ISSR) polymorphism associated with the development of intermediate leaves. An analysis of nuclear ribosomal internal transcribed spacers ITS1–2 sequences revealed several ribotypes, which indicated the genetic heterogeneity of studied plants and indirectly confirmed the hybrid origin of some of them. Sterile plants originated from crossing of R. circinatus and R. penicillatus were discovered in the Skroblus River; however, identification of the parental species was impeded by the polymorphism detected. For this reason, cytological studies were performed and allowed confirmation of the hybrid origin of these plants.

Genetic diversification of acanthocephalans of the genus Floridosentis Ward 1953 (Acanthocephala: Neoechinorhynchidae), parasites of mullets from the Americas.

Adult worms of the genus Floridosentis are endoparasites of marine fishes of the genus Mugil and are broadly distributed in the Americas. Currently, Floridosentis includes two species, F. mugilis, distributed in the Gulf of Mexico and along the Atlantic Ocean coast, and F. pacifica, restricted to the Pacific Ocean coast. The aim of this study was to explore the species limit of both species of the genus Floridosentis, collected in 37 localities in eight countries: Mexico, Guatemala, El Salvador, Honduras, Nicaragua, Costa Rica, Ecuador and Venezuela. We sequenced 253 specimens to build a comprehensive dataset for three genes: the cytochrome c oxidase subunit I (cox 1) from mitochondrial DNA, the internal transcribed spacers ITS1 and ITS2 including the 5.8S gene (ITS region), and the D2+D3 domains of the large subunit (LSU) of nuclear DNA. Maximum likelihood and Bayesian analyses with the cox 1 and concatenated (cox 1+ITS+LSU) datasets were conducted. Two species delimitation methods were implemented, the Automatic Barcode Gap Discovery (ABGD), and Bayesian species delimitation (BPP), plus a haplotype network inferred with 253 specimens, allowing us to validate two nominal species of Floridosentis., F. mugilis, plus one linage distributed in the Gulf of Mexico and along the Atlantic Ocean coast, and F. pacifica, plus two additional lineages distributed along the Pacific Ocean coast. All these lineages are shared by both species of mullet (Mugil curema and M. cephalus). The currents in the Atlantic Ocean, Pacific Ocean and Gulf of Mexico, in combination with the biology of the definitive hosts, have played a key role in the distribution of the two nominal species and of the three lineages of Floridosentis across the Americas.

Identification of species in the genus Nitraria L. (Nitrariaceae) based on nucleotide variability of nuclear ribosomal DNA.

Intragenomic polymorphism of ITS1 and ITS2 of nuclear ribosomal DNA sequences was analysed in 33 samples belonging to the Nitraria species N. schoberi, N. sibirica, and N. komarovii. The nucleotide variability of the ITS region was detected in the Nitraria species as single-nucleotide substitutions (mainly transitions) and single-nucleotide deletion. Information about the nucleotide variability of fragments is given for the first time by us. The ITS1-5.8S-ITS2 region contained 17 phylogenetically informative single-nucleotide polymorphisms. Eleven single-nucleotide substitutions (transitions, C/T) were detected in ITS1. The ITS2 spacer contained 273–274 bp and was more conservative. A total of 5 phylogenetically informative single-nucleotide polymorphisms (4 transitions: C/T, G/A, one transversion: G/C), one single-nucleotide deletion (T/–) were detected in ITS2. The average GC content was 61.5 %. The GC content was lower in N. sibirica (59.2 %) than in N. schoberi and N. komarovii (62.7 %). It has been shown that the shorter ITS2 is a suitable molecular marker separating these species, due to the low interspecific variability and simultaneous available intraspecific variability. Phylogenetic ML and BI trees constructed separately for the ITS1 and ITS2 spacers, as well as separately for the full-size ITS region and the ITS2 spacer, were congruent. The results obtained on the intraspecific differentiation of N. sibirica revealed two main ribotypes among the samples of this species: the main Siberian sibirica-ribotype and the main Kazakh sibiricaribotype. Geographical features of the distribution of N. sibirica ribotypes, as well as the presence of significant differences between the main Siberian and Kazakh sibirica-ribotypes (3 single-nucleotide substitutions) indicated significant inter-population differences and taxonomic heterogeneity of N. sibirica. Most likely, the processes of homogenization of nuclear ribosomal DNA of N. sibirica samples, the origin of which is associated with hybridization and speciation, are currently continuing.

The Patterns and Puzzles of Genetic Diversity of Endangered Freshwater Mussel Unio crassus Philipsson, 1788 Populations from Vistula and Neman Drainages (Eastern Central Europe)

Mussels of the family Unionidae are important components of freshwater ecosystems. Alarmingly, the International Union for Conservation of Nature and Natural Resources Red List of Threatened Species identifies almost 200 unionid species as extinct, endangered, or threatened. Their decline is the result of human impact on freshwater habitats, and the decrease of host fish populations. The Thick Shelled River Mussel Unio crassus Philipsson, 1788 is one of the examples that has been reported to show a dramatic decline of populations. Hierarchical organization of riverine systems is supposed to reflect the genetic structure of populations inhabiting them. The main goal of this study was an assessment of the U. crassus genetic diversity in river ecosystems using hierarchical analysis. Different molecular markers, the nuclear ribosomal internal transcribed spacer ITS region, and mitochondrial DNA genes (cox1 and ndh1), were used to examine the distribution of U. crassus among-population genetic variation at multiple spatial scales (within rivers, among rivers within drainages, and between drainages of the Neman and Vistula rivers). We found high genetic structure between both drainages suggesting that in the case of the analyzed U. crassus populations we were dealing with at least two different genetic units. Only about 4% of the mtDNA variation was due to differences among populations within drainages. However, comparison of population differentiation within drainages for mtDNA also showed some genetic structure among populations within the Vistula drainage. Only one haplotype was shared among all Polish populations whereas the remainder were unique for each population despite the hydrological connection. Interestingly, some haplotypes were present in both drainages. In the case of U. crassus populations under study, the Mantel test revealed a relatively strong relationship between genetic and geographical distances. However, in detail, the pattern of genetic diversity seems to be much more complicated. Therefore, we suggest that the observed pattern of U. crassus genetic diversity distribution is shaped by both historical and current factors i.e. different routes of post glacial colonization and history of drainage systems, historical gene flow, and more recent habitat fragmentation due to anthropogenic factors.

Gut microbiome of the emerald ash borer, Agrilus planipennis Fairmaire, and its relationship with insect population density.

The gut microbial communities of beetles play crucial roles in their adaptive capacities. Environmental factors such as temperature or nutrition naturally affect the insect microbiome, but a shift in local conditions like the population density on a host tree could also lead to changes in the microbiota. The emerald ash borer (EAB), Agrilus planipennis Fairmaire, is an exotic wood borer that causes environmental and economic damage to ash trees in North America. This study aimed to describe the taxonomic structure of the EAB gut microbiome and explore its potential relationship with borer population size. The number of EAB adults collected per tree through a 75km transect from an epicenter allowed the creation of distinct classes of population density. The Gammaproteobacteria and Ascomycota predominated in bacterial and fungal communities respectively, as determined by sequencing of the bacterial 16S rRNA gene and the fungal internal transcribed spacer ITS2. Species richness and diversity of the bacterial community showed significant dependence on population density. Moreover, α-diversity and β-diversity analysis revealed some indicator amplicon sequence variants suggesting that the plasticity of the gut microbiome could be related to the EAB population density in host trees.

Lapaea (Plantaginaceae, Gratioleae), a new genus endemic to the Espinhaço Range (Brazil) with a remarkable red-flowered new species

Stemodia sensu lato is one of the largest genera of the Gratioleae (Plantaginaceae). Recent phylogenetic studies corroborated the paraphyly of Stemodia in its traditional circumscription and suggested the segregation of S. stellata from this genus. This species belongs to a morphologically cohesive group along with S. cipoensis, S. harleyi and S. lobata, all endemic to the Espinhaço Range, eastern Brazil. In this study, we tested the monophyly and interspecific relationships of this group by reconstructing its phylogeny, based on sequences of three cpDNA regions (rps16 and trnL introns and the trnL–trnF intergenic spacer), and one nrDNA region (ITS1 spacer), using Maximum parsimony and Bayesian inference. The monophyly of the 'S. stellata group' was confirmed, and its segregation from Stemodia corroborated. We propose Lapaea as a new genus to accommodate its species, with subsequent four new combinations: L. cipoensis, L. harleyi, L. lobata, and L. stellata. Additionally, we describe and illustrate L. rubriflora, a remarkable red-flowered new species, based on morphological and micromorphological evidence. Lastly, a taxonomic revision of the species recognized in Lapaea is presented, providing an identification key, photos, descriptions, distribution map and notes on their morphology, geographic distribution, habitat, ecology, and conservation status. Lapaea is restricted to the Espinhaço Range comprising five threatened species, each of them with a narrow distribution and phylogenetic relationships consistent with biogeographic provinces.

Corrigendum: In the Wake of the Ongoing Mass Mortality Events: Co-occurrence of Mycobacterium, Haplosporidium and Other Pathogens in Pinna nobilis Collected in Italy and Spain (Mediterranean Sea)

Following the Mass Mortality Events (MMEs) of the pen shell P. nobilis in Campania region and Sicily, a survey of moribund P. nobilis specimens was also conducted in other Italian regions (Campania, Tuscany, Sardinia and Apulia) and Spain (Catalunya). Histopathological and molecular examination of 27 specimens of P. nobils revealed different types of pathogens associated with tissue lesions, morbidity and mortality. Presence of Mycobacterium, Vibrio species, Haplosporidium pinnae and Perkinsus sp. were detected, differently distributed into the areas. The Mycobacterium sp., previously reported in Campania and Sicily samples, was observed in all the analysed areas and individuals, associated to systemic inflammatory lesions. In Spain, H. pinnae was observed in 36% of cases, always associated to the Mycobacterium sp.. Molecular study using hsp65 genes and Internal Transcriber Spacer ITS support that a new species of Mycobacteria is infecting P. nobilis, close to M. triplex and belonging to the group of M. simiae complex with M. sherrisi. Presence of Perkinsus spp. resembling P. mediterraneus was observed in 2 out of 13 Italian individuals whose presence should be addressed as potential risk for shellfish aquaculture of the area. Vibrio spp. were also detected in some case. The preliminary results of this study suggest that Mycobacterium sp., Vibrio spp., H. pinnae and Perkinsus sp. cooperate to disease pathogenesis, being Mycobacterium and Haplosporidium most of the time involved. Vigilant inspection of those areas where MME is now starting, along with continuous systematic surveys, are crucial to define the spatiotemporal progress of mortality and the role of every single pathogen in the disease outcome.

<p><strong>Morphological and molecular evidence for two new species of <em>Absidia </em>from Neotropic soil</strong></p>

During a survey of the diversity of Mucorales in Atlantic Forest and upland rainforest soils in northeastern Brazil, two strains belonging to Absidia were isolated. These strains are morphologically and molecularly (nuc rDNA internal transcribed spacer ITS1–5.8–ITS2 and D1–D2 domains of nuc 28S rDNA) distinct from other Absidia species. Absidia cornuta sp. nov. presents exclusively cylindrical sporangiospores and up to three projections on columellae. Absidia pernambucoensis sp. nov. presents up to two apical projections on columellae and produces two types of sporangiospores: cylindrical and slightly cuneiform. Based on morphological and phylogenetic evidence, two new species of Absidia are proposed. An identification key for the species of Absidia with cylindrical sporangiospores found in the Neotropics is provided.