Keith A. Seifert

Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case

DNA barcoding systems employ a short, standardized gene region to identify species. A 648-bp segment of mitochondrial cytochrome c oxidase 1 (CO1) is the core barcode region for animals, but its utility has not been tested in fungi. This study began with an examination of patterns of sequence divergences in this gene region for 38 fungal taxa with full CO1 sequences. Because these results suggested that CO1 could be effective in species recognition, we designed primers for a 545-bp fragment of CO1 and generated sequences for multiple strains from 58 species of Penicillium subgenus Penicillium and 12 allied species. Despite the frequent literature reports of introns in fungal mitochondrial genomes, we detected introns in only 2 of 370 Penicillium strains. Representatives from 38 of 58 species formed cohesive assemblages with distinct CO1 sequences, and all cases of sequence sharing involved known species complexes. CO1 sequence divergences averaged 0.06% within species, less than for internal transcribed spacer nrDNA or β-tubulin sequences (BenA). CO1 divergences between species averaged 5.6%, comparable to internal transcribed spacer, but less than values for BenA (14.4%). Although the latter gene delivered higher taxonomic resolution, the amplification and alignment of CO1 was simpler. The development of a barcoding system for fungi that shares a common gene target with other kingdoms would be a significant advance.

Identification and nomenclature of the genus Penicillium.

Penicillium is a diverse genus occurring worldwide and its species play important roles as decomposers of organic materials and cause destructive rots in the food industry where they produce a wide range of mycotoxins. Other species are considered enzyme factories or are common indoor air allergens. Although DNA sequences are essential for robust identification of Penicillium species, there is currently no comprehensive, verified reference database for the genus. To coincide with the move to one fungus one name in the International Code of Nomenclature for algae, fungi and plants, the generic concept of Penicillium was re-defined to accommodate species from other genera, such as Chromocleista, Eladia, Eupenicillium, Torulomyces and Thysanophora, which together comprise a large monophyletic clade. As a result of this, and the many new species described in recent years, it was necessary to update the list of accepted species in Penicillium. The genus currently contains 354 accepted species, including new combinations for Aspergillus crystallinus, A. malodoratus and A. paradoxus, which belong to Penicillium section Paradoxa. To add to the taxonomic value of the list, we also provide information on each accepted species MycoBank number, living ex-type strains and provide GenBank accession numbers to ITS, β-tubulin, calmodulin and RPB2 sequences, thereby supplying a verified set of sequences for each species of the genus. In addition to the nomenclatural list, we recommend a standard working method for species descriptions and identifications to be adopted by laboratories working on this genus.

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.

Quantifying microbial communities with 454 pyrosequencing: does read abundance count?

Pyrosequencing technologies have revolutionized how we describe and compare complex microbial communities. In 454 pyrosequencing data sets, the abundance of reads pertaining to taxa or phylotypes is commonly interpreted as a measure of genic or taxon abundance, useful for quantitative comparisons of community similarity. Potentially systematic biases inherent in sample processing, amplification and sequencing, however, may alter read abundance and reduce the utility of quantitative metrics. Here, we examine the relationship between read abundance and biological abundance in a sample of house dust spiked with known quantities and identities of fungi along a dilution gradient. Our results show one order of magnitude differences in read abundance among species. Precision of quantification within species along the dilution gradient varied from R2 of 0.96–0.54. Read‐quality based processing stringency profoundly affected the abundance of one species containing long homopolymers in a read orientation‐biased manner. Order‐level composition of background environmental fungal communities determined from pyrosequencing data was comparable with that derived from cloning and Sanger sequencing and was not biased by read orientation. We conclude that read abundance is approximately quantitative within species, but between‐species comparisons can be biased by innate sequence structure. Our results showed a trade off between sequence quality stringency and quantification. Careful consideration of sequence processing methods and community analyses are warranted when testing hypotheses using read abundance data.

Progress towards DNA barcoding of fungi

The use of DNA sequences for identifying fungi and fungus‐like organisms predates the DNA barcoding movement by at least 10 years. A brief overview of the mycological shift from phenotypic to molecular taxonomy is provided. Exploration of the animal barcode marker, cytochrome oxidase 1, by Canadian mycologists has been fruitful for some fungi, but intron issues and lack of resolution in other taxa prevent its universal application. The momentum established by 15 years of research on the fungal nuclear ribosomal internal transcribed spacer (ITS) sequences will lead to a proposal to the Consortium for the Barcode of Life on the adoption of this marker as the fungal barcode. Existing mycological research networks should facilitate the rapid development of DNA barcoding of fungi once the marker issue is settled. Some available online fungal identification databases are briefly described.

Indoor fungal composition is geographically patterned and more diverse in temperate zones than in the tropics.

Fungi are ubiquitous components of indoor human environments, where most contact between humans and microbes occurs. The majority of these organisms apparently play a neutral role, but some are detrimental to human lifestyles and health. Recent studies that used culture-independent sampling methods demonstrated a high diversity of indoor fungi distinct from that of outdoor environments. Others have shown temporal fluctuations of fungal assemblages in human environments and modest correlations with human activity, but global-scale patterns have not been examined, despite the manifest significance of biogeography in other microbial systems. Here we present a global survey of fungi from indoor environments (n = 72), using both taxonomic and phylogeny-informative molecular markers to determine whether global or local indoor factors determine indoor fungal composition. Contrary to common ecological patterns, we show that fungal diversity is significantly higher in temperate zones than in the tropics, with distance from the equator being the best predictor of phylogenetic community similarity. Fungal composition is significantly auto-correlated at the national and hemispheric spatial scales. Remarkably, building function has no significant effect on indoor fungal composition, despite stark contrasts between architecture and materials of some buildings in close proximity. Distribution of individual taxa is significantly range- and latitude-limited compared with a null model of randomized distribution. Our results suggest that factors driving fungal composition are primarily global rather than mediated by building design or function.

Phylogeny, identification and nomenclature of the genus Aspergillus

Aspergillus comprises a diverse group of species based on morphological, physiological and phylogenetic characters, which significantly impact biotechnology, food production, indoor environments and human health. Aspergillus was traditionally associated with nine teleomorph genera, but phylogenetic data suggest that together with genera such as Polypaecilum, Phialosimplex, Dichotomomyces and Cristaspora, Aspergillus forms a monophyletic clade closely related to Penicillium. Changes in the International Code of Nomenclature for algae, fungi and plants resulted in the move to one name per species, meaning that a decision had to be made whether to keep Aspergillus as one big genus or to split it into several smaller genera. The International Commission of Penicillium and Aspergillus decided to keep Aspergillus instead of using smaller genera. In this paper, we present the arguments for this decision. We introduce new combinations for accepted species presently lacking an Aspergillus name and provide an updated accepted species list for the genus, now containing 339 species. To add to the scientific value of the list, we include information about living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, calmodulin, β-tubulin and RPB2 sequences. In addition, we recommend a standard working technique for Aspergillus and propose calmodulin as a secondary identification marker.

Phylogeny and nomenclature of the genus Talaromyces and taxa accommodated in Penicillium subgenus Biverticillium

The taxonomic history of anamorphic species attributed to Penicillium subgenus Biverticillium is reviewed, along with evidence supporting their relationship with teleomorphic species classified in Talaromyces. To supplement previous conclusions based on ITS, SSU and/or LSU sequencing that Talaromyces and subgenus Biverticillium comprise a monophyletic group that is distinct from Penicillium at the generic level, the phylogenetic relationships of these two groups with other genera of Trichocomaceae was further studied by sequencing a part of the RPB1 (RNA polymerase II largest subunit) gene. Talaromyces species and most species of Penicillium subgenus Biverticillium sensu Pitt reside in a monophyletic clade distant from species of other subgenera of Penicillium. For detailed phylogenetic analysis of species relationships, the ITS region (incl. 5.8S nrDNA) was sequenced for the available type strains and/or representative isolates of Talaromyces and related biverticillate anamorphic species. Extrolite profiles were compiled for all type strains and many supplementary cultures. All evidence supports our conclusions that Penicillium subgenus Biverticillium is distinct from other subgenera in Penicillium and should be taxonomically unified with the Talaromyces species that reside in the same clade. Following the concepts of nomenclatural priority and single name nomenclature, we transfer all accepted species of Penicillium subgenus Biverticillium to Talaromyces. A holomorphic generic diagnosis for the expanded concept of Talaromyces, including teleomorph and anamorph characters, is provided. A list of accepted Talaromyces names and newly combined Penicillium names is given. Species of biotechnological and medical importance, such as P. funiculosum and P. marneffei, are now combined in Talaromyces. Excluded species and taxa that need further taxonomic study are discussed. An appendix lists other generic names, usually considered synonyms of Penicillium sensu lato that were considered prior to our adoption of the name Talaromyces. Taxonomic novelties: Taxonomic novelties: New species – Talaromyces apiculatus Samson, Yilmaz & Frisvad, sp. nov. New combinations and names – Talaromyces aculeatus (Raper & Fennell) Samson, Yilmaz, Frisvad & Seifert, T. albobiverticillius (H.-M. Hsieh, Y.-M. Ju & S.-Y. Hsieh) Samson, Yilmaz, Frisvad & Seifert, T. allahabadensis (B.S. Mehrotra & D. Kumar) Samson, Yilmaz & Frisvad, T. aurantiacus (J.H. Mill., Giddens & A.A. Foster) Samson, Yilmaz, & Frisvad, T. boninensis (Yaguchi & Udagawa) Samson, Yilmaz, & Frisvad, T. brunneus (Udagawa) Samson, Yilmaz & Frisvad, T. calidicanius (J.L. Chen) Samson, Yilmaz & Frisvad, T. cecidicola (Seifert, Hoekstra & Frisvad) Samson, Yilmaz, Frisvad & Seifert, T. coalescens (Quintan.) Samson, Yilmaz & Frisvad, T. dendriticus (Pitt) Samson, Yilmaz, Frisvad & Seifert, T. diversus (Raper & Fennell) Samson, Yilmaz & Frisvad, T. duclauxii (Delacr.) Samson, Yilmaz, Frisvad & Seifert, T. echinosporus (Nehira) Samson, Yilmaz & Frisvad, comb. nov. T. erythromellis (A.D. Hocking) Samson, Yilmaz, Frisvad & Seifert, T. funiculosus (Thom) Samson, Yilmaz, Frisvad & Seifert, T. islandicus (Sopp) Samson, Yilmaz, Frisvad & Seifert, T. loliensis (Pitt) Samson, Yilmaz & Frisvad, T. marneffei (Segretain, Capponi & Sureau) Samson, Yilmaz, Frisvad & Seifert, T. minioluteus (Dierckx) Samson, Yilmaz, Frisvad & Seifert, T. palmae (Samson, Stolk & Frisvad) Samson, Yilmaz, Frisvad & Seifert, T. panamensis (Samson, Stolk & Frisvad) Samson, Yilmaz, Frisvad & Seifert, T. paucisporus (Yaguchi, Someya & Udagawa) Samson & Houbraken T. phialosporus (Udagawa) Samson, Yilmaz & Frisvad, T. piceus (Raper & Fennell) Samson, Yilmaz, Frisvad & Seifert, T. pinophilus (Hedgcock) Samson, Yilmaz, Frisvad & Seifert, T. pittii (Quintan.) Samson, Yilmaz, Frisvad & Seifert, T. primulinus (Pitt) Samson, Yilmaz & Frisvad, T. proteolyticus (Kamyschko) Samson, Yilmaz & Frisvad, T. pseudostromaticus (Hodges, G.M. Warner, Rogerson) Samson, Yilmaz, Frisvad & Seifert, T. purpurogenus (Stoll) Samson, Yilmaz, Frisvad & Seifert, T. rademirici (Quintan.) Samson, Yilmaz & Frisvad, T. radicus (A.D. Hocking & Whitelaw) Samson, Yilmaz, Frisvad & Seifert, T. ramulosus (Visagie & K. Jacobs) Samson, Yilmaz, Frisvad & Seifert, T. rubicundus (J.H. Mill., Giddens & A.A. Foster) Samson, Yilmaz, Frisvad & Seifert, T. rugulosus (Thom) Samson, Yilmaz, Frisvad & Seifert, T. sabulosus (Pitt & A.D. Hocking) Samson, Yilmaz & Frisvad, T. siamensis (Manoch & C. Ramírez) Samson, Yilmaz & Frisvad, T. sublevisporus (Yaguchi & Udagawa) Samson, Yilmaz & Frisvad, T. variabilis (Sopp) Samson, Yilmaz, Frisvad & Seifert, T. varians (G. Sm.) Samson, Yilmaz & Frisvad, T. verruculosus (Peyronel) Samson, Yilmaz, Frisvad & Seifert, T. viridulus Samson, Yilmaz & Frisvad.

Leptographium wingfieldii introduced into North America and found associated with exotic Tomicus piniperda and native bark beetles

Leptographium wingfieldii is a well-known fungal associate of the pine shoot beetle, Tomicus piniperda, in Europe. This fungus is pathogenic to pines and is an important cause of blue-stain in the sapwood of infested trees. Tomicus piniperda was first found in a Christmas tree plantation in Ohio, USA, 1992, but isolation of the fungi associated with these intercepted insects was not attempted. Fungal strains resembling L. wingfieldii were recently isolated from pines attacked by T. piniperda, Dendroctonus valens and Ips pini in the northeastern United States. These strains were morphologically similar to the ex-type and other reference strains of L. wingfieldii. Strains were also compared based on sequences of the partial ITS ribosomal DNA operon, beta-tubulin and elongation factor 1-alpha (EF-1alpha) genes. Based on these DNA sequence comparisons, reference strains of European L. wingfieldii were conspecific with North American strains from pines attacked by T. piniperda, D. valens and I. pini. A single strain from Canada, collected in 1993 near the Ontario border with the USA, shortly after the discovery of T. piniperda in that area and tentatively identified as L. wingfieldii, was also included in this study. Its identification was confirmed, suggesting that L. wingfieldii has been present in this region and probably over the whole range of the insects distribution for at least a decade. This represents the first record of L. wingfieldii associated with the introduced and damaging pine shoot beetle T. piniperda in North America. It shows that the fungus is well established and can become associated with other native bark beetles that attack stressed and/or dying trees. The occurrence and spread of this highly pathogenic fungus associated with North American bark beetles should be monitored.

An overview of the taxonomy, phylogeny, and typification of nectriaceous fungi in Cosmospora, Acremonium, Fusarium, Stilbella, and Volutella

A comprehensive phylogenetic reassessment of the ascomycete genus Cosmospora (Hypocreales, Nectriaceae) is undertaken using fresh isolates and historical strains, sequences of two protein encoding genes, the second largest subunit of RNA polymerase II (rpb2), and a new phylogenetic marker, the larger subunit of ATP citrate lyase (acl1). The result is an extensive revision of taxonomic concepts, typification, and nomenclatural details of many anamorph- and teleomorph-typified genera of the Nectriaceae, most notably Cosmospora and Fusarium. The combined phylogenetic analysis shows that the present concept of Fusarium is not monophyletic and that the genus divides into two large groups, one basal in the family, the other terminal, separated by a large group of species classified in genera such as Calonectria, Neonectria, and Volutella. All accepted genera received high statistical support in the phylogenetic analyses. Preliminary polythetic morphological descriptions are presented for each genus, providing details of perithecia, micro- and/or macro-conidial synanamorphs, cultural characters, and ecological traits. Eight species are included in our restricted concept of Cosmospora, two of which have previously documented teleomorphs and all of which have Acremonium-like microconidial anamorphs. A key is provided to the three anamorphic species recognised in Atractium, which is removed from synonymy with Fusarium and epitypified for two macroconidial synnematous species and one sporodochial species associated with waterlogged wood. Dialonectria is recognised as distinct from Cosmospora and two species with teleomorph, macroconidia and microconidia are accepted, including the new species D. ullevolea. Seven species, one with a known teleomorph, are classified in Fusicolla, formerly considered a synonym of Fusarium including members of the F. aquaeductuum and F. merismoides species complex, with several former varieties raised to species rank. Originally a section of Nectria, Macroconia is raised to generic rank for five species, all producing a teleomorph and macroconidial anamorph. A new species of the Verticillium-like anamorphic genus Mariannaea is described as M. samuelsii. Microcera is recognised as distinct from Fusarium and a key is included for four macroconidial species, that are usually parasites of scale insects, two of them with teleomorphs. The four accepted species of Stylonectria each produce a teleomorph and micro- and macroconidial synanamorphs. The Volutella species sampled fall into three clades. Pseudonectria is accepted for a perithecial and sporodochial species that occurs on Buxus. Volutella s. str. also includes perithecial and/or sporodochial species and is revised to include a synnematous species formerly included in Stilbella. The third Volutella-like clade remains unnamed. All fungi in this paper are named using a single name system that gives priority to the oldest generic names and species epithets, irrespective of whether they are originally based on anamorph or teleomorph structures. The rationale behind this is discussed.