Structural Diversity of Aspergillus ~Section Nigri ! Spores Marta Filipa Simões, Cledir Santos, and Nelson Lima* IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal Abstract: The taxonomy of fungal species, similar to that of many other microorganisms, suffers frequent revisions due to the discovery of new species and to the development and gathering of characterization data and morphological information. Morpho-taxonomy helps in the identification of many species. This work presents the macro, micro-morphological, and spectral mass analyses for phenotypic characterization of 13 species of Aspergillus section Nigri, showing that the characterization of spores ~conidia! by scanning electron microscopy can be used as a tool to discriminate key morphological characteristics and separate closely related fungi. These results were corroborated by colony plates, stereomicroscopy, light microscopy, and spectral mass data. Key words: Aspergillus section Nigri, fungal taxonomy, morpho-taxonomy, spore characterization I NTRODUCTION Species of Aspergillus section Nigri ~known as black as- pergilli! have been extensively used for various biotechno- logical purposes and are among the best-studied fungi. Many are responsible for biodeterioration of diverse com- modities, including food ~Abarca et al., 2004!. The Nigri section has evolved continuously, and the last taxonomy revision includes 26 different taxa ~Varga et al., 2011!. However, because of molecular problems detected on b-tubulin DNA primers by Hubka and Loralik ~2012!, Aspergillus japonicus should be treated as a synonym of Aspergillus violaceofuscus, reducing the number to 25 differ- ent taxa. Similarly, Aspergillus fijiensis, described by Varga et al. ~2011! as a new species, was found to be the already known Aspergillus brunneoviolaceus. Meanwhile, two more species were described by Jurjevic ´ et al. ~2012!, which means that we currently accept 27 different taxa. The identification of species is a fundamental goal in microbiology. Information about each microorganism, such as morphological description, physiological and biochemi- cal properties, ecological roles, and societal risks or benefits, is a key element in this process. The concept of a distinct species is clearly abstract; delimitations are very difficult and often not consensual. Identification of fungi, even for experts, can be time- consuming and is hampered by frequent revisions of the taxonomic schemes. In addition, each taxonomic group has specialized literature, terminology, and characters. Some practical solutions have been designed to overcome these limitations with very limited success ~Paterson et al., 2004, 2006!. This occurs to the extent that identifications can only be undertaken with a degree of reliability, by a small num- ber of scientists skilled in the “art.” The black aspergilli are considered a taxonomically challenging group of fungi. It is difficult to recognize some of these species. Several approaches have been developed to this end, such as new taxonomic schemes using extensive molecular analysis ~de Vries et al., 2005; Varga et al., 2011!, and all available information is valuable in differentiating each strain. Therefore, a more complete and polyphasic approach allows better results in fungal taxonomy studies ~Varga et al., 2011; Simões et al., 2013!. The aim of this study was to contribute additional information on the exist- ing strains of the section Nigri from the genus Aspergillus. MATERIALS AND METHODS Thirteen strains belonging to Aspergillus section Nigri were selected and supplied by the Micoteca da Universidade do Minho ~MUM! fungal culture collection ~Table 1!. For the macro-morphological analysis, the selected strains were grown on potato dextrose agar ~PDA, OXOID CM0139, Basingstoke Hampshire, UK!, malt extract agar ~MEA, malt extract 2%, mycological peptone 0.1%, glucose 2%, and agar 2%!, and Czapek yeast extract agar ~CYA, sucrose 2%, yeast extract 0.5%, NaNO 3 0.3%, KCl 0.05%, MgSO 4 {7H 2 O 0.05%, FeSO 4 {7H 2 O 0.001%, K 2 HPO 4 0.1%, and agar 2%!. To guarantee the same medium depth in all of the plates and a standardized growth, 20 mL of medium was poured into standard ~90 mm! sterile and disposable Petri dishes. Each plate was inoculated at three points, equidistant from the center, and incubated in the dark at 258C for 7 days. To prevent stray colonies on the plates, inoculation was made from a spore suspension on a solution of 0.2% agar with 0.05% tween 80. For microscopy analysis the strains were grown for 3 or 4 days on MEA. Digital images of colonies were obtained on a register recording system for fungal plates that consists of a dedi- cated macro-photo stand with a Canon EOS 5D Mark II camera. The camera was operated with a remote shutter release device equipped with a 150-mm interchangeable Sigma ~St. Louis, MO, USA! lens APO MACRO F2.8EX DG HSM. The equipment consisted of a Polaroid MP4 + land camera stand where the camera was held horizontally at bull’s eye level and pointing down toward the culture plates Received December 26, 2012; accepted April 29, 2013 *Corresponding author. E-mail: nelson@ie.uminho.pt Microsc. Microanal. 19, 1151–1158, 2013 doi:10.1017/S1431927613001712 Microscopy AND Microanalysis © MICROSCOPY SOCIETY OF AMERICA 2013