Aspergillus niger contains the cryptic phylogenetic species A. awamori

f u n g a l b i o l o g y 1 1 5 ( 2 0 1 1 ) 1 1 3 8 e1 1 5 0

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Aspergillus niger contains the cryptic phylogenetic species A. awamori
nos VARGAb,d, Giancarlo PERRONEa,*, Gaetano STEAa, Filomena EPIFANIa, Ja Jens C. FRISVADc, Robert A. SAMSONd a

Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Via G. Amendola 122/O, 70126 Bari, Italy Department of Microbiology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, K€oz
ep fasor 52, Hungary c Center for Microbial Biotechnology, Department of Systems Biology, Building 221, Technical University of Denmark, DK-2800 Kgs Lyngby, Denmark d CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands b

article info

abstract

Article history:

Aspergillus section Nigri is an important group of species for food and medical mycology,

Received 10 January 2011

and biotechnology. The Aspergillus niger ‘aggregate’ represents its most complicated taxo-

Received in revised form

nomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergil-

14 July 2011

lus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus

Accepted 15 July 2011

lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described

Available online 23 July 2011

by Nakazawa, has been compared taxonomically with other black aspergilli and recently it

Corresponding Editor:

has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated

Joseph W. Spatafora

from portions of three genes coding for the proteins b-tubulin (benA), calmodulin (CaM ), and the translation elongation factor-1 alpha (TEF-1a) of a population of A. niger strains iso-

Keywords:

lated from grapes in Europe revealed the presence of a cryptic phylogenetic species within

AFLP

this population, A. awamori. Morphological, physiological, ecological and chemical data

Aspergillus niger aggregate

overlap occurred between A. niger and the cryptic A. awamori, however the splitting of

Extrolites

these two species was also supported by AFLP analysis of the full genome. Isolates in

Multilocus approach

both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B2, and they

Phylogenetic species

also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-g-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies. ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Species assigned to Aspergillus section Nigri (Gams et al. 1985) occupy a wide spectrum of habitats in animal and plant environments, and they are economically important both as harmful or useful microorganisms. They can contaminate foods and feeds at different stages including pre- and

postharvest stages, processing, and handling (Kozakiewicz 1989; Abarca et al. 2004; Samson et al. 2004). By contrast, they are also frequently used in the fermentation industry for the production of organic acids, enzymes, vitamins, and antibiotics (Varga et al. 2000). Section Nigri species are also candidates for genetic manipulation in the biotechnology industries since Aspergillus niger used under certain industrial

* Corresponding author. Tel.: þ39 080 5929363; fax: þ39 080 5929874. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.07.008

A. awamori a cryptic phylogenetic species

conditions has been granted generally regarded as safe (GRAS) status by the Food and Drug Administration of the US government. Schuster et al. (2002) in their review discussed widely on the possible safe/unsafe status of A. niger and its closely related morphologically indistinguishable species. However, it was suggested to avoid the industrial use of strains proved to produce undesirable metabolites (i.e. ochratoxins or fumonisins). In principle only A. niger sensu stricto has been given GRAS status, and only in connection with particular industrial processes (so a species cannot be given GRAS status in its own right), and for example the GRAS status could be given to the Japanese strains of Aspergillus awamori in connection with koji processes. In the last 10 years, several studies have been focused on this group of organisms due to their role as causative agents of black rot of grapes, which subsequently may result in ochratoxin A (OTA) contamination of grapes, grapes by-products,  es et al. 2002; dried vine fruits, coffee, and cocoa (Caban Samson et al. 2004). Furthermore strains of A. niger have the capability to produce fumonisins and they may represent a new toxicological risk since the production has been recently proved for several A. niger isolates coming from culture collections, coffee beans, and grapes and since the natural contamination by FB2 of must and wine has also been reported (Frisvad et al. 2007; Logrieco et al. 2009; Noonim et al. 2009; Mogensen et al. 2010). However, black aspergilli are one of the more difficult groups concerning classification and identification. The taxonomy of Aspergillus section Nigri has been studied by many taxonomists and several schemes have been proposed (Abarca et al. 2004; Varga et al. 2006; Samson et al. 2007b). Recently in their review on diagnostic tools for identify black aspergilli, Samson et al. (2007b) provided different schemes and suggested a polyphasic approach for identification using molecular analysis, morphology and growth on specific media at different temperatures, extrolite production, and Ehrlich reaction. After various revisions and amendments, the number of accepted species within this section is 19, including the recently described species Aspergillus ibericus, Aspergillus brasiliensis, Aspergillus aculeatus, Aspergillus sclerotiicarbonarius, and Aspergillus uvarum (Serra et al. 2006; Varga et al. 2007; Noonim et al. 2008; Perrone et al. 2008). Considering the species belonging to the A. niger complex/ aggregate, eight species have been assigned to it: A. niger, Aspergillus tubingensis, Aspergillus acidus, A. brasiliensis (Kusters-van
gne
gneau et al. 1993; Varga et al. 1993, Someren et al. 1991; Me 1994, 2007; Parenicova et al. 1997, 2001; Accensi et al. 1999), Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis (Samson et al. 2004), and Aspergillus vadensis (de Vries et al. 2005). Aspergillus awamori was first described by Nakazawa (1907) based on the strain IFO 4033, and was said to be the most favourable mould for the koji of the awamori alcoholic fermentation. In his study of black aspergilli, Murakami (1979) separated the A. niger group with a A. niger series and A. pulverulentus series from the Black Koji-mold group consisting of a A. aureus series and a A. awamori series. Murakami characterized the A. awamori series by small and brown conidial heads, nitrite-assimilating, and high amylase production. However the strain selected as neotype is from Rio de Janeiro in Brazil (CBS 557.65 ¼ WB 4948) and has no connection to

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awamori fermentations. A. awamori has been cited numerous times in the literature, and has been used in several kinds of fermentations. The status of A. awamori has been revised several times with various synonyms i.e. A. pseudoniger or A. pseudocitricus (Mosseray 1934), A. usamii (Sakaguci et al. 1951), and A. niger var. awamori (Al-Musallam 1980). More recently it has been treated as a synonym of A. niger by Samson et al. (2004). During a molecular characterization of A. niger aggregate isolates collected from grapes in Europe, we identified a uniform separate cluster by AFLP analysis which included the neotype strain of A. awamori (ITEM 4509 ¼ CBS 557.65). This preliminary observation led us to further analyze this population by using molecular, biochemical, and biological data to better circumscribe this group of isolates, to investigate its phylogenetic relationships within the A. niger ‘aggregate’ species and to determine whether A. niger contains distinct evolutionary lineages/species. Species concepts and the criteria to recognize species are much discussed and controversial topics, and they have become more important since the discovery that species recognized by phenotypic characters or reproductive isolation commonly contains multiple genetically differentiated clades qualified as phylogenetic species (Dettman et al. 2003). In this respect, the use of concordance of multiple gene genealogies to recognize species boundaries, namely phylogenetic species recognition (PSR), has led in recent years to recognize multiple cryptic, phylogenetic species within single morphological or biological species and these have had a tendency to be geographically distinct. Taylor et al. (2000) and Dettman et al. (2003) have promoted the use of multiple gene genealogies to recognize boundaries of such lineages (GCPRS ¼ Genealogical concordance phylogenetic species recognition). According to this approach, trees of multiple genes have the same topology due to fixations of previously polymorphic loci following genetic isolation. In principle it should only be appropriate for sexually reproducing species, but technically, it is applicable to all organisms, so it is becoming popular among mycologist (Taylor et al. 2006). However, an omnispective (Blackwelder 1977) or polyphasic approach (Vandamme et al. 1996) to taxonomy is in reality what has been used for species recognition throughout the history of taxonomy, and such an approach include all kinds of characters. For these reasons, a multilocus sequence typing (MLST) approach was applied to evaluate phylogenetic relationships of the A. niger population by using partial sequences of the b-tubulin, calmodulin, and the nuclear translation elongation factor-1 alpha (TEF-1a) genes. Data were combined with extrolite profiles, together with morphological and physiological traits.

Material and methods Fungal isolates A total of 80 isolates were analyzed in this study, including the type strains of Aspergillus niger and Aspergillus awamori and 63 strains grouped as A. niger by morphological and preliminary molecular analysis (Perrone et al. 2006) isolated from grapes, with the exception of ITEM 3856, which was isolated from figs; nine representative strains of A. niger ‘aggregate’ chosen

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G. Perrone et al.

Table 1 e List of the Aspergillus Sect. Nigri strains used in this study. Isolate

Species

T

ITEM 4509 ITEM 3856 ITEM 4502 ITEM 4541 ITEM 4551 ITEM 4552 ITEM 4686 ITEM 4689 ITEM 4717 ITEM 4730 ITEM 4771 ITEM 4774 ITEM 4775 ITEM 4777 ITEM 4778 ITEM 4847 ITEM 4851 ITEM 4853 ITEM 4858 ITEM 4859 ITEM 4863 ITEM 4945 ITEM 4947 ITEM 4951 ITEM 4983 ITEM 5018 ITEM 5240 ITEM 5255 ITEM 5257 ITEM 5266 ITEM 5267 ITEM 5268 ITEM 5272 ITEM 5277 ITEM 5280 ITEM 5283 ITEM 5289 ITEM 6122 ITEM 6123 ITEM 6126 ITEM 6127 ITEM 6128 ITEM 6129 ITEM 6138 ITEM 6140 ITEM 6141 ITEM 6142 ITEM 6143 ITEM 6144 ITEM 7093 ITEM 7096 ITEM 7097 ITEM 7098 ITEM 7460 ITEM 7468 ITEM 7486 ITEM 7500 ITEM 7501 ITEM 4501T ITEM 4547 ITEM 5276

A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. awamori A. niger A. niger A. niger

GenBank accession no.a

Origin

Unknown Dry Figs, Market, Turkey Unknown Grapes, Portugal Grapes, Portugal Grapes, Portugal Grapes, Spain Grapes, Spain Grapes, Bari, Apulia, Italy Grapes, Bari, Apulia, Italy Grapes, Portugal Grapes, Portugal Grapes, Portugal Grapes, Portugal Grapes, Portugal Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Spain Grapes, Spain Grapes, Spain Grapes, Israel Grapes, Italy Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Greece Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Portugal Grapes, Portugal Grapes, Portugal Grapes, Portugal Grapes, Portugal Grapes, Portugal Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Tannic acid fermentation USA White must, Portugal Grapes, Greece

Calmodulin

b-tubulin

TEF-1a

AJ964874 FN394666 FN394678 (I) II FN394670 (V) FN394668 (III) I I I FN394667 (II) I III I FN394671 V I I I I I I II I I I I II I I I V FN394669 (IV) I II I IV II IV IV III I I I II I I III III II II III I I II I I III III AY585536 (I) I FN394673 (II)

AY820001 (I) I I I I I I I I FN395675 I I FN394674 (II) I I I II II II I II I I FN394676 I II I II I I II I I I II I I I I I I I I I II I I I I I I I I I II I I I AJ964872 (I) I I

FN665395 (I) FN665394 FN665399 I FN665392 (III) VI II II IV FN665397 (IV) I VI IV FN665402 (VI) III VI I I IV I IV III II I I IV NS I FN665396 (II) III FN665393 VI III FN665401 I VI I FN665400 (V) V VI II NS FN665398 III III III VI VI VI III VI I I I I II VI VI FN665404 (I) I I

A. awamori a cryptic phylogenetic species

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Table 1 e (continued) Isolate

ITEM 7090 ITEM 7091 ITEM 7092 ITEM 7496 ITEM 7497 CBS 139.52b CBS 117.51 ITEM 7048T ITEM 7040T ITEM 4500 ITEM 4503T ITEM 4507 ITEM 4508 CBS 115.52 CBS 111.34 ITEM 7555T ITEM 7561T ITEM 7557 ITEM 7559T

Species

A. niger A. niger A. niger A. niger A. niger A. niger A. niger A. brasiliensis A. tubingensis A. tubingensis A. carbonarius A. acidus A. acidus A. acidus A. acidus A. costaricaensis A. vadensis A. lacticoffeatus A lacticoffeatus

GenBank accession no.a

Origin

Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Grapes, Italy Kuro-koji, Japan Japan Soil, Brazils Unknown Unknown Paper Japan Japan Kuro-koji, Japan Japan Soil, Costa Rica air, Egypt Beans of coffee arabica, Venezuela Beans of coffee robust, Indonesia

Calmodulin

b-tubulin

TEF-1a

FN394672 (III) III III I I I II AM295175 AJ964876 e AJ964873 AM419749 e e e EU163268 EU163269 e EU163270

I I I FN394677 (II) II I I AY820006 AY820007 e AY585532 AY585533 AY585534 e e AY820014 AY585531 AY819999 AY819998

FN665403 (II) II II FN665657 (III) III I I FN665411 FN665407 e FN665412 e FN665410 e e FN665409 FN665408 FN665405 FN665406

T

¼ Type strain. a Identical sequences among the same species are designated by the same Roman numeral. b CBS ¼ Centraalbureau voor Scimmelcultures, Utrecht, The Netherlands.

to represent genetic diversity within this group (Perrone et al. 2006, 2007) and a strain of A. carbonarius as outgroup (Table 1). The analysis also included four putative A. awamori isolates utilized in Japanese koji fermentation. All the isolates were identified morphologically and subjected to molecular and biochemical studies involving sequences, AFLP data, extrolites, morphological and physiological analysis.

Isolation and analysis of nucleic acids Fungal strain growth and relative DNA isolation were done according to Perrone et al. (2006). Amplification of part of the b-tubulin gene (benA) was performed using the primers Bt2a and Bt2b (Glass & Donaldson 1995). Amplifications of the partial calmodulin (CaM ) gene were set up as described previously (Perrone et al. 2004). Two primer pairs were designed in conserved regions found aligning the TEF-1a gene of Aspergillus fumigatus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus niger, Aspergillus clavatus, Aspergillus terreus, Aspergillus flavus downloaded from http://www.cadre-genomes.org.uk/. The primers were designed using the Primer Express software (Applied Biosystems, Foster City, CA), to operate at relatively high annealing temperatures (59  C), thereby preventing the coamplification of non-specific target of DNA. The primer sequences: A-TEF_F: 50 -CCTTCAAGTACGCYTGGGTTC-30 ; 0 A-TEF_R: 5 -TTCTTGGAGTCACCGGCAA-30 , the size of the amplicon is about 750 bp. Sequence analysis was performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit for both strands. All the sequencing reactions were purified by gel filtration through Sephadex G-50 (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in doubledistilled water and analyzed on the ‘ABI PRISM 3100 Genetic Analyzer’ (Applied Biosystems).

Extrolite analysis Extrolites were analyzed by HPLC using alkylphenone retention indices and diode array UV-VIS detection as described by Frisvad & Thrane (1987), with modifications as described by Smedsgaard (1997). Standards of ochratoxin A and B, aflavinine, asperazine, austdiol, fumonisin B2, B4 and B6, kotanin, asterric acid, secalonic acid D, neoxaline, roquefortine C & D from the collection at Department of Systems Biology, Technical University of Denmark were used for comparison with the extrolites from the species under study. Other metabolites were identified using the HPLCeMSD method of Nielsen & Smedsgaard (2003), including high resolution mass spectrometry for confirmation of production of malformins, funalenone, aurasperones and other naphtho-g-pyrones, tensidols, and pyranonigrin A (Nielsen et al. 2009).

AFLP analysis Seventy-four strains, the same studied by sequence analysis with the exception of ITEM 4730, ITEM 4507, and of the four Japanes koji moulds (CBS 111.34, CBS 115.52, CBS 117.51, CBS 139.52) were subjected to AFLP analysis (Table 1). Fungal strain growth and relative DNA isolation were done according to Perrone et al. (2006). Fluorescent AFLP was performed as described in the AFLP microbial fingerprinting kit protocol (Applied Biosystems Division, Foster City, CA). Three separate primer combinations were utilized for the selective amplifications: EcoRIþAC and MseIþCC; EcoRIþAT and MseIþCG; EcoRIþAC and MseIþCA. GeneScan-500 (ROX) was used as internal size standard (Applied Biosystems). The product was separated by capillary electrophoresis on an ‘ABI PRISM 310 Genetic Analyzer’

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Fig 1 e Phylogenetic trees based on b-tubulin sequence data of 29 taxa belonging to A. niger ‘aggregate’ group. Numbers above branches are bootstrap values. Only values above 70 % are indicated. The evolutionary history was inferred using the maximum parsimony (A) and the neighbor-joining method (B).

(Applied Biosystems). After electrophoresis, the pattern was extracted with GeneScan collection version 3.1.2 software (Applied Biosystems) and the fingerprints were automatically analyzed with Genotyper software (Applied Biosystems). To test reproducibility of the method, DNA of five strains was isolated from three replicate cultures and tested separately in triplicate. DNA of remaining strains was tested in duplicate. Peak height thresholds were set at 200. Genotyper software (Applied Biosystems) was set to medium smoothing. Bands of the same size in different individuals were assumed to be homologous and to represent the same allele. Bands of different size were treated as independent loci with two alleles. Data were analyzed with the ‘AFLP Manager database’ developed by ACGT BioInformatica S.r.l. (via Principe Amedeo 347 e 70100 BARI) and were exported in a binary format with ‘1’ for presence of the peak and ‘0’ for its absence. For clustering two different analyses were performed: fragments between 100 and 500 bp and between 200 and 500 bp were analyzed with NTSYS software by using the Dice similarity coefficient and clustered by the unweighted pair group method (UPGMA) (Nei & Li 1979). The clustering was validated by 1000 replicates of the bootstrap analysis using the WinBoot computer package (Yap & Nelson 1996). A. carbonarius ITEM 4503T was used as an outgroup in these experiments.

Analysis of sequence data The alignment of the partial b-tubulin, calmodulin and TEF-1a genes sequence data were performed using the software

package BioNumerics 5.1 from Applied Maths and manual adjustment for improvement were made by eye where necessary. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 (Tamura et al. 2007). Phylogenetic trees were prepared by the neighbor-joining method (Saitou & Nei 1987). The evolutionary distances were computed using the Tamura-Nei method of the package and are in the units of the number of base substitutions per site (Tamura & Nei 1993). All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). Bootstrap values were calculated from 1000 replications of the bootstrap procedure using programs within MEGA 4 package which refers to tests of the reliability of an inferred tree (Felsenstein 1985, 1995). Maximum parsimony analysis was also performed for all datasets using the MEGA 4 package. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. The MP trees were obtained using the Close-Neighbor-Interchange algorithm with search level 3 in which the initial trees were obtained with the random addition of sequences (ten replicates). The trees are drawn to scale, with branch lengths calculated using the average pathway method and are in the units of the number of changes over the whole sequence (Nei & Kumar 2000). All positions containing gaps and missing data were eliminated from the dataset (Complete Deletion option), A. carbonarius ITEM 4503T was used as an

A. awamori a cryptic phylogenetic species

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Fig 2 e Phylogenetic trees based on calmodulin sequence data of 29 taxa belonging to A. niger ‘aggregate’ group. Numbers above branches are bootstrap values. Only values above 70 % are indicated. The evolutionary history was inferred using the maximum parsimony (A) and the neighbor-joining method (B).

outgroup in these experiments. A tree possessing only branches that received MP bootstrap properties 70 % was chosen to represent each of the three loci. This criterion prohibited poorly supported nonmonophyly at one locus from undermining well supported monophyly at another locus.

Morphological analysis The morphological identification was done according to the criteria of Samson et al. (2007a). For macromorphological observations, Czapek yeast autolysate (CYA), malt extract autolysate (MEA) agar and Czapek agar (CZA) (Samson et al. 2004), and G25N agar (Pitt 1979) were used. Czapek agar with 20 % sucrose (CZ20) and malt and yeast extract agar with 40 % sucrose (M40Y) were made as described (Raper & Fennell 1965). Growth rates were studied and compared with reference strains as follows: on CYA at 5, 25, and 37  C, on G25N, CZA, and MEA at 25  C in the dark, all for 7 d. Cultures were also grown on CZ20 and M40Y for 7 d at 25  C in the dark to assess growth on media with reduced water activity. For assess their acidic reaction and growth they were also grown on creatineesucrose agar (CREA) (Frisvad 1985). For micromorphological observations, microscopic mounts were made in lactic acid from MEA colonies and a drop of alcohol was added to remove air bubbles and excess conidia. Scanning electron microscopy was done with uncoated frozen samples and micrographs consisted out of 30 averaged fast scans (SCAN 2 mode) in a JEOL 5600LV scanning electron microscope (JEOL, Tokyo, Japan) equipped with an Oxford CT1500 Cryostation.

Nucleotide sequence accession numbers Sequences of the benA, CaM, and TEF-1a partial regions of the studied strains have been submitted to GenBank and assigned the accession numbers listed in Table 1 and in Figs 1e3. Strains having identical sequences are grouped under the accession same numbers in Table 1.

Results Morphological, physiological and ecological data Isolates of Aspergillus niger and Aspergillus awamori have overlapping features concerning conidium size, ornamentation, stipe ornamentation, stipe length, and conidium colour. Both species contain mutant isolates that have more brownish conidium colours. The strains now grouped as A. awamori produced conidia which were mostly globose and finely to distinct roughened. Some industrial strains had almost smooth conidia. The conidial dimensions varied from 3.5 to 5.5 mm. Vesicles were 45e85 mm in diameter. These morphological characters are identical to the morphological structures of typical A. niger strains (Samson et al. 2007b) Isolates of A. niger and A. awamori had the same ranges of growth rates on the media CYA at 5, 25, and 37  C, on G25N, CZA, MEA at 25  C in the dark, and also at reduced water activity on CZ20, M40Y at 25  C. They had the same strong acid

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Fig 3 e Phylogenetic trees based on TEF-1a sequence data of 29 taxa belonging to A. niger ‘aggregate’ group. Numbers above branches are bootstrap values. Only values above 70 % are indicated. The evolutionary history was inferred using the maximum parsimony (A) and the neighbor-joining method (B).

production on CREA substrate. Strains of A. niger and A. awamori both have been found on grapes, from fermentations, and other sources and from countries such as Japan, Brazil, Italy, Spain, and USA, so there appears to be no geographic or habitat separation of the two species.

Extrolite data Extrolite profiles of isolates of Aspergillus niger and Aspergillus awamori were similar, and there were no extrolites unique to any of them (Table 2). Nearly all isolates of the two species produced pyranonigrin A, tensidol B, funalenone, and naphthog-pyrones (with the exception of isolates formerly called Aspergillus lacticoffeatus, which did not produce naphtho-g-pyrones). A high proportion of A. niger produced kotanins, while a smaller proportion of A. awamori produced kotanins. Malformins were produced by many isolates in each group. Ochratoxin A was produced by some isolates of A. niger (ITEM 7090, ITEM 7091, ITEM 7092) and some isolates of A. awamori (ITEM 7098, ITEM 7096, ITEM 4552, and traces of OTA by ITEM 6144). Fumonisins B2 and B4 were produced by isolates of both A. niger (ITEM 7091, ITEM 7092) and A. awamori (ITEM 7098 and CBS 555.65).

Sequence and AFLP data We examined the genetic relatedness of the putative 62 Aspergillus awamori isolates and of some atypical Aspergillus niger isolates to other black aspergilli belonging to the A. niger

‘aggregate’ (Table 1), analyzing nucleotide sequences of partial benA, CaM, and TEF-1a genes. The sequence diversity among the A. awamori strains led us to identify eight different groups (haplotypes) of sequences in CaM, four in benA and eleven in TEF-1a, then we selected 15 representative isolates of sequence diversity in the loci analyzed (Table 1); they were used in the phylogenetic analysis. The same analysis on the atypical A. niger strains led us to define three haplotypes used in the final dataset for the phylogenetic analysis. The mean genetic distance, using the Tamura & Nei (1993) model, was calculated on the pooled data of the three loci; among phylogenetic species within the A. niger ‘aggregate’ complex was 0.040, with the closest phylogenetic species A. awamorieA. niger and A. nigereAspergillus lacticoffeatus having a genetic distance of 0.011 and 0.002 respectively; and the more distant Aspergillus brasiliensis having a mean genetic distance of 0.061. For the outgroup species A. carbonarius, the genetic distance from the A. niger ‘aggregate’ complex was 0.141. The phylogenetic analysis was run on the representative strains showing differences in sequence alignment, then a total of 15 A. awamori strains and four of A. niger strains were selected for the analysis, together with the ten strains representative of the other species belonging to A. niger ‘aggregate’. The phylogenetic analysis was conducted firstly on the three single locus alignments and successively the combined alignment of the three loci was analyzed for inferring the organismal phylogeny. The molecular variability differed considerably among the three loci, and the CaM locus produced the

A. awamori a cryptic phylogenetic species

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Table 2 e Production of extrolites by Aspergillus niger. Species

Aspergillus niger

‘Phylospecies’

Aspergillus niger Aspergillus awamori

Fumonisins (B2, B4, B6) Ochratoxin A Malformins Pyranonigrin A Tensidol B Funalenone Kotanins Naphtho-g-pyrones Citric acid ‘KUTZ’ ‘CORAL’ ‘DERH’ ‘CCO’ Pyrophen Antafumicins Asperazine Neoxaline and roquefortines Secalonic acids Aspergillimide Asterric acid

Aspergillus niger

þ þ þ þ þ þ þ þ þ

(75 %)a (25 %)a (>50 %)c (100 %)e (>75 %)e (>75 %)e (>75 %)e (100 %)e (100 %)d þ þ þ þ þ  (100 %)  (100 %)  (100 %)

(60 %)b (20 %)b (>50 %)c (100 %)e (>75 %)e (>75 %)e (>75 %)e (100 %)e (100 %)d þ þ þ þ þ  (100 %)  (100 %)  (100 %)

 (100 %)  (100 %)  (100 %)

 (100 %)  (100 %)  (100 %)

þ þ þ þ þ þ þ þ þ

a Based on four isolates from this study. The percentage of fumonisin producers in A. niger is approximately 70 % and of ochratoxin A producers approximately 5 % (Frisvad personal communication). b Based on five isolates from this study. The percentage of fumonisin producers in the A. awamori clade is approximately 60 % and of ochratoxin A producers approximately 5 % (Frisvad, personal communication). c According the LCeMS analysis, more than 50 % of all isolates of both A. niger and clade A. awamori produce malformins. d Based on literature and culture collection data, all isolates of A. niger and A. awamori produce citric acid. e Based on examination of 78 strains of A. niger and A. awamori (Frisvad, personal communication).

largest number of informative nucleotide characters (Table 3). The benA dataset included 428 sites, that are effectively 406 characters excluding sites with gaps/missing data, with 307 conserved and 109 variable characters of which 36 are parsimony informative. The neighbor-joining tree based on partial benA gene sequences (sum of branch length ¼ 0.350) has the same topology as that obtained by the maximum parsimony

analysis (Fig 1) with a bootstrap test (1000 replicates). The CaM dataset included 561 sites (540 characters), with 431 conserved and 121 variable characters of which 52 are parsimony informative. The neighbor-joining tree of CaM (sum of branch length ¼ 0.294) has the same topology of the tree obtained by the maximum parsimony analysis (Fig 2, bootstrap test of 1000 replicates). The TEF-1a dataset included 666 sites (657 characters),, with 588 conserved and 71 variable characters of which 34 are parsimony informative. The neighbor-joining tree obtained (sum of branch length ¼ 0.156) has the same topology that obtained by the maximum parsimony analysis (Fig 3, bootstrap test of 1000 replicates). Finally a tree produced from a MP heuristic search using the combined alignment of 1655 characters from all three loci is shown in Fig 4. The neighbor-joining analysis based on the combined alignment of the three loci showed the same topology than the MP phylogenetic analysis with the optimal tree with the sum of branch length ¼ 0.251; the analysis made separately and in combination of the three loci confirmed the same relationships among species, and clearly separated the 15 strains of A. awamori from the A. niger strains. Tree statistics from analyses of the 3-gene dataset are summarized in Table 3; 27 haplotype were identified by sequence analysis and 15 belong to A. awamori population evidencing the high variability in sequence of this species. Based on the phylogenetic analysis A. awamori fullfills the requirements for species recognition under the GCPSR. In addition, four putative strains of A. awamori from Japan, used in the koji industrial fermentation, were analyzed by GCPSR methods; none of the strains belonged to the A. awamori phylospecies (Table 1). Strains CBS 139.52 and 117.51 belonged to A. niger and strains CBS 115.52 and 111.34 to Aspergillus acidus (data not shown), indicating that Japanese industrial strains were misidentified. Extrolites profile of these strains confirmed their GCPSR identification, CBS 115.52 produces antafumicins, asperazine, pyranonigrin A, naphtho-g-pyrones, funalenone, ‘SPUT’, ‘VERN’, ‘DERH’, ‘KUTZ’, CBS 117.51 is a very poor producer of secondary metabolites (poor sporulation), and produces one indol-alkaloid and nigragillin; both strains are no OTA and fumonisins producers. CBS 139.52 produces ochratoxin A (but not fumonisin), ‘DERH’ and naphtho-gammapyrones, while CBS 111.34 produces no OTA and no fumonisin. Additionally, the genome of an industrial strain so-called ‘A. awamori’ (NRBC 4314 ¼ RIB 2604) has been sequenced by Machida et al. (2010). However, based on ITS sequence data,

Table 3 e Tree statistics for each partition.a Sites Charactersb Variable Mutations Haplotypes MPTs MPT sites Length CaM benA TEF-1a Combinedd

561 428 666 1655

540 406 657 1603

121 109 71 301

139 121 94 354

15 11 20 27

8060 9046 3084 386

154 129 109 402

CI

0.800 0.937 0.862 0.763

RI

0.846 0.953 0.906 0.915

PIC/bp % 9.62 8.88 5.17 7.6

Bootstrap support (%)c A. awamori

A. niger

96 <70 <70 99

93 <70 <70 91

a CaM, calmodulin; benA, b-Tubulin; TEF-1a, translation elongation factor-1a; MPTs, most parsimonious trees; CI, consistency index; RI, retention index; PIC, parsimony informative character. b Characters are all the sites excluding sites with gaps/missing data. c Based on maximum parsimony bootstrap support. d The combined dataset consisted of the alignment of the three partial genes sequenced.

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Fig 4 e Phylogenetic trees produced from the combined sequence data of the three loci (CaM, benA, TEF-1a) of 29 taxa belonging to A. niger ‘aggregate’ group. Numbers above branches are bootstrap values. Only values above 70 % are indicated. The evolutionary history was inferred using the maximum parsimony (A) and the neighbor-joining method (B).

this isolate actually belongs to Aspergillus tubingensis (data not shown). The A. awamori isolates also formed a well-defined cluster on the UPGMA tree obtained by AFLP data (Fig 5) relative to two primers combination assays. Our data indicate that these isolates are well separated from the other A. niger aggregate species, and in particular this group of strains consistently clustered together and separately from the most closely related species, A. niger. Specific polymorphisms within and between species were observed by AFLP. Each primer combination consistently distinguished the eight different species of Aspergillus considered to belong to A. niger ‘aggregate’. The similarity found among the species analyzed was between 20 % and 45 % (Fig 5). All the strains belonging to the same species shared more than 50 % of peaks, A. awamori consists of 57 strains clustering at 58 % similarity, these strains showed an intraspecific polymorphism resulting in four main groups (I, II, III, and IV, see Fig 5). Group I consisted of eight A. awamori strains, which clustered together at a similarity of 65 %, group II included 26 strains and the type strain (ITEM 4509) at a similarity of 63 %, group III included eleven strains at a similarity of 72 %, and group IV, showed higher polymorphism, and included nine strains at a similarity of 60 %; finally three strains, ITEM 4847, 4541, and 5240 were outside the four main groups but were found to belong to the A. awamori species. The bootstrap

analysis made on AFLP data supported the division among A. awamori and A. niger, and also among the other species of the A. niger ‘aggregate’; while the intraspecific clusters of the A. awamori strains (groups IeIV) were not supported by the bootstrap analysis (Fig 5). No relationship between the UPGMA groups and the source/origin of the A. awamori strains was found, neither between these groups and the bootstrapped subgroup of A. awamori identified by the pooled sequence data (Fig 4). Both molecular approaches confirmed the same clustering of the A. awamori strains; all the 58 strains belonging to the ‘awamori’ cluster by AFLP were consistently grouped as A. awamori by sequence analysis (Figs 1e4). The bootstrap values were high (>95 %) in calmodulin, while they were less consistent in b-tubulin, and in TEF-1a phylogenetic trees; the topology was also confirmed by the consensus tree calculated both with the neighbor-joining and maximum parsimony method. However, the combined data of the three loci support most strongly the separation of these two species (bootstrap value: 99, Fig 4).

Sequence-fixed differences between Aspergillus awamori and Aspergillus niger Aspergillus awamori is morphologically indistinguishable from A. niger but some fixed nucleotide differences between these two species could be useful for their identification. Below are

A. awamori a cryptic phylogenetic species

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Fig 5 e Dendrogram of representative A. niger ‘aggregate’ strains together with A. awamori isolates based on cluster analysis with the UPGMA method using the Dice genetic distance coefficient on AFLP data obtained with two primer pairs generated by NTSYS software. Numbers on branches are percentage values from bootstrap analysis (1000 replicates). The arrow indicates the type strain of A. awamori.

listed the sequence positions and differences of A. awamori species compared to A. niger in the three regions sequenced: 1) calmodulin: 50 (T), 73 (C), 94e95 (CT), 101e102 (CG), 409 (T) 2) b-tubulin: 267 (T), 274 (A) 3) translation elongation factor-1: 49 (G), 53 (A)

Discussion We report the discovery of a cryptic black Aspergillus species (phylospecies), Aspergillus awamori, which meet the

requirements for species recognition under the highly conservative genealogical non-discordance criterion for GCPSR (Dettman et al. 2003). In GCPSR a clade is recognized as a species when supported as reciprocally monophyletic by bootstrap analyses in some or all independent gene genealogies, and when monophyly is not contradicted by bootstrap analyses in any other data partition. Differently, conflicts among independent gene topologies can be caused by recombination between individuals within a species, and transition from concordance to conflict determines the limits of species. More recently, GCPSR approach has been used to identify genetically isolated lineages and species in a number of different fungi including

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basidiomycetes (Johannesson & Stenlid 2003) and ascomycetes (O’Donnell et al. 2004, 2008; Dettman et al. 2006; Peterson 2008). In this respect the phylogenetic trees obtained separately with the three loci and by the combined loci confirmed the topology of the trees, the consensus trees were supported both in the maximum parsimony analysis and in the neighbor-joining method (Figs 1e4). The three gene genealogies examined indicate genetic isolation between Aspergillus niger and A. awamori, providing evidence of a distinct species under the PSR concept. Combined-evidence phylogeny trees generated by both the MP and NJ methods had a topology similar to those generated from single loci, further supporting the genetic uniqueness of A. awamori. These results are not in agreement with phenotypic or ecological data including extrolite profiles (Table 2), which cannot be used to separate the ‘awamori’ group from the A. niger species, so phenotypical data are not in accordance with the clear phylogenetic distinction obtained by the multilocus approach. A similar example is known in Fusarium graminearum, which has been subdivided into ten phylospecies, which cannot be distinguished phenotypically (O’Donnell et al. 2004, 2008). This formal subdivision into ten phylospecies has not been accepted by other Fusarium experts such as Leslie et al. (2007) and Leslie & Bowden (2008). Apart from sexual crosses, Leslie and coworkers used AFLP to evaluate the strength of the hypothesis that the ten Fusarium phylospecies were really different. One of the ‘rule of thumb’ criteria they used was that less than 40 % AFLP similarity indicated different species, while more than 60 % AFLP similarity indicated that isolates were in the same species. Values between 40 % and 60 % AFLP similarity were more difficult to evaluate and most of the phylospecies in Fusarium graminearum had similarities in that range (Leslie et al. 2007), thus they did not accept the phylospecies in Fusarium graminearum at this point in time. However, Leslie et al. (2007) also provided evidence that this criterion did not fit well in the Fusarium fujikuroi/Fusarium proliferatum species pair, where strains often fall in the 40e60 % similarity range that is inconclusive for species identity. In the black aspergilli obviously different species such as A. carbonarius and Aspergillus costaricaensis have a AFLP similarity well below 40 %, but the similarity between A. awamori and A. niger is 48 % (Fig 5), indicating a case where it is doubtful whether A. awamori and A. niger are two different species. However, A. awamori is grouped at an AFLP similarity close to 60 %. For the time being A. awamori could be called a cryptic, sibling, or phylogenetic species. Such cases should be discussed in the taxonomical community, so a stable solution to this problem can be proposed. The idea of reintroducing subspecies, varieties or other subspecific levels has not been explored much in mycology, and in principle invokes the same problems as species, if any of those subspecific levels were accepted broadly, but varieties and subspecies are used sparingly in mycology. Some ‘sibling’ species of Aspergillus described recently, such as Aspergillus lentulus, as a sibling of A. fumigatus (Balajee et al. 2005) have later been shown to have many phenotypic differences (Samson et al. 2007a), and thus can easily be distinguished, both using phenotypic and molecular data. Until a solution to the ‘phylospecies’ problem has been found we recommend to use a technical polyphasic approach to taxonomy, and would thus accept A. niger as the ‘real’ species and, for the time being, mention A. awamori as a ‘cryptic phylogenetic species’, or a phylogenetically distinct sister species of A. niger.

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However, in a very recent review on new and revisited species of Aspergillus section Nigri, Varga et al. (2011) evidenced some physiological differences among these two sibling species; in particular most A. awamori isolates are not able to grow on 2-deoxy-D-glucose as sole carbon source, in contrast with A. niger isolates. Aspergillus lacticoffeatus is an interesting case where phenotypic data (cappuccino-brown conidia, no production of naphtho-g-pyrones, and smooth conidia) are in concordance with low AFLP similarity to A. niger, but not concurring with sequencing that probably would call for placement of A. lacticoffeatus as a brown mutant of A. niger. This was also confirmed by Varga et al. (2011). Some isolates of A. niger, A. lacticoffeatus and the phylospecies A. awamori can produce two potentially carcinogenic mycotoxins, ochratoxin A, and fumonisin B2 and no other fungal species can produce this combination of mycotoxins. It is important which species can produce these mycotoxins. Using a polyphasic approach one would suggest that A. niger and A. lacticoffeatus produced the two mycotoxins, a multilocus gene sequencing approach would indicate that the mycotoxins were produced by A. niger and A. awamori, and an AFLP approach would indicate that A. niger and A. lacticoffeatus produced the two toxins. However, the GCPSR criterion, used in the present study has proven to be a useful and pragmatic tool for assessing sibling species limits, because concordance of multiple gene genealogies provides a means for evaluating the significance of gene flow between groups on an evolutionary timescale. The gene flow among closely related species was also recently discussed by Pal et al. (2007) who demonstrated the presence of MAT and heterokaryon genes in the asexual black aspergilli. In addition, this study demonstrates the power of PSR to reveal cryptic diversity that may be missed, also because morphological and biological species recognition are not applicable to all organisms, and PSR appears to be the most effective for revealing species diversity (Dettman et al. 2003). On the other hand, the polyphasic approach that is claimed by Vandamme et al. (1996) and Samson et al. (2007a,b) for the species recognition, enhance the knowledge of biodiversity and characteristics of fungi and addresses even better the issue of species versus individuals in the species (Peterson 2008). Again, probably genetic isolation precedes the divergence of character states, whether due to drift or selection. So it is not expected that recently genetically isolated species will show immediate phenotypic differences, although over the time they should, and this could be the case of the closely related sibling species A. niger, A. awamori and A. lacticoffeatus.

Acknowledgement This work was partially supported by EC KBBE-2007-222690-2 MYCORED.

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