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Phylogeny of Xerophilic Aspergilli (Subgenus Aspergillus) and Taxonomic Revision of Section Restricti

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Phylogeny of Xerophilic Aspergilli (Subgenus Aspergillus) and Taxonomic Revision of Section Restricti

F Sklenář et al. Stud Mycol.

Abstract

Aspergillus section Restricti together with sister section Aspergillus (formerly Eurotium) comprises xerophilic species, that are able to grow on substrates with low water activity and in extreme environments. We adressed the monophyly of both sections within subgenus Aspergillus and applied a multidisciplinary approach for definition of species boundaries in sect. Restricti. The monophyly of sections Aspergillus and Restricti was tested on a set of 102 isolates comprising all currently accepted species and was strongly supported by Maximum likelihood (ML) and Bayesian inferrence (BI) analysis based on β-tubulin (benA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) loci. More than 300 strains belonging to sect. Restricti from various isolation sources and four continents were characterized by DNA sequencing, and 193 isolates were selected for phylogenetic analyses and phenotypic studies. Species delimitation methods based on multispecies coalescent model were employed on DNA sequences from four loci, i.e., ID region of rDNA (ITS + 28S), CaM, benA and RPB2, and supported recognition of 21 species, including 14 new. All these species were also strongly supported in ML and BI analyses. All recognised species can be reliably identified by all four examined genetic loci. Phenotype analysis was performed to support the delimitation of new species and includes colony characteristics on seven cultivation media incubated at several temperatures, growth on an osmotic gradient (six media with NaCl concentration from 0 to 25 %) and analysis of morphology including scanning electron microscopy. The micromorphology of conidial heads, vesicle dimensions, temperature profiles and growth parameters in osmotic gradient were useful criteria for species identification. The vast majority of species in sect. Restricti produce asperglaucide, asperphenamate or both in contrast to species in sect. Aspergillus. Mycophenolic acid was detected for the first time in at least six members of the section. The ascomata of A. halophilicus do not contain auroglaucin, epiheveadride or flavoglaucin which are common in sect. Aspergillus, but shares the echinulins with sect. Aspergillus.

Keywords: Aspergillus canadensis Visagie, Yilmaz, F. Sklenar & Seifert; Aspergillus clavatophorus F. Sklenar, S.W. Peterson & Hubka; Aspergillus destruens Zalar, F. Sklenar, S.W. Peterson & Hubka; Aspergillus domesticus F. Sklenar, Houbraken, Zalar & Hubka; Aspergillus glabripes F. Sklenar, Ž. Jurjević & Hubka; Aspergillus hordei F. Sklenar, S.W. Peterson & Hubka; Aspergillus infrequens F. Sklenar, S.W. Peterson & Hubka; Aspergillus magnivesiculatus F. Sklenar, Zalar, Ž. Jurjević & Hubka; Aspergillus pachycaulis F. Sklenar, S.W. Peterson, Ž. Jurjević & Hubka; Aspergillus penicillioides; Aspergillus pseudogracilis F. Sklenar, Ž. Jurjević & Hubka; Aspergillus restrictus; Aspergillus reticulatus F. Sklenar, Ž. Jurjević, S.W. Peterson & Hubka; Aspergillus salinicola Zalar, F. Sklenar, Visagie & Hubka; Aspergillus tardicrescens F. Sklenar, Houbraken, Zalar, & Hubka; Aspergillus villosus F. Sklenar, S.W. Peterson & Hubka; Eurotium; food spoilage; indoor fungi; linear discriminant analysis; multigene phylogeny; multispecies coalescent model; sick building syndrome; xerophilic fungi.

Figures

Fig. 1
Fig. 1
A 90 % majority consensus tree of the subgenus Aspergillus inferred with Maximum likelihood analysis based on benA, CaM and RPB2 loci (partitioning scheme and substitution models are listed in Table 3). The data set contained 102 strains and 1902 characters, of which 929 characters were variable and 864 were parsimony informative. Support values represent maximum likelihood bootstrap/ Bayesian posterior probability values, 100 % bootstrap values and 1.00 posterior probability are designated by asterisk *. The ex-type isolates are designated by a superscript T. Hamigera avellanea (NRRL 1938) was used as outgroup.
Fig. 2
Fig. 2
Maximum likelihood phylogenetic tree of the subgenus Aspergillus inferred from partitioned analysis of concatenated dataset (benA, CaM and RPB2) with Maximum likelihood method and presented in radial format.
Fig. 3
Fig. 3
Schematic representation of results of species delimitation methods in A. restrictus, A. conicus and A. vitricola clades (108 isolates). The results of multilocus method (STACEY) are compared to results of single-locus methods (bGMYC, PTP, GMYC, ABGD). Results from different methods are depicted with coloured bars highlighting congruence across methods. Displayed tree comes from STACEY analysis and is used solely for the comprehensive presentation of the results from different methods.
Fig. 4
Fig. 4
Schematic representation of results of species delimitation methods in A. penicillioides clade (86 isolates). The results of multilocus method (STACEY) are compared to results of single-locus methods (bGMYC, PTP, GMYC, ABGD). Results from different methods are depicted with coloured bars highlighting congruence across methods. Displayed tree comes from STACEY analysis and is used only used solely for the comprehensive presentation of the results from different methods.
Fig. 5
Fig. 5
Bayesian species tree based on sequence data from four loci of 193 isolates inferred by *BEAST with posterior probabilities appended to nodes. Terminal branches represent delimited species (each comprises all isolates of respective species).
Fig. 6
Fig. 6
Species tree inferred with *BEAST visualized by using DensiTree (Bouckaert 2010). All trees created in the analysis (except burn-in phase) are displayed. Trees with the most common topology are highlighted by blue colour, trees with the second most common topology by red colour, trees with the third most common topology by pale green and all other trees by dark green.
Fig. 7
Fig. 7
Bayesian tree depicting the relationship of species from Aspergillus section Restricti based on four loci (partitioning scheme and substitution models are listed in Table 3). Total length of the concatenated alignment was 2093 characters, with 837 variable and 668 parsimony informative sites. Support values represent Bayesian posterior probability/maximum likelihood bootstrap values, 100 % bootstrap values and 1.00 posterior probability are designated by asterisk *. The ex-type isolates are designated by bold font and superscript T. Hamigera avellanea (NRRL 1938) was used as outgroup.
Fig. 7
Fig. 7
Bayesian tree depicting the relationship of species from Aspergillus section Restricti based on four loci (partitioning scheme and substitution models are listed in Table 3). Total length of the concatenated alignment was 2093 characters, with 837 variable and 668 parsimony informative sites. Support values represent Bayesian posterior probability/maximum likelihood bootstrap values, 100 % bootstrap values and 1.00 posterior probability are designated by asterisk *. The ex-type isolates are designated by bold font and superscript T. Hamigera avellanea (NRRL 1938) was used as outgroup.
Fig. 8
Fig. 8
Neighbour Joining tree created in MEGA 7 with 1000 bootstrap replicates based on ITS from 454-pyrosequences obtained from house dust samples (n = 1061; Amend et al. 2010) and from this study (n = 188). The tree is rooted with Hamigera avellanea. Monophyletic groups/species are collapsed and shown as proportional triangles. The information about country of origin pertains to and is given only for species found in house dust.
Fig. 8
Fig. 8
Neighbour Joining tree created in MEGA 7 with 1000 bootstrap replicates based on ITS from 454-pyrosequences obtained from house dust samples (n = 1061; Amend et al. 2010) and from this study (n = 188). The tree is rooted with Hamigera avellanea. Monophyletic groups/species are collapsed and shown as proportional triangles. The information about country of origin pertains to and is given only for species found in house dust.
Fig. 9
Fig. 9
Results of linear discriminant analysis (LDA) performed in R 3.3.1 based on micromorphological measurements of all individuals assigned into species complexes. Ellipses represent 95 % confidence interval and arrows represent the contribution of each character to the axes. The length (l) and width (w) of conidia, diameter of stipes and vesicles were measured separately from the center and the edge of colonies.
Fig. 10
Fig. 10
Results of linear discriminant analysis (LDA) performed in R 3.3.1 based on micromorphological measurements of species from A. restrictus, A. conicus and A. vitricola clade. Dashed ellipses represent 95 % confidence interval, full circles euclidean distances. Analyses involving only two species (B, D, F) are represented by probability density function. 1 – A. restrictus; 2 – A. caesiellus; 3 – A. pachycaulis; 4 – A. conicus; 5 – A. domesticus; 6 – A. destruens; 7 – A. villosus; 8 – A. gracilis; 9 – A. pseudogracilis; 10 – A. vitricola; 11 – A. glabripes.
Fig. 11
Fig. 11
Results of linear discriminant analysis (LDA) performed in R 3.3.1 based on micromorphological measurements of species from A. penicillioides clade. Dashed ellipses represent 95 % confidence interval, full circles euclidean distances. Analyses involving only two species (B, D, F) are represented by probability density function. 1 – A. penicillioides; 2 – A. tardicrescens; 3 – A. clavatophorus; 4 – A. magnivesiculatus; 5 – A. hordei; 6 – A. reticulatus; 7 – A. salinicola; 8 – A. canadensis; 9 – A. infrequens.
Fig. 12
Fig. 12
Osmotic gradient growth curves of species from A. restrictus, A. conicus and A. vitricola clades. Growth curves were created using local regression (LOESS) in R 3.3.1, grey zones represent 95 % confidence intervals. 1 – A. restrictus; 2 – A. caesiellus; 3 – A. pachycaulis; 4 – A. conicus; 5 – A. domesticus; 6 – A. destruens; 7 – A. villosus; 8 – A. gracilis; 9 – A. pseudogracilis; 10 – A. vitricola; 11 – A. glabripes.
Fig. 13
Fig. 13
Osmotic gradient growth curves of species from A. penicillioides clade. Growth curves were created using local regression (LOESS) in R 3.3.1, grey zones represent 95 % confidence intervals. 1 – A. penicillioides; 2 – A. tardicrescens; 3 – A. clavatophorus; 4 – A. magnivesiculatus; 5 – A. hordei; 6 – A. reticulatus; 7 – A. salinicola; 8 – A. canadensis; 9 – A. infrequens.
Fig. 14
Fig. 14
Aspergillus caesiellus. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 15
Fig. 15
Aspergillus canadensis. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 16
Fig. 16
Aspergillus clavatophorus. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 17
Fig. 17
Aspergillus conicus. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 18
Fig. 18
Aspergillus destruens. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 19
Fig. 19
Aspergillus domesticus. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 20
Fig. 20
Aspergillus glabripes. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 21
Fig. 21
Aspergillus gracilis. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 22
Fig. 22
Aspergillus halophilicus. A. Colonies after 14 d at 25 °C: left to right, obverse CZA70S, reverse CZA70S, M40Y and MEA + 10 % NaCl. B–E. Cleistothecia. F, G. Asci. H, I. Ascospores. J, K. SEM (ascospores). Scale bars: D–I = 10 μm; J, K = 2 μm.
Fig. 23
Fig. 23
Aspergillus hordei. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 24
Fig. 24
Aspergillus infrequens. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 25
Fig. 25
Aspergillus magnivesiculatus. A. Colonies: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 26
Fig. 26
Aspergillus pachycaulis. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 27
Fig. 27
Aspergillus penicillioides. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 28
Fig. 28
Aspergillus pseudogracilis. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 29
Fig. 29
Aspergillus restrictus. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 30
Fig. 30
Aspergillus reticulatus. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 31
Fig. 31
Aspergillus salinicola. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 32
Fig. 32
Aspergillus tardicrescens. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 33
Fig. 33
Aspergillus villosus. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 34
Fig. 34
Aspergillus vitricola. A. Colonies after 14 d at 25 °C: top row left to right, obverse M40Y, CYA, CY20S and DG18; bottom row left to right, reverse M40Y, MEA, M60Y and MEA + 10 % NaCl. B, C. Conidiophores. D, E. Conidial heads. F. Conidia. G. Conidia in SEM. H. Stipe in SEM. Scale bars: B, C, F = 10 μm; G, H = 2 μm.
Fig. 35
Fig. 35
Different kinds of food and commodities on which members of section Restricti are to be found. A–F. Various bakery products: A. Chocolate chip cookies (“A. penicillioides” by morphology – isolate not included in this study; isolated together with A. cibarius). B. Mouldy bread. C. Madeleines (isolate UBOCC-A-115045 – A. reticulatus). D. French savaroise (isolate UBOCC-A-115043 – A. salinicola). E. French mini cakes (isolate UBOCC-A-115048 – A. domesticus). F. Mouldy cake (mixed spoilage with Penicillium and Eurotium spp.). G. Grain containing diverse fungal populations similar to nuts, spices and seeds. H. Cigars (“A. penicillioides” by morphology – isolate not included in this study).
Fig. 36
Fig. 36
Overview of some substrates and habitats associated with members of section Restricti. A. Solar salterns at the Adriatic coast, Sečovlje, Slovenia (isolate EXF 226 – A. salinicola). B. Indoor environment, a source of numerous species: one of the sampling sites in Canada where house dust was collected. C. Water damaged wall surface, dry at the time of sampling (isolate DTO 073-H6 – A. tardicrescens). D. Mouldy chair backrest (isolate DTO 231-B9 – A. domesticus). E. Textiles (“A. penicillioides” by morphology – isolate not included in this study). F. Textile water hose imported from China (“A. penicillioides” and Cladosporium sp. by morphology – isolates not included in this study). G. Leather baseball gloves (isolate CCF 5500 – A. penicillioides and CCF 5514 – A. restrictus). H. Leather armrest of a dentist chair (isolate DTO 316-A7 – A. tardicrescens). I. Skeleton of Elephas maximus (Asian elephant) (“A. penicillioides” by morphology – isolate not included in this study). J. Mouldy sclerotium of Corallocytostroma ornicopreoides imported from Australia (isolate CCF 3364 – A. restrictus).
Fig. 37
Fig. 37
Deteriorated paintings and books from archives. A–H. Deteriorated painting from Capuchin monastery of St. Francis, Ajdovščina, Slovenia (A) and Musée des beaux Arts in Brest, France (B–H); isolated species: A. destruens, A. domesticus, A. conicus, A. glabripes, A. magnivesiculatus, A. reticulatus, A. salinicola, A. tardicrescens, A. villosus, A. vitricola. I–M. Deteriorated books from archives; isolated species: A. vitricola and A. domesticus.

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