ITS Barcoding Research Articles

Fungal DNA barcoding.

Fungi are ubiquitous in both natural and human-made environments. They play important roles in the health of plants, animals, and humans, and in broad ecosystem functions. Thus, having an efficient species-level identification system could significantly enhance our ability to treat fungal diseases and to monitor the spatial and temporal patterns of fungal distributions and migrations. DNA barcoding is a potent approach for rapid identification of fungal specimens, generating novel species hypothesis, and guiding biodiversity and ecological studies. In this mini-review, I briefly summarize (i) the history of DNA sequence-based fungal identification; (ii) the emergence of the ITS region as the consensus primary fungal barcode; (iii) the use of the ITS barcodes to address a variety of issues on fungal diversity from local to global scales, including generating a large number of species hypothesis; and (iv) the problems with the ITS barcode region and the approaches to overcome these problems. Similar to DNA barcoding research on plants and animals, significant progress has been achieved over the last few years in terms of both the questions being addressed and the foundations being laid for future research endeavors. However, significant challenges remain. I suggest three broad areas of research to enhance the usefulness of fungal DNA barcoding to meet the current and future challenges: (i) develop a common set of primers and technologies that allow the amplification and sequencing of all fungi at both the primary and secondary barcode loci; (ii) compile a centralized reference database that includes all recognized fungal species as well as species hypothesis, and allows regular updates from the research community; and (iii) establish a consensus set of new species recognition criteria based on barcode DNA sequences that can be applied across the fungal kingdom.

Analysis of Metagenomics Next Generation Sequence Data for Fungal ITS Barcoding: Do You Need Advance Bioinformatics Experience?

During the last few decades, most of microbiology laboratories have become familiar in analyzing Sanger sequence data for ITS barcoding. However, with the availability of next-generation sequencing platforms in many centers, it has become important for medical mycologists to know how to make sense of the massive sequence data generated by these new sequencing technologies. In many reference laboratories, the analysis of such data is not a big deal, since suitable IT infrastructure and well-trained bioinformatics scientists are always available. However, in small research laboratories and clinical microbiology laboratories the availability of such resources are always lacking. In this report, simple and user-friendly bioinformatics work-flow is suggested for fast and reproducible ITS barcoding of fungi.

Molecular evidence of undescribed Ceratonova sp. (Cnidaria: Myxosporea) in the freshwater polychaete, Manayunkia speciosa, from western Lake Erie

We used PCR to screen pooled individuals of Manayunkia speciosa from western Lake Erie, Michigan, USA for myxosporean parasites. Amplicons from positive PCRs were sequenced and showed a Ceratonova species in an estimated 1.1% (95% CI=0.46%, 1.8%) of M. speciosa individuals. We sequenced 18S, ITS1, 5.8S, ITS2 and most of the 28S rDNA regions of this Ceratonova sp., and part of the protein-coding EF2 gene. Phylogenetic analyses of ribosomal and EF2 sequences showed the Lake Erie Ceratonova sp. is most similar to, but genetically distinct from, Ceratonova shasta. Marked interspecific polymorphism in all genes examined, including the ITS barcoding genes, along with geographic location suggests this is an undescribed Ceratonova species. COI sequences showed M. speciosa individuals in Michigan and California are the same species. These findings represent a third parasite in the genus Ceratonova potentially hosted by M. speciosa.

Establishment of a standard reference material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures

ABSTRACTThe majority of the population in the Philippines relies on herbal products as their primary source for their healthcare needs. After the recognition of Vitex negundo L. (lagundi) as an important and effective alternative medicine for cough, sore throat, asthma and fever by the Philippine Department of Health (DOH), there was an increase in the production of lagundi-based herbal products in the form of teas, capsules and syrups. The efficiency of these products is greatly reliant on the use of authentic plant material, and to this day no standard protocol has been established to authenticate plant materials. DNA barcoding offers a quick and reliable species authentication tool, but its application to plant material has been less successful due to (1) lack of a standard DNA barcoding loci in plants and (2) poor DNA yield from powderised plant products. This study reports the successful application of DNA barcoding in the authentication of five V. negundo herbal products sold in the Philippines. Also, the first standard reference material (SRM) herbal library for the recognition of authentic V. negundo samples was established using 42 gene accessions of ITS, psbA-trnH and matK barcoding loci. Authentication of the herbal products utilised the SRM following the BLASTn and maximum-likelihood (ML) tree construction criterion. Barcode sequences were retrieved for ITS and psbA-trnH of all products tested and the results of the study revealed that only one out of five herbal products satisfied both BLASTn and ML criterion and was considered to contain authentic V. negundo. The results prompt the urgent need to utilise DNA barcoding in authenticating herbal products available in the Philippine market. Authentication of these products will secure consumer health by preventing the negative effects of adulteration, substitution and contamination.

Chilenopeptins A and B, Peptaibols from the Chilean Sepedonium aff. chalcipori KSH 883.

The Chilean Sepedonium aff. chalcipori strain KSH 883, isolated from the endemic Boletus loyo Philippi, was studied in a polythetic approach based on chemical, molecular, and biological data. A taxonomic study of the strain using molecular data of the ITS, EF1-α, and RPB2 barcoding genes confirmed the position of the isolated strain within the S.chalcipori clade, but also suggested the separation of this clade into three different species. Two new linear 15-residue peptaibols, named chilenopeptins A (1) and B (2), together with the known peptaibols tylopeptins A (3) and B (4) were isolated from the semisolid culture of strain KSH 883. The structures of 1 and 2 were elucidated on the basis of HRESIMS(n) experiments in conjunction with comprehensive 1D and 2D NMR analysis. Thus, the sequence of chilenopeptin A (1) was identified as Ac-Aib(1)-Ser(2)-Trp(3)-Aib(4)-Pro(5)-Leu(6)-Aib(7)-Aib(8)-Gln(9)-Aib(10)-Aib(11)-Gln(12)-Aib(13)-Leu(14)-Pheol(15), while chilenopeptin B (2) differs from 1 by the replacement of Trp(3) by Phe(3). Additionally, the total synthesis of 1 and 2 was accomplished by a solid-phase approach, confirming the absolute configuration of all chiral amino acids as l. Both the chilenopeptins (1 and 2) and tylopeptins (3 and 4) were evaluated for their potential to inhibit the growth of phytopathogenic organisms.

Sexual and asexual states of some endophytic Phialocephala species of Picea

Unidentified DNA sequences in isolation-based or culture-free studies of conifer endophytes are a persistent problem that requires a field approach to resolve. An investigation of foliar endophytes of Picea glauca, P. mariana, P. rubens and Pinus strobus in eastern Canada, using a combined field, morphological, cultural and DNA sequencing approach, resulted in the frequent isolation of Phialocephala spp. and the first verified discovery of their mollisia-like sexual states in the field. Phialocephala scopiformis and Ph. piceae were the most frequent species isolated as endophytes from healthy conifer needles. Corresponding Mollisia or mollisioid sexual states for Ph. scopiformis, Ph. piceae and several undescribed species in a clade containing Ph. dimorphospora were collected in the sampling area and characterized by analysis of the nuc internal transcribed spacer rDNA (ITS) and gene for the largest subunit of RNA polymerase II (RPB1) loci. Four novel species and one new combination in a clade containing Ph. dimorphospora, the type of Phialocephala, are presented, accompanied by descriptions of apothecia and previously undocumented synanamorphs. An epitype culture and corresponding reference sequences for Phialocephala dimorphospora are proposed. The resulting ITS barcodes linked with robust taxonomic species concepts are an important resource for future research on forest ecosystems and endophytes.

Sulzbacheromyces caatingae: notes on its systematics, morphology and distribution based on ITS barcoding sequences

AbstractSulzbacheromyces is a recently erected genus in Lepidostromatales, differing from Lepidostroma in the crustose thallus. After the initial discovery of S. caatingae, the only species to be found in Brazil so far, a large quantity of additional data and ITS barcoding sequences for this taxon from a much broader geographical range and different habitats was collected. Phylogenetic analysis under a maximum likelihood framework demonstrated that all specimens are genetically uniform, showing no variation in their ITS, suggesting that S. caatingae has a wide ecological amplitude beyond the Brazilian Caatinga and Atlantic Forest biomes. Detailed descriptions and illustrations of the species are presented, including a map showing the distribution of S. caatingae in the Brazilian semi-arid region and the north-eastern Atlantic rainforest.

Erratum to: Four new Penicillium species isolated from the fynbos biome in South Africa, including a multigene phylogeny of section Lanata-Divaricata

A survey of the fynbos biome of South Africa resulted in the isolation and characterization of 61 distinct Penicillium species. ITS barcodes place six of these in section Lanata-Divaricata. Based on morphology and multigene phylogenies, species were identified as P. oxalicum, P. skrjabinii, and four that were previously unknown. Multigene phylogenies resolve the new species into three consistent clades. Species from these clades were compared morphologically, and the new species are described here as P. annulatum, P. curticaule, P. malacosphaerulum, and P. ortum. Penicillium annulatum (CBS 135126T) characteristically produces colonies on Czapek yeast autolysate (CYA) and yeast extract sucrose (YES) agar that sporulate in annular patterns and grow faster on CYA at 37 °C than those of closely related species, except for the faster growing P. rolfsii. Penicillium curticaule (CBS 135128T) most closely resembles P. raperi, but grows more restrictedly on CYA at 37 °C and produces shorter phialides. Penicillium ortum (CBS135668T) grows faster on CYA at 37 °C than P. cremeogriseum, its closest relative. Penicillium malacosphaerulum (CBS 135120T) produces cleisthothecia that give colonies on most media a yellowish straw color, similar to those of P. reticulisporum. However, faster growth on CYA at 37 °C distinguishes the new species from P. reticulisporum.

Four new Penicillium species isolated from the fynbos biome in South Africa, including a multigene phylogeny of section Lanata-Divaricata

A survey of the fynbos biome of South Africa resulted in the isolation and characterization of 61 distinct Penicillium species. ITS barcodes place six of these in section Lanata-Divaricata. Based on morphology and multigene phylogenies, species were identified as P. oxalicum, P. skrjabinii, and four that were previously unknown. Multigene phylogenies resolve the new species into three consistent clades. Species from these clades were compared morphologically, and the new species are described here as P. annulatum, P. curticaule, P. malacosphaerulum, and P. ortum. Penicillium annulatum (CBS 135126T) characteristically produces colonies on Czapek yeast autolysate (CYA) and yeast extract sucrose (YES) agar that sporulate in annular patterns and grow faster on CYA at 37 °C than those of closely related species, except for the faster growing P. rolfsii. Penicillium curticaule (CBS 135128T) most closely resembles P. raperi, but grows more restrictedly on CYA at 37 °C and produces shorter phialides. Penicillium ortum (CBS135668T) grows faster on CYA at 37 °C than P. cremeogriseum, its closest relative. Penicillium malacosphaerulum (CBS 135120T) produces cleisthothecia that give colonies on most media a yellowish straw color, similar to those of P. reticulisporum. However, faster growth on CYA at 37 °C distinguishes the new species from P. reticulisporum.

The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia spp.).

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and improve knowledge about species distributions.

Microscopy- or DNA-based analyses: Which methodology gives a truer picture of stream-dwelling decomposer fungal diversity?

Abstract We assessed aquatic hyphomycete diversity in autumn and spring on oak leaves decomposing in five streams along a gradient of eutrophication in the Northwest of Portugal. Diversity was assessed through microscopy-based (identification by spore morphology) and DNA-based techniques (Denaturing Gradient Gel Electrophoresis and 454 pyrosequencing). Pyrosequencing revealed five times greater diversity than DGGE. About 21% of all aquatic hyphomycete species were exclusively detected by pyrosequencing and 26% exclusively by spore identification. In some streams, more than half of the recorded species would have remained undetected if we had relied only on spore identification. Nevertheless, in spring aquatic hyphomycete diversity was higher based on spore identification, probably because many species occurring in this season are not yet connected to ITS barcodes in genetic databases. Pyrosequencing was a powerful tool for revealing aquatic hyphomycete diversity on decomposing plant litter in streams and we strongly encourage researchers to continue the effort in barcoding fungal species.

Two widespread green Neottia species (Orchidaceae) show mycorrhizal preference for Sebacinales in various habitats and ontogenetic stages.

Plant dependence on fungal carbon (mycoheterotrophy) evolved repeatedly. In orchids, it is connected with a mycorrhizal shift from rhizoctonia to ectomycorrhizal fungi and a high natural (13)C and (15)N abundance. Some green relatives of mycoheterotrophic species show identical trends, but most of these remain unstudied, blurring our understanding of evolution to mycoheterotrophy. We analysed mycorrhizal associations and (13)C and (15)N biomass content in two green species, Neottia ovata and N. cordata (tribe Neottieae), from a genus comprising green and nongreen (mycoheterotrophic) species. Our study covered 41 European sites, including different meadow and forest habitats and orchid developmental stages. Fungal ITS barcoding and electron microscopy showed that both Neottia species associated mainly with nonectomycorrhizal Sebacinales Clade B, a group of rhizoctonia symbionts of green orchids, regardless of the habitat or growth stage. Few additional rhizoctonias from Ceratobasidiaceae and Tulasnellaceae, and ectomycorrhizal fungi were detected. Isotope abundances did not detect carbon gain from the ectomycorrhizal fungi, suggesting a usual nutrition of rhizoctonia-associated green orchids. Considering associations of related partially or fully mycoheterotrophic species such as Neottia camtschatea or N. nidus-avis with ectomycorrhizal Sebacinales Clade A, we propose that the genus Neottia displays a mycorrhizal preference for Sebacinales and that the association with nonectomycorrhizal Sebacinales Clade B is likely ancestral. Such a change in preference for mycorrhizal associates differing in ecology within the same fungal taxon is rare among orchids. Moreover, the existence of rhizoctonia-associated Neottia spp. challenges the shift to ectomycorrhizal fungi as an ancestral pre-adaptation to mycoheterotrophy in the whole Neottieae.

The largest type study of <I>Agaricales</I> species to date: bringing identification and nomenclature of <I>Phlegmacium</I> (<I>Cortinarius</I>) into the DNA era

Cortinarius is a species-rich and morphologically challenging genus with a cosmopolitan distribution. Many names have not been used consistently and in some instances the same species has been described two or more times under separate names. This study focuses on subg. Phlegmacium as traditionally defined and includes species from boreal and temperate areas of the northern hemisphere. Our goals for this project were to: i) study type material to determine which species already have been described; ii) stabilize the use of Friesian and other older names by choosing a neo- or epitype; iii) describe new species that were discovered during the process of studying specimens; and iv) establish an accurate ITS barcoding database for Phlegmacium species. A total of 236 types representing 154 species were studied. Of these 114 species are described only once whereas 40 species had one ore more synonyms. Of the names studied only 61 were currently represented in GenBank. Neotypes are proposed for 21 species, and epitypes are designated for three species. In addition, 20 new species are described and six new combinations made. As a consequence ITS barcodes for 175 Cortinarius species are released.

Some new DNA barcodes of aquatic hyphomycete species

Aquatic hyphomycetes play a key role in organic matter processing in freshwaters. Traditionally, species have been identified through the morphology of their conidia, but mycelia can fail to sporulate, making aquatic hyphomycetes a group where DNA barcoding is crucial. We generated ITS barcodes for nine aquatic hyphomycetes without published ITS sequences that, together with all published sequences, were used to construct a neighbor-joining tree. In general, the new barcoded species grouped with species of the same genus, but Tricladium and Triscelophorus species were interspersed among different clades of the tree, confirming the multiple origins of aquatic hyphomycetes.

Aspergillus, Penicillium and Talaromyces isolated from house dust samples collected around the world

As part of a worldwide survey of the indoor mycobiota, dust was collected from nine countries. Analyses of dust samples included the culture-dependent dilution-to-extinction method and the culture-independent 454-pyrosequencing. Of the 7 904 isolates, 2 717 isolates were identified as belonging to Aspergillus, Penicillium and Talaromyces. The aim of this study was to identify isolates to species level and describe the new species found. Secondly, we wanted to create a reliable reference sequence database to be used for next-generation sequencing projects. Isolates represented 59 Aspergillus species, including eight undescribed species, 49 Penicillium species of which seven were undescribed and 18 Talaromyces species including three described here as new. In total, 568 ITS barcodes were generated, and 391 β-tubulin and 507 calmodulin sequences, which serve as alternative identification markers.

A phylogenetic revision of HawaiianPseudocyphellariasensu lato (lichenized Ascomycota: Lobariaceae) reveals eight new species and a high degree of inferred endemism

Molecular sequence data of the ITS barcoding locus were employed to clarify the taxonomy of Hawaiian species of Pseudocyphellaria s.lat., including the recently segregated genera Crocodia and Podostictina. Based on published checklists and taxonomic and nomenclatural revisions, Hawaii was supposed to harbor seven widespread species, with zero degree of endemism: P. argyracea, P. aurata, P. crocata, P. dissimulata, P. intricata, P. pickeringii and P. punctillaris. Our results show that there are at least 12 species in the archipelago (one each in Crocodia and Podostictina, and ten in Pseudocyphellaria s.str.). Of the previously reported species, only the presence of Crocodia aurata was confirmed. Reports of Pseudocyphellaria crocata refer to no less than six species, four of them new to science: P. biliana Moncada, Reidy & Lücking, P. longiloba Moncada, Reidy & Lücking, P. philipiana Moncada, Reidy & Lücking, and P. pomaikaiana Moncada, Reidy & Lücking. Pseudocyphellaria hawaiiensis H. Magn. and P. xanthosticta (Pers.) Moncada & Lücking, comb. nov. [ = Sticta crocata f. sandwicensis Zahlbr.] are reinstated, whereas P. crocata s.str. does not occur in Hawaii. Pseudocyphellaria perpetua McCune & Miądl., described from North America, is established as a synonym of P. hawaiiensis. Reports of P. argyracea, P. intricata and P. punctillaris refer to three newly described species, P. confusa Moncada, Reidy & Lücking, P. gallowayana Moncada, Reidy & Lücking and P. macroisidiata Moncada, Reidy & Lücking, and the reinstated P. horridula (Zahlbr.) H. Magn. Material identified as P. flavicans and P. pickeringii is not identical with either species but represents another new taxon, P. cliffordiana Moncada, Reidy & Lücking. Based on these findings, the degree of inferred endemism in Hawaiian Pseudocyphellaria s.lat. is 75%, higher than suggested even in early treatments. The following further six new combinations are proposed—Podostictina ardesiaca (D.J. Galloway) Moncada & Lücking, comb. nov., P. berberina (G. Forst.) Moncada & Lücking, comb. nov., P. compar (Nyl.) Moncada & Lücking, comb. nov., P. degelii (D.J. Galloway & P. James) Moncada & Lücking, comb. nov., P. flavicans (Hook. f. & Taylor) Moncada & Lücking, comb. nov. and P. pickeringii (Tuck.) Moncada & Lücking, comb. nov.

Two new Talaromyces species from soil in Thailand

Two new Talaromyces species, T. thailandensis and T. tratensis, isolated from forest soil in Trat in Thailand are described. ITS barcodes were used to show the relationships between the newly described species and other Talaromyces taxa. β-tubulin and RPB1 sequences were used for the detailed analysis and the phylogenetic data showed that Talaromyces tratensis resolved in a clade closely related to T. rotundus, T. phialosporus and T. tardifaciens, which contain species that typically display restricted growth. Talaromyces thailandensis was resolved in different clades for each gene in a position closely related to T. macrosporus and T. flavus where they can be distinguished by length of stipe and shape of conidia.

Phylogenetic relationships and the newly discovered sexual state of Talaromyces flavovirens, comb. nov.

Typical Talaromyces ascomata were observed on dry Quercus suber leaf litter amongst the characteristic synnemata of Penicillium aureocephalum, and they appear to represent the sexual state of the latter species. The species is a synonym of the older Lasioderma flavovirens, and we propose the new combination Talaromyces flavovirens. Lectotype and epitype specimens are designated for this name. The defining characters of the asexual state include yellow, short-stalked, mycetozoan-like synnemata with an unusual, almost closed terminal head of penicillate conidiophores intermixed with sinuous hyphae, and dark green conidia. Ascomata could not be induced in culture, but PCR amplifications of mating-type genes indicate the species is heterothallic. In nature, ascocarp initials appear to be antheridia coiled around clavate ascogonia, similar to those of T. flavus, and the thick-walled, spiny ascospores are also similar to those of T. flavus. ITS barcodes and β-tubulin sequences place T. flavovirens in a clade with T. apiculatus, T. flavus, T. funiculosus, T. galapagensis, T. pinophilus, T. macrosporus, and seven other species.

DNA barcoding in <I>Mucorales</I>: an inventory of biodiversity

The order Mucorales comprises predominantly fast-growing saprotrophic fungi, some of which are used for the fermentation of foodstuffs but it also includes species known to cause infections in patients with severe immune or metabolic impairments. To inventory biodiversity in Mucorales ITS barcodes of 668 strains in 203 taxa were generated covering more than two thirds of the recognised species. Using the ITS sequences, Molecular Operational Taxonomic Units were defined by a similarity threshold of 99 %. An LSU sequence was generated for each unit as well. Analysis of the LSU sequences revealed that conventional phenotypic classifications of the Mucoraceae are highly artificial. The LSU- and ITS-based trees suggest that characters, such as rhizoids and sporangiola, traditionally used in mucoralean taxonomy are plesiomorphic traits. The ITS region turned out to be an appropriate barcoding marker in Mucorales. It could be sequenced directly in 82 % of the strains and its variability was sufficient to resolve most of the morphospecies. Molecular identification turned out to be problematic only for the species complexes of Mucor circinelloides, M. flavus, M. piriformis and Zygorhynchus moelleri. As many as 12 possibly undescribed species were detected. Intraspecific variability differed widely among mucorealean species ranging from 0 % in Backusella circina to 13.3 % in Cunninghamella echinulata. A high proportion of clinical strains was included for molecular identification. Clinical isolates of Cunninghamella elegans were identified molecularly for the first time. As a result of the phylogenetic analyses several taxonomic and nomenclatural changes became necessary. The genus Backusella was emended to include all species with transitorily recurved sporangiophores. Since this matched molecular data all Mucor species possessing this character were transferred to Backusella. The genus Zygorhynchus was shown to be polyphyletic based on ITS and LSU data. Consequently, Zygorhynchus was abandoned and all species were reclassified in Mucor. Our phylogenetic analyses showed, furthermore, that all non-thermophilic Rhizomucor species belong to Mucor. Accordingly, Rhizomucor endophyticus was transferred to Mucor and Rhizomucor chlamydosporus was synonymised with Mucor indicus. Lecto-, epi- or neotypes were designated for several taxa.

Delimitation and characterisation of <I>Talaromyces purpurogenus </I> and related species

Taxa of the Talaromyces purpurogenus complex were studied using a polyphasic approach. ITS barcodes were used to show relationships between species of the T. purpurogenus complex and other Talaromyces species. RPB1, RPB2, β-tubulin and calmodulin sequences were used to delimit phylogenetic species in the complex. These data, combined with phenotypic characters, showed that the complex contains four species: T. purpurogenus, T. ruber comb. nov. and two new species T. amestolkiae sp. nov. and T. stollii sp. nov. The latter three species belong to the same clade and T. purpurogenus is located in a phylogenetic distant clade. The four species all share similar conidiophore morphologies, but can be distinguished by macromorphological characters. Talaromyces ruber has a very distinct colony texture on malt extract agar (MEA), produces bright yellow and red mycelium on yeast extract sucrose agar (YES) and does not produce acid on creatine sucrose agar (CREA). In contrast, T. amestolkiae and T. stollii produce acid on CREA. These two species can be differentiated by the slower growth rate of T. amestolkiae on CYA incubated at 36 °C. Furthermore, T. stollii produces soft synnemata-like structures in the centre of colonies on most media. Extrolite analysis confirms the distinction of four species in the T. purpurogenus complex. The red diffusing pigment in T. purpurogenus is a mixture of the azaphilone extrolites also found in Monascus species, including N-glutarylrubropunctamine and rubropunctatin. Talaromyces purpurogenus produced four different kinds of mycotoxins: rubratoxins, luteoskyrin, spiculisporic acid and rugulovasins and these mycotoxins were not detected in the other three species.