Three new species of Agaricus from Baiyun Mountain, Guangzhou, China.

Agaricus is a species-rich genus with more than 600 species around the world. In this work, three new species, Agaricus cacainus, A. baiyunensis, and A. praeclarefibrillosus are described from the specimens collected at Baiyun Mountain, Guangzhou, China, a subtropical area with a monsoon maritime climate, based on phylogenetic analyses and morphological examinations of internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), D1/D2 domains of the large subunit of ribosomal DNA (28S), and a part of translation elongation factor 1-alpha (TEF1). Agaricus cacainus in A. sect. Amoeni is characterized by a parabolic to applanate, slightly depressed pileus covered with chocolate brown, appressed, triangular squamules against white background, a white, furfuraceous stipe, an unchanging context when cut, a fragile and evanescent annulus, usually 4- or 2-spored basidia, and mostly pyriform cheilocystidia. Agaricus baiyunensis in A. sect. Minores has a pileus with a slightly truncate top covered with light brown, downy-wooly fibrillose scales and a light yellowish stipe with membranous annulus. Agaricus praeclarefibrillosus in A. sect. Brunneopicti is characterized by a pileus surface with brownish, triangular, recurved scales and longitudinally splitting lines toward margin, a cottony stipe with white, tiny, recurved fibrils, a single annulus, and variously shaped cheilocystidia, with sparsely ornamented basidiospores. The detailed comparison of their morphological characteristics with closely related species is provided.

Geometric morphometric analysis of spore shapes improves identification of fungi.

Morphology of organisms is an essential source of evidence for taxonomic decisions and understanding of ecology and evolutionary history. The geometric structure (i.e., numeric description of shape) provides richer and mathematically different information about an organism's morphology than linear measurements. A little is known on how these two sources of morphological information (shape vs. size) contribute to the identification of organisms when implied simultaneously. This study hypothesized that combining geometric information on the outline with linear measurements results in better species identification than either evidence alone can provide. As a test system for our research, we used the microscopic spores of fungi from the genus Subulicystidium (Agaricomycetes, Basidiomycota). We analyzed 2D spore shape data via elliptic Fourier and principal component analyses. Using flexible discriminant analysis, we achieved the highest species identification success rate for a combination of shape and size descriptors (64.7%). The shape descriptors alone predicted species slightly better than size descriptors (61.5% vs. 59.1%). We conclude that adding geometric information on the outline to linear measurements improves the identification of the organisms. Despite the high relevance of spore traits for the taxonomy of fungi, they were previously rarely analyzed with the tools of geometric morphometrics. Therefore, we supplement our study with an open access protocol for digitizing and summarizing fungal spores' shape and size information. We propagate a broader use of geometric morphometric analysis for microscopic propagules of fungi and other organisms.

Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination.

There is a critical need for new antifungal drugs; however, the lack of available fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a differentiation process absent in humans that could harbor uncharacterized fungus-specific targets. To capitalize on this possibility, we developed novel phenotypic assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we carried out a high-throughput screen of ∼75,000 drug-like small molecules and identified and characterized 191 novel inhibitors of spore germination, many of which also inhibited yeast replication and demonstrated low cytotoxicity against mammalian cells. Using an automated, microscopy-based, quantitative germination assay (QGA), we discovered that germinating spore populations can exhibit unique phenotypes in response to chemical inhibitors. Through the characterization of these spore population dynamics in the presence of the newly identified inhibitors, we classified 6 distinct phenotypes based on differences in germination synchronicity, germination rates, and overall population behavior. Similar chemical phenotypes were induced by inhibitors that targeted the same cellular function or had shared substructures. Leveraging these features, we used QGAs to identify outliers among compounds that fell into similar structural groups and thus refined relevant structural moieties, facilitating target identification. This approach led to the identification of complex II of the electron transport chain as the putative target of a promising structural cluster of germination inhibitory compounds. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics. IMPORTANCE Fungal pathogens cause 1.5 million deaths annually, and there is a critical need for new antifungal drugs. However, humans and fungi are very similar on a molecular level, and so many drugs that kill fungi also damage human cells, leading to extreme side effects, including death. The lack of fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a process absent in humans that could harbor fungus-specific targets. To capitalize on this possibility, we developed new assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we identified and characterized 191 novel inhibitors of spore germination. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics.

Can wood-decaying urban macrofungi be identified by using fuzzy interference system? An example in Central European Ganoderma species.

Ganoderma is a cosmopolitan genus of wood-decaying basidiomycetous macrofungi that can rot the roots and/or lower trunk. Among the standing trees, their presence often indicates that a hazard assessment may be necessary. These bracket fungi are commonly known for the crust-like upper surfaces of their basidiocarps and formation of white rot. Six species occur in central European urban habitats. Several of them, such as Ganoderma adspersum, G. applanatum, G. resinaceum and G. pfeifferi, are most hazardous fungi causing extensive horizontal stem decay in urban trees. Therefore, their early identification is crucial for correct management of trees. In this paper, a fast technique is tested for the determination of phytopathologically important urban macrofungi using fuzzy interference system of Sugeno type based on 13 selected traits of 72 basidiocarps of six Ganoderma species and compared to the ITS sequence based determination. Basidiocarps features were processed for the following situations: At first, the FIS of Sugeno 2 type (without basidiospore sizes) was used and 57 Ganoderma basidiocarps (79.17%) were correctly determined. Determination success increased to 96.61% after selecting basidiocarps with critical values (15 basidiocarps). These undeterminable basidiocarps must be analyzed by molecular methods. In a case, that basidiospore sizes of some basidiocarps were known, a combination of Sugeno 1 (31 basidiocarps with known basidiospore size) and Sugeno 2 (41 basidiocarps with unknown basidiospore size) was used. 84.72% of Ganoderma basidiocarps were correctly identified. Determination success increased to 96.83% after selecting basidiocarps with critical values (11 basidiocarps).

Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison.

Tilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

Propagation and characterization of viable arbuscular mycorrhizal fungal spores within maize plant (Zea mays L.).

BACKGROUND: The harmful effect of chemical fertilizer application on human health and the environment as a modern method of meeting the food demand of the increasing world population demands an urgent alternative that is environmentally friendly, which will pose no harm to human health and the environment. Arbuscular mycorrhizal fungi (AMF) are beneficial soil microorganisms that provide various ecological functions in increasing soil fertility and enhancing plant growth. This present study aimed to propagate, characterize and examine the effect of viable arbuscular mycorrhizal fungal spores on maize (Zea mays L) hosts using molecular methods. The propagation of AMF in the host plant using sterile soil and vermiculite was conducted in the greenhouse. RESULT: The effect of AMF inoculation revealed a significant difference (P > 0.05) in maize growth, root colonization and AMF spore count when compared with the control. In all the parameters measured in this study, all the AMF spores propagated had a positive effect on the maize plant over the control, with the highest value mostly recorded in Rhizophagus irregularis AOB1. The molecular characterization of the spore using a specific universal primer for Glomeromycota established the success of the propagation process, which enhanced the classification of the AMF species into Rhizophagus irregularis OAB1, Glomus mosseae OAB2 and Paraglomus occultum OAB3. CONCLUSION: This finding will be a starting point in producing arbuscular mycorrhizal inoculum as a biofertilizer to enhance plant growth promotion. © 2021 Society of Chemical Industry.

Four new species of Talaromyces section Talaromyces discovered in China.

Four new Talaromyces species without any close relatives are reported here, namely, T. aureolinus (ex-type AS3.15865 T), T. bannicus (ex-type AS3.15862 T), T. penicillioides (ex-type AS3.15822 T), and T. sparsus (ex-type AS3.16003 T). Morphologically, T. aureolinus is unique in producing orange-yellow mycelium and gymnothecia, singly borne asci, and ellipsoidal, spiny ascospores. Talaromyces bannicus is characterized by the slow growth rate, polymorphic conidiophores, inconsistent stipe lengths, and pyriform to ellipsoidal, echinulate conidia. Talaromyces penicillioides is distinguished by good growth and sporulation on malt extract agar (MEA) and yeast extract sucrose agar (YES) media, resembling the colony appearances of certain Penicillium species, and appressed biverticillate and occasionally monoverticillate penicilli bearing globose to ellipsoidal, echinulate conidia. Talaromyces sparsus has wide, submerged colony margins with sparse aerial mycelium, and conidial areas overlaid with yellow-green, sterile hyphae on MEA medium. These four new species are well supported by individual phylogenetic trees based on ß-tubulin (BENA), calmodulin (CALM), DNA-dependent RNA polymerase II second largest subunit (RPB2), and internal transcribed spacer region (ITS) gene sequences and the tree of the concatenated BENA-CALM-RPB2 sequence.

Two new species and one new record of Xenosporium with ellipsoidal or ovoid conidia from Taiwan.

Three species of Xenosporium producing ellipsoidal or ovoid conidia were collected from decaying culms of Miscanthus floridulus submerged in freshwater streams of Alishan area, Chiayi County, Taiwan. Xenosporium formosiforme and X. parvulum are described and illustrated as new species. Xenosporium africanum is described in this paper as a new record for Taiwan. The phylogenetic relationships of Xenosporium species and related taxa were sought by comparing the sequences of their internal transcribed spacer (ITS) barcode, 28S rDNA, and RNA polymerase II second largest subunit (RPB2) gene segments. Phylogenetic analysis of these Xenosporium species supports their placement in the Tubeufiaceae. A synopsis of Xenosporium species producing ellipsoidal or ovoid conidia is given. The criteria used to differentiate species are discussed.

Four new species of Agaricus subgenus Spissicaules from China.

Agaricus subgenus Spissicaules is widely distributed in the world. In this study, 114 specimens were included in multigene phylogenetic analyses that allowed a better circumscription of the four sections in A. subg. Spissicaules. Three new species from China, A. brunneovariabilis, A. beijingensis, and A. planiceps, are described and placed in different sections. The invalidly described A. catenariocystidiosus is validated here as an additional new species. Comparisons between the sequences of the three closely related species A. thiersii, A. parasubrutilescens, and A. linzhiensis support the distinction between these three species despite the paraphyly of A. linzhiensis, which results from its high intraspecific variability with numerous heteromorphisms.

Steccherinum tenuissimum and S. xanthum spp. nov. (Polyporales, Basidiomycota): New species from China.

Two new wood-inhabiting fungal species, Steccherinum tenuissimum and S. xanthum spp. nov. are described based on a combination of morphological features and molecular evidence. Steccherinum tenuissimum is characterized by an annual growth habit, resupinate basidiomata with an odontioid hymenial surface, a dimitic hyphal system with clamped generative hyphae, strongly encrusted cystidia and basidiospores measuring 3-5 × 2-3.5 µm. Steccherinum xanthum is characterized by odontioid basidiomata and a monomitic hyphal system with generative hyphae bearing clamp connections and covering by crystals, colourless, thin-walled, smooth, IKI-, CB-and has basidiospores measuring 2.7-5.5 × 1.8-4.0 µm. Sequences of the ITS and nLSU nrRNA gene regions of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and Bayesian inference methods. The phylogenetic analyses based on molecular data of ITS + nLSU sequences showed that two new Steccherinum species felled into the residual polyporoid clade. Further investigation was obtained for more representative taxa in Steccherinum based on ITS + nLSU sequences, which demonstrated that S. tenuissimum and S. xanthum were sister to S. robustius with high support (100% BP, 100% BS and 1.00 BPP).

Morphological and genetic characteristics of the novel entomopathogenic fungus Ophiocordyceps langbianensis (Ophiocordycipitaceae, Hypocreales) from Lang Biang Biosphere Reserve, Vietnam.

An entomopathogenic fungus newly named Ophiocordyceps langbianensis was collected from Lang Biang Biosphere Reserve, located in Lam Dong Province, Vietnam. It is characterized as a species of Ophiocordyceps (Ophiocordycipitaceae, Hypocreales) having the unique characteristics of a cylindrical fertile part and several branched apical appendices. Each ascospore develops as two swollen, constricted part-spores. A phylogenetic analysis of multiple genes, including nrLSU, nrSSU, Rpb1, ITS and Tef, supported its systematic position in the genus of Ophiocordyceps; it is related to O. brunneipunctata. Based on morphological and phylogenetic analyses, O. langbianensis was confirmed as a new species from Vietnam.

Atypical chlamydoconidium-producing Trichophyton tonsurans strains from Ceará State, Northeast Brazil: investigation of taxonomy by phylogenetic analysis and biofilm susceptibility.

Chlamydoconidium-producing Trichophyton tonsurans strains isolated in Northeastern Brazil have morphological features different from the classic description of this dermatophyte species. This study investigated the phylogenetic relationship of chlamydoconidium-producing T. tonsurans strains isolated in Northeastern Brazil. Also, the effect of terbinafine and farnesol on mature biofilms of T. tonsurans strains was evaluated. The mass spectra of T. tonsurans strains were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The ITS and LSU loci regions of rDNA and the partial ß-tubulin gene were sequenced and the phylogenetic tree was analysed. The effects of terbinafine and farnesol on mature T. tonsurans biofilms were evaluated through the analysis of metabolic activity, quantification of biomass and observation by scanning electron microscopy. MALDI-TOF MS spectra of the chlamydoconidium-producing T. tonsurans strains differed from the spectrum of the control strain (ATCC 28942), presenting an intense ion peak at m/z 4155 Da. Phylogenetic tree analysis showed that the chlamydoconidium-producing strains isolated in Northeastern Brazil are allocated to a single cluster, differing from strains isolated from other countries. As for mature T. tonsurans biofilms, farnesol reduced biomass and metabolic activity by 64.4 and 65.9 %, respectively, while terbinafine reduced the biomass by 66.5 % and the metabolic activity by 69 %. Atypical morphological characteristics presented by chlamydoconidium-producing T. tonsurans strains result from phenotypic plasticity, possibly for adaptation to environmental stressors. Also, farnesol had inhibitory activity against T. tonsurans biofilms, demonstrating this substance can be explored for development of promising anti-biofilm drugs against dermatophytes.

Interleukin-8 gene expression and apoptosis induced by Salmonella Typhimurium in the presence of Bacillus probiotics in the epithelial cell.

AIMS: This study aimed to evaluate the effects of three Bacillus probiotics on Salmonella Typhimurium, and interleukin-8 (IL-8) gene expression in the co-culture of the Bacillus and the pathogen in vitro. METHODS AND RESULTS: Bacillus subtilis, Bacillus indicus and Bacillus coagulans were initially turned to spore and heat-inactivated forms. The cellular damages of the probiotics on the HT-29 cells were investigated individually and in combination with S. Typhimurium using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorescence assays. To extract cell free supernatants (CFS) of the probiotics, they were cultured in selective media. The inhibitory activity of CFSs were then assayed against the pathogen. The gene expression of IL-8 of the HT-29 cells was evaluated by real-time PCR in all the groups. The results showed that the CFSs of three probiotics could inhibit the growth of S. Typhimurium by more than 50%. Inhibitory effects of B. indicus and B. subtilis CFSs were related to the production of pepsin-sensitive compounds, except B. coagulans in which the high inhibitory effect was due to organic acids. The spores of the three probiotics and the heat-inactivated forms of B. subtilis and B. coagulans could reduce the cytotoxicity of S. Typhimurium. The cell viability also increased applying both forms probiotics against the pathogen. In all co-culture groups, the IL-8 gene expression induced by S. Typhimurium was reduced. CONCLUSIONS: The three Bacillus probiotics can be considered as proper candidates for the prevention and treatment of S. Typhimurium food poisoning. SIGNIFICANCE AND IMPACT OF THE STUDY: Applying probiotics as live bacteria is universally noted in foods. This study tried to discover the effects of Bacillus probiotics in the form of spore or even heat-killed bacteria against S. Typhimurium and evaluate ratio of IL-8 gene expression in cell culture. The most effective Bacillus probiotic will be recommended. This approach will help to use probiotics as nonvegetative cells in foods to fight gastrointestinal pathogens.

Conidiobolus lunulus, a new entomophthoralean species isolated from leafcutter ants.

Entomophthoralean fungi with pathogenic abilities to infect social insects are rare. Here, we describe a fungus isolated from leafcutter ants. Morphologically, the fungus has spherical primary conidia and two types of microconidia: one with the same shape as the primary conidia and another with an elliptical to half-moon shape. The fungus also produces villose conidia known previously only from Conidiobolus coronatus. A multilocus phylogenetic analysis was performed with nuc rDNA sequences from three regions (28S, 18S, and internal transcribed spacer [ITS]). Our isolates are distinguished as a new species, described here as Conidiobolus lunulus, and is more closely related to C. brefeldianus than to C. coronatus, despite the greater morphological resemblance to the latter. Morphological differences, unique phylogenetic placement, and isolation from an altogether new host support this finding. This is the first record of an entomophthoralean species isolated from leafcutter ants.

Characterization and biocontrol potential of a naturally occurring isolate of Metarhizium pingshaense infecting Conogethes punctiferalis.

An entomopathogenic fungus was isolated from an infected larva of Conogethes punctiferalis (Guenée) (Crambidae: Lepidoptera), a highly polyphagous pest recorded from more than 120 plants and widely distributed in Asia and Oceanic countries. The fungus was identified as Metarhizium pingshaense Q.T. Chen & H.L. Guo (Ascomycota: Hypocreales) based on morphological characteristics and molecular studies. Scanning electron microscopic studies were conducted to study the infection of C. punctiferalis by M. pingshaense. Bioassay studies with purified conidial suspension proved that the isolate was highly virulent to C. punctiferalis, causing more than 86 % mortality to fifth instar larvae at 1 × 108 spores/mL, under laboratory conditions. The median lethal concentration (LC50) of the fungus against late instar larvae was 9.1 × 105 conidia/mL and the median survival time (MST) of late instar larvae tested at the doses of 1 × 108 and 1 × 107 conidia/mL were 4.7 and 6.4 days, respectively. The optimal temperature for fungal growth and sporulation was found to be 25 ± 1 °C. This is the first report of M. pingshaense naturally infecting C. punctiferalis. Isolation of a highly virulent strain of this fungus holds promise towards development of a potential mycoinsecticide against this pest.

Identification of toxic mold species through Raman spectroscopy of fungal conidia.

We use a 785 nm shifted excitation Raman difference (SERDS) technique to measure the Raman spectra of the conidia of 10 mold species of especial toxicological, medical, and industrial importance, including Stachybotrys chartarum, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and others. We find that both the pure Raman and fluorescence signals support the hypothesis that for an excitation wavelength of 785 nm the Raman signal originates from the melanin pigments bound within the cell wall of the conidium. In addition, the major features of the pure Raman spectra group into profiles that we hypothesize may be due to differences in the complex melanin biosynthesis pathways. We then combine the Raman spectral data with neural network models to predict species classification with an accuracy above 99%. Finally, the Raman spectral data of all species investigated is made freely available for download and use.

Four phylogenetic species of ergot from Canada and their characteristics in morphology, alkaloid production, and pathogenicity.

Four ergot species (Claviceps ripicola, C. quebecensis, C. perihumidiphila, and C. occidentalis) were recognized based on analyses of DNA sequences from multiple loci, including two housekeeping genes, RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1-α), and a single-copy ergot alkaloid synthesis gene (easE) encoding chanoclavine I synthase oxidoreductase. Morphological features, ergot alkaloid production, and pathogenicity on five common cereal crops of each species were evaluated and presented in taxonomic descriptions. A synoptic key was also provided for identification.

Periplasma, gen. nov., a new oomycete lineage with isogamous sexual reproduction.

This paper describes the taxonomy, developmental morphology, and phylogeny of Periplasma isogametum, a new monotypic member of the Leptomitales (Oomycota). In phylogenetic trees inferred from concatenated and separate nuc 18S rDNA (18S) and nuc 28S rDNA (28S) sequences, P. isogametum forms a well-supported clade related to but distinct from Apodachlya, a member of the Leptomitales. The organism is a holocarpic facultative saprotroph of moribund aquatic insects but grows well on a variety of mycological media in which it produces large eucarpic hyphae with a peripheral layer of protoplasm surrounding a central vacuole. In zoosporogenesis, a peripheral network of zoospore initials collapses to the center of the zoosporangium and is partitioned into individual heterokont zoospores. Sexual reproduction involves morphological isogamy in which male gametes produce elongate fertilization tubes, which fuse with female gametes, which are subsequently converted into thick-walled oospores. Developmental morphology is detailed in photomicrographs and pointillism drawings.

The new truffle genus Babosia and a new species of Stouffera from semiarid grasslands of Hungary.

Truffles with distinct morphological and anatomical features were collected during a study of hypogeous fungi of semiarid sandy grasslands of the Great Hungarian Plain in Hungary, representing the westernmost localities of the Eurasian steppe belt. None of the ascomata were collected near ectomycorrhizal plant species, and none were identified as ectomycorrhizal during previous surveys in the collection area. We studied morphoanatomical characteristics of these truffles with light and scanning electron microscopy and investigated their phylogenetic positions based on analyses of different nuclear loci. The truffles were found to represent two novel lineages that grouped with the Marcelleina-Peziza gerardii clade of the Pezizaceae. One formed a distinct lineage, for which we propose a new genus Babosia with a new species Babosia variospora characterized by diverse spore ornamentation varying even within one ascus. The truffles in the other lineage clustered with the rarely collected American truffle Stouffera longii and share with it similar spore ornamentation and habitat features. However, our material differs from S. longii by geographic origin, the quick and strong coloration of the ascomata to dark gray at cut surface or bruised area, varying spore number in asci, and smaller spore size; thus, we describe it as a new species, Stouffera gilkeyae.

Tuber iranicum, sp. nov., a truffle species belonging to the Excavatum clade.

Truffles in the genus Tuber are hypogeus fungi that have a worldwide distribution. Despite this, knowledge about their diversity in the Middle East is very limited. In recent years, large quantities of truffles have been imported from Iran for being sold in Italy. While analyzing certain commercial batches of T. aestivum from Iran, we found some ascomata that resembled T. excavatum but had macro- and micromorphological features that were distinct from this species. They were subglobose, or depressed to slightly irregular, with a conspicuous basal cavity, grayish brown, brown, or pinkish gray, with a minutely papillose peridium. The gleba was pinkish gray in youth, brown at maturity, marbled with cream branched veins. Ascospores were broadly ellipsoid, with an irregular reticulum and distinctive long crests along the longitudinal axis, up to 9 µm high. Analysis of internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences showed that these specimens form a monphyletic and well-supported taxon within the Excavatum clade. Morphological and molecular analyses supported the proposal of the new species T. iranicum.