Mycoscience

, Volume 53, Issue 6, pp 433–445

The correlation among molecular phylogenetics, morphological data, and growth temperature of the genus Emericella, and a new species

Authors

    • Medical Mycology Research CenterChiba University
  • Reiko Tanaka
    • Medical Mycology Research CenterChiba University
  • Yoshikazu Horie
    • Medical Mycology Research CenterChiba University
  • Yan Hui
    • Department of DermatologyThe First Hospital of Xinjiang Medical University
  • Paride Abliz
    • Department of DermatologyThe First Hospital of Xinjiang Medical University
  • Takashi Yaguchi
    • Medical Mycology Research CenterChiba University
Full Paper

DOI: 10.1007/s10267-012-0188-x

Cite this article as:
Matsuzawa, T., Tanaka, R., Horie, Y. et al. Mycoscience (2012) 53: 433. doi:10.1007/s10267-012-0188-x
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Abstract

The species of the genus Emericella have been classified and identified on the basis of morphological features. However, the phylogenetic relationships in this genus have not been investigated. To clarify the relationships according to molecular phylogenetics, morphological characteristics, and growth temperature regimens in Emericella, multilocus sequencing analysis based on recent Aspergillus taxonomy was carried out. Various characteristic species formed individual clades, and maximum growth temperature reflected the phylogenetics. Emericella species exhibit various ascospore characteristics, although some species do not have distinct ascospore ornamentation. Species that have smooth-walled ascospores with two equatorial crests are polyphyletic. Here, Emericella pachycristata is described and illustrated as a new species. Its ascospores are similar to those of E. nidulans. These species produce smooth-walled ascospores, but the equatorial crests of E. pachycristata are thicker than those of E. nidulans. On the phylogenetic trees, E. pachycristata is closely related to E. rugulosa, which produces ascospores with ribbed convex surfaces. Thus, E. pachycristata is considered to be a new species both morphologically and phylogenetically.

Keywords

Emericella pachycristataMorphological featuresNew taxonPhysiological characteristics

Introduction

Aspergillus nidulans (Eidam) G. Winter is related to the teleomorphic genus Emericella Berk. It has been used as a model filamentous fungus to investigate secondary metabolism and signal transduction pathways (Keller et al. 1994; Brown et al. 1996; Kato et al. 2003; Keller 2006). The accumulated data, methods, and techniques of A. nidulans can be directly applied to Emericella. Emericella species have the ability to produce structurally unique metabolites or induce their production (Malmstrom 1999; Malmstrom et al. 2002; Oh et al. 2007). The genus includes a few species that produce aflatoxins and are not part of the species of Aspergillus section Flavi (Frisvad and Samson 2004; Frisvad et al. 2004; Cary et al. 2005; Zalar et al. 2008). In addition, several species of this genus are reported to be etiological agents in various infections (de Hoog et al. 2000; Horre et al. 2002; Dotis et al. 2003; Gugnani et al. 2004; Balajee et al. 2007). This genus has been classified and identified on the basis of morphological characteristics. Horie (1980) reevaluated the classification of Emericella species on the basis of ascospore ornamentation by scanning electron microscopy (SEM). Since then, the species of this genus have been mainly classified according to this criterion.

Horie has investigated Emericella and related species in Chinese soils since 1996 and has documented E. miyajii, E. appendiculata, and E. qinqixianii as new species (Horie 1996, 1998, 2000). In 2004, E. venezuelensis was reported as a new species on the basis of ascospore ornamentation and aflatoxin B1 production (Frisvad and Samson 2004). Moreover, four new species of Emericella, one of which produces aflatoxin B1, have been recently reported (Zalar et al. 2008).

Although morphological characteristics are the most important factors in classifying fungi, they depend on subtle and subjective criteria. In modern descriptions of novel species, traditional morphological characteristics and more objective criteria are combined. These criteria include multilocus sequencing analysis, growth temperature regimens, and extrolite patterns; examination of the combination of these is termed “polyphasic analysis” (Hong et al. 2005, 2008; Yaguchi et al. 2007).

The species of the genus Emericella have previously been classified according to the characteristics of their ascospores, but detailed phylogenetic analysis of the genus has never been performed. In this study, we examined Emericella species isolated from Chinese soils and identified new species. These strains were similar to Aspergillus nidulans var. roseus Boeck and Kastner and a sterigmatocystin-producing variant of Emericella reported by Klich et al. (2001). In addition, we performed multilocus sequencing analysis on the basis of recent Aspergillus taxonomy (Samson et al. 2007) and attempted to clarify the relationships among the multilocus sequencing analysis, morphological characteristics, and growth temperature regimens in the genus Emericella.

Materials and methods

Strains in this study

The strains used were preserved at Medical Mycology Research Center, Chiba University (IFM) and the Natural History Museum and Institute, Chiba, Japan (CBM), or were purchased from the Centraalbureau voor Schimmelcultures (CBS), American Type Culture Collection (ATCC), or International Mycological Institute (IMI). Some strains were supplied from the Southern Regional Research Center, Agricultural Research Service, USDA (SRRC; by Dr. Maren Klich). The strains are listed in Table 1.
Table 1

List of Emericella species in this study and its maximal growth temperature

Taxon

Strain number

Origin

GenBank accession number

Maximal growth temperature (°C)

β-Tubulin

Calmodulin

Actin

E. acristata (Fennell & Raper) Horie

CBS 119.55T

USA (fabric)

AB248304

AB476805

AB476768

48

E. appendiculata Horie & Li

CBM-FA-865T

China (soil)

AB248345

AB476806

AB476769

<40

E. appendiculata (=E. filifera Zalar, Frisvad & Samson)

CBS 113636T

Slovenia (salt water)

EF428372e

EU443973e

AB524371

 

E. astellata (Fennell & Raper) Horie

CBS 134.55T

Ecuador (plant)

AB248330

AB476807

AB476770

<40

E. aurantiobrunnea (Atkins, Hindson & Russell) Malloch & Cain

IMI 74897T

Australia (haversack)

AB248306

AB476808

AB476771

<40

E. bicolor Christensen & States

CBS 425.77T

USA (soil)

AB375872

AB476809

AB524366

<40

E. cleistominuta Mehrotra & Prasad

IMI 131554T

India (soil)

AB248331

AB476810

AB476772

48

E. corrugata Udagawa & Horie

CBM-FA-73T

Thailand (soil)

AB248351

AB476811

AB476773

48

E. dentata (D. K. Sandhu & R. S. Sandhu) Horie

IMI 126693T

India (human)

AB248337

AB476812

AB524367

48

E. desertorum Samson & Mouch

CBS 653.73T

Egypt (soil)

AB248332

AB524034

AB524368

42

E. discophora Samson, Zalar & Frisvad

CBS 469.88T

Spain (soil)

AY339999e

EU443970e

AB524369

 

E. echinulata (Fennell & Raper) Horie

CBM-FA-663

Unknown

AB248354

AB524035

AB524370

48

E. falconensis Horie, Miyaji, Nishimura & Udagawa

CBM-FA-82

Venezuela (soil)

AB248346

AB524036

AB476774

45

E. foeniculicola Udagawa

IFM 54188T

China (soil)

AB524357

AB524037

AB476775

<40

E. foveolata Horie

IFM 42015T

India (herbal drug)

AB248310

AB524038

AB524372

48

E. fruticulosa (Raper & Fennell) Malloch & Cain

CBS 650.73

Egypt (soil)

AB248311

AB524039

AB476776

45

E. heterothallica (Kwon-Chung, Fennell & Raper) Malloch & Cain

ATCC 16847T

Costa Rica (soil)

AB248329

EF652411b

AB524373

<40

E. indica Stchigel & Guarro

IMI 378525T

India (soil)

AY339988a

>42d

E. miyajii Horie

CBM-FA-833NT

Unknown

AB243110

AB524040

AB476777

48

E. montenegroi Horie, Miyaji & Nishimura

CBM-FA-669T

Brazil (soil)

AB248312

AB524041

AB476778

48

E. navahoensis Christensen & States

CBS 351.81T

USA (soil)

AB248333

AB524042

AB476779

45

E. nidulans (Eidam) Vuillemin

CBS 589.65T

Belgium (unknown)

AB524358

AB524043

AB476780

48

E. nidulans (Eidam) Vuillemin

IFM 51356

Japan (human)

AB375874

AB524044

AB476781

48

E. nidulans (Eidam) Vuillemin

IFM 55368

China (soil)

AB375873

AB524045

AB476782

48

E. nidulans var. lata (Thom & Raper) Subramanian

CBS 492.65T

Unknown

AB248334

AB524046

AB476783

48

E. olivicola Frisvad, Zalar & Samson

CBS 119.37T

Italy (decaying fruit)

AY339996e

EU443986e

AB524374

 

E. omanensis Horie & Udagawa

CBM-FA-700T

Oman (soil)

AB248347

AB524047

AB524375

45

E. pachycristata Matsuzawa, Horie & Yaguchi

IFM 55265T

China (soil)

AB375875

AB524062

AB476795

48

E. pachycristata Matsuzawa, Horie & Yaguchi

IFM 55259

China (soil)

AB375876

AB524063

AB476796

48

E. pachycristata Matsuzawa, Horie & Yaguchi

IFM 55260

China (soil)

AB375877

AB524064

AB476797

48

E. pachycristata Matsuzawa, Horie & Yaguchi

IFM 55261

China (soil)

AB375878

AB524065

AB476798

48

E. pachycristata Matsuzawa, Horie & Yaguchi

IFM 55262

China (soil)

AB375879

AB524066

AB476799

48

E. pachycristata Matsuzawa, Horie & Yaguchi

IFM 55263

China (soil)

AB375880

AB524067

AB476800

48

E. pachycristata Matsuzawa, Horie & Yaguchi

IFM 55264

China (soil)

AB375881

AB524068

AB476801

48

E. parvathecia (Raper & Fennell) Malloch & Cain

IMI 139280T

USA (human)

AB243111

AB524048

AB476784

48

E. pluriseminata Stchigel & Guarro

NBRC 33028T

India (soil)

AB524359

AB524049

AB476785

>37c

E. purpurea Samson & Mouchacca

CBS 754.74T

Egypt (soil)

AB248315

AB524050

AB476786

<40

E. qinqixianii Horie, Abliz & Li

CBM-FA-866T

China (soil)

AB524360

AB524051

AB476787

<40

E. quadrilineata (Thom & Raper) Benjamin

IMI 89351NT

USA (soil)

AB248335

AB524052

AB476788

48

E. rugulosa (Thom & Raper) Benjamin

CBS 133.60T

Brazil (soil)

AB524361

AB524053

AB476789

48

E. rugulosa var. lazulina Horie, Miyaji & Nishimura

CBM-FA-710T

Brazil (soil)

AB248319

AB524054

AB476790

48

E. similis Horie, Udagawa, Abdullah & Al-Bader

IFM 54235T

Iraq (soil)

AB248321

AB524055

AB524376

48

E. spectabilis Christensen & Raper

CBS 429.77T

USA (soil)

AB248320

AB524056

AB476791

<40

E. stella-maris Zalar, Frisvad & Samson

CBS113638T

Slovenia (salt water)

EF428367e

EU443978e

AB524377

 

E. striata (Rai, Tewari & Mukerji) Malloch & Cain

IMI 163899

India (plant seed)

AB248322

AB524057

AB524378

48

E. sublata Horie

IFM 42029T

Japan (herbal drug)

AB248323

AB524058

AB476792

48

E. undulata Kong & Qi

CBM-FA-715T

China (soil)

AB248324

EU443989d

AB476793

<40

E. unguis Malloch & Cain

ATCC 16812T

USA (shoe)

AB248325

AB524059

AB524379

<40

E. variecolor Berkeley & Broome

NBRC 32302T

India (plant seed)

AB524362

AB524060

AB476794

<40

E. venezuelensis Frisvad & Samson

CBS 868.97T

Venezuela (sponge)

AY339998a

>37a

E. violacea (Fennell & Raper) Malloch & Cain

CBS 138.55T

Ghana (soil)

AB248336

AB524061

AB524380

48

Emericella sp.

SRRC1398

USA (soil)

AB524363

AB524069

AB476802

48

Emericella sp.

SRRC1402

USA (soil)

AB524364

AB524070

AB476803

48

Aspergillus nidulans var. roseus & Kastner (E. nidulans var. roseus)

ATCC 58397

USA (soil)

AB524365

AB524071

AB476804

48

Aspergillus clavatus Desmazières

CBS 514.65

 

AB489851

AB489852

AB489853

 

aData of Frisvad et al. (2004)

bData of Peterson (2008)

cData of Stchigel and Guarro (1997)

dStchigel et al. (1999)

eData of Zalar et al. (2008)

Incubation and observation

Each strain was grown in incubators at 25 °C or 37 °C for 14 days on Czapek (CZA) or malt extract (MEA) agar. After incubation, colonies were examined using a light microscope (LM) or scanning electron microscope (SEM) (Hitachi S-800, Tokyo, Japan). Colony colors were designated according to the Methuen Handbook of Colour (Kornerup and Wanscher 1978).

Growth studies

The maximum growth temperatures of all Emericella species were determined according to the method of Balajee et al. (2005): 10 μl of conidial suspension (105 conidia or ascospores/ml sterile distilled water) was placed onto the center of an MEA plate, which was then incubated at 40 °, 42 °, 45 °, or 48 °C for 7 days. The presence or absence of fungal growth at the end of the incubation period was recorded.

DNA extraction and sequencing analysis

DNA was extracted from all examined strains with a DNA extraction kit (Dr. GenTLE; Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. The parts of the β-tubulin (benA), calmodulin, and actin genes were amplified using primer pairs Bt2a and Bt2b (Glass and Donaldson 1995), cmd5 and cmd6 (Hong et al. 2005), and act-512F and ACT-783R (Carbone and Kohn 1999), respectively. Polymerase chain reaction (PCR) products were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 3130ABI Genetic Analyzer (Applied Biosystems) according to the manufacturer’s instructions.

Molecular phylogenetic analysis

DNA sequences were edited using ATGC version 4 sequence assembly software (Genetyx, Tokyo, Japan), and sequence alignment was analyzed using Clustal X software (Thompson et al. 1997). Maximum parsimony (MP) analysis (Fitch 1977) was determined by heuristic search with random addition sequences, branch swapping by tree bisection–reconnection (TBR), and MAXTREES set at 20,000, using PAUP* 4b10 (Swofford 2002). The relative robustness of the individual branches was estimated by bootstrapping (Felsenstein 1985), with 1,000 replicates using heuristic search and branch swapping by TBR and MAXTREES set at 100. For neighbor-joining (NJ) analysis (Saitou and Nei 1987), the distances between base sequences were calculated using Kimura’s two-parameter model (Kimura 1980).

Results

Multilocus sequencing analysis of the genus Emericella

Partial DNA sequences of the β-tubulin, calmodulin, and actin genes were determined in the strains used in this study. All sequences were deposited in the DNA Data Bank of Japan (DDBJ), and the accession numbers are listed in Table 1. The phylogenetic tree of the β-tubulin gene (Fig. 1) yielded 57 equally parsimonious trees based on 97 parsimony informative characters, 361 steps in length, with a consistency index (CI) of 0.447 and a retention index (RI) of 0.761. The phylogenetic tree of the calmodulin gene sequences (Fig. 2) yielded 44 equally parsimonious trees based on 147 parsimony informative characters, 651 steps in length, with a CI of 0.464 and an RI of 0.762. Last, the phylogenetic tree of the actin gene sequences (Fig. 3) yielded 21 parsimonious trees based on 129 parsimony informative characters, 532 steps in length, with a CI of 0.513 and an RI of 0.773. No differences were found between tree topologies from MP and NJ analyses (NJ trees not shown) of the β-tubulin, calmodulin, and actin genes. The three trees based on the three loci were similar. There was a correlation between molecular phylogenetics and morphological data on the phylogenetic tree of the three genes. The ascospores of E. nidulans have smooth convex walls with two equatorial crests, whereas those of E. dentata (D.K. Sandhu & R.S. Sandhu) Y. Horie have smooth convex walls with two dentate equatorial crests; these two species formed a clade (Figs. 1, 2, 3; clade I). Moreover, various characteristic species formed individual clades: E. sublata and E. montenegroi, which have ascospores with broad equatorial crests (Figs. 1, 2, 3; clade II); E. quadrilineata, E. parvathecia, E. miyajii, and E. acristata, which have ascospores with four equatorial crests (Figs. 1, 2, 3; clade III); E. rugulosa, E. rugulosa var. lazulina and E. cleistominuta, which have ascospores with ribbed convex surfaces (Figs. 1, 2, 3; clade IV); E. violacea and E. similis, which have ascospores with cancellous convex surfaces (Figs. 1, 2, 3; clade VI); E. appendiculata, E. qinqixianii, and E. filifera, which produce ascospores with appendaged threads (Figs. 1, 2, 3; clade VII); and E. variecolor, E. astellata, E. venezuelensis, E. stella-maris, and E. olivicola, which produce stellate ascospores or ascospores with widely waved equatorial crests (Figs. 1, 2, 3; clade VIII).
https://static-content.springer.com/image/art%3A10.1007%2Fs10267-012-0188-x/MediaObjects/10267_2012_188_Fig1_HTML.gif
Fig. 1

One of 57 equally parsimonious trees obtained from analysis of the β-tubulin gene using PAUP. Trees were 361 steps in length with a consistency index (CI) of 0.447 and a retention (RI) of 0.761. Numbers above or below nodes represent bootstrap values >50 % (of 1,000 bootstrap replications)

https://static-content.springer.com/image/art%3A10.1007%2Fs10267-012-0188-x/MediaObjects/10267_2012_188_Fig2_HTML.gif
Fig. 2

One of 44 equally parsimonious trees obtained from analysis of the calmodulin gene using PAUP. Trees were 651 steps in length with a CI of 0.464 and an RI of 0.762. Numbers above or below nodes represent bootstrap values >50 % (of 1,000 bootstrap replications)

https://static-content.springer.com/image/art%3A10.1007%2Fs10267-012-0188-x/MediaObjects/10267_2012_188_Fig3_HTML.gif
Fig. 3

One of 21 equally parsimonious trees obtained from analysis of the actin gene using PAUP. Trees were 532 steps in length with a CI of 0.513 and an RI of 0.773. Numbers above or below nodes represent bootstrap values >50 % (of 1,000 bootstrap replications)

The maximum growth temperatures of all strains are listed in Table 1. Approximately half the species in this genus were able to grow up to 48 °C. Emericella nidulans, E. rugulosa, and E. echinulata were typical species capable of growing at 48 °C. Six species of this genus (except E. indica) grew at 45 °C. Emericella undulata, E. appendiculata, E. qinqixianii, and the other nine species of this genus grew at temperatures less than 40 °C. On the dendrograms of all genes, the species of Emericella were clearly separated into three groups with respect to maximum growth temperature. As a result, correlations among molecular phylogenetics, morphological data, and growth temperature were apparent.

Taxonomic position of Emericella pachycristata

We found seven strains isolated from Chinese soils (IFM 55259–55265) in clade V (Figs. 1, 2, 3). On the phylogenetic trees of the three genes, these strains were closely related to E. rugulosa. Emericella pachycristata formed individual clades supported by high bootstrap values. Although E. rugulosa produces ascospores with ribbed convex surfaces, E. pachycristata does not: it shows ascospore morphology similar to that of E. nidulans (Fig. 4e, f). However, E. pachycristata differed from E. nidulans with respect to the thickness of the equatorial crests. The equatorial crests of ascospores of E. pachycristata were thicker than those of E. nidulans. Therefore, E. pachycristata is considered to be phylogenetically distinct from E. nidulans. Here, we propose this species as a novel species, Emericella pachycristata sp. nov.
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Fig. 4

Emericella pachycristata sp. nov. a Aspergillum (light microscopy: LM). b Conidia (LM). c Asci (LM). d Ascospores (LM). e Ascospores (scanning electron microscopy: SEM). f Ascospores of E. nidulans IFM 55368 (SEM). Barsad 10 μm; ef 5 μm

Taxonomy

Emericella pachycristata Matsuzawa, Horie & Yaguchi sp. nov. Figs. 4 and 5
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Fig. 5

Emericella pachycristata sp. nov. a Asci. b Ascospores. c Aspergillum. d Conidia. Barsa, c 10 µm; b, d 5 µm

MB 564572

Coloniae in agaro maltoso expansae restrictae, ascomata abundanter producentia, conidiogenesis abundanter dilute aurantiacae vel griseo-viridia vel hebes virides; reversum brunneo-aurantiacum.

Ascomata, rabro-purpurea vel atro rubro-brunnea, globosa vel subglobosa, 210–320 μm diametro, cum cellulis dictis “hülle” numerosis, crassitunicatis, globosis vel ovoideis, 15–25 × 12.5–25 μm circumcinctis; peridium griseo-flavum, membranaceum. Asci 8-spori, globosi vel subglobosi vel ovoidei, 9–11 × 8.5–10 μm, evanescentes. Ascosporae dilute rubidae vel rubro-brunneae, lenticulares, 4–5 × 3.5–4 μm, cristis aequatorialibus duabus praeditae, parte convexa laevi. Status anamorphus: Aspergillus pachycristatus.

Holotypus. IFM 55265. colonia exsiccata in culturea ex solo, in Pichan, Xinjiang, Sina, VIII-2006, a T. Yaguchi isolata et ea collectione fungorum Medical Mycology Reserch Center, Chiba University (IFM) conservata.

Anamorphosis. Aspergilluspachycristatus sp. nov. Capitula conidica griseo-olivacea, radiantia vel brevi-columnaria 35–75 × 30–40 μm, conidiophora ex mycelio basali oriunda, usque 175 μm longa, ad medium 3.5–6 μm crassa, laevia; vesiculae hemisphaericae vel ampulliformes, 7.5–12.5 μm diametro. Aspergilla in summa 2/3 vel 1/3 vesicula insidentia, biseriata; metulae griseo-brunneae 5–7 × 3.5–5 μm; phialides griseo-brunneae 5–9 × 2.5–3.5 μm. Conidia hyalina vel dilute griseo-viridia, globosa vel subglobosa, 3–4 μm, echinulata. Status teleomorphus: Emericellapachycristata.

Colonies on Czapek’s solution agar growing restrictedly, attaining a diameter of 20–21 mm in 14 days, floccose, consisting of a thin mycelial felt and loose aerial hyphae, ascomata and conidial heads few in number, orange white (5A2–6A2, after Kornerup and Wanscher 1978); reverse greyish orange (6B3) to brownish orange (6C5).

Colonies on malt agar growing restrictedly, attaining a diameter of 22–23 mm in 14 days, consisting of a dense basal mycelium and loose aerial hyphae, ascomata abundantly produced, conidial heads abundantly produced, pale orange (6A3) to greyish green (29D5) or deep green (30D8); reverse brownish orange (6C6) to brown (6E7).

At 37 °C, growth rate better than at 25 °C, and with increased production of ascomata. Ascomata, reddish purple to dark reddish brown, globose to subglobose, 210–320 μm in diameter, surrounded by hyaline to pale yellowish brown, globose to ovate, thick-walled hülle cells measuring 15–25 × 12.5–25 μm; peridium grayish yellow, membranaceous, consisting of a angular cells. Asci 8-spored, globose to subglobose or ovate, 9–11 × 8.5–10 μm, evanescent. Ascospores at first hyaline to pale yellowish brown, then becoming dull red to reddish brown at maturity, lenticular, spore bodies 4–5 × 3.5–4 μm, provided with two equatorial crests measuring 1.0 μm wide, with convex surfaces smooth.

Conidial heads grayish olive, radiate to short columnar, 35–75 × 30–40 μm. Conidiophores grayish brown to reddish brown, smooth, arising from the basal mycelium or aerial hyphae, up to 175 μm long, 3.5–6 μm diameter at the middle, vesicles grayish brown, hemispherical to flask shaped, 7.5–12.5 μm diameter with metulae covering the upper 2/3–1/3 of surfaces. Aspergilla biseriate; metulae grayish brown 5–7 × 3.5–5 μm; phialides grayish brown, 5–9 × 2.5–3.5 μm. Conidia hyaline to pale greenish gray, globose to subglobose, 3–4 μm diameter, echinulate.

Etymology. From Latin, pachy- = thick- and cristata = crest, referring to the ascospore ornamentation.

Specimen examined. IFM 55265 (holotype), a dried culture derived from an isolate of vineyard soil, Pichan (Shanshan), Pichan Prefecture, Xinjiang Uygur autonomous region, China, collected by Paride Abliz, isolated and developed by T. Yaguchi in the laboratory of the Department of Dermatology, Xinjiang Medical University, Urumuqi, as isolate strain No. E-100, August 2006. The living culture was deposited in NITE Biological Resource Center (NBRC) as NBRC 104790.

Discussion

Emericella species exhibit various ascospore phenotypes, although several species do not exhibit remarkable ascospore phenotypes (Fig. 6). In this study, we indicated that species having smooth-walled ascospores with two equatorial crests are polyphyletic groups. The typical species correspond to E. nidulans, E. sublata, E. fruticulosa, E. falconensis, E. spectabilis, E. foeniculicola, E. aurantiobrunnea, and E. astellata. Emericella nidulans has smooth-walled ascospores with two equatorial crests. However, E. sublata has ascospores with two broad equatorial crests and is distinguishable from E. nidulans based on the width of its equatorial crests (Horie 1979). Emericella spectabilis radically differs from E. nidulans with respect to conidiophore size, hülle cell prominence, and color in mass (vinaceous in E. spectabilis) (Christensen 1978). Emericella foeniculicola differs from E. nidulans with respect to anamorph morphology (Udagawa and Muroi 1979). Emericella aurantiobrunnea does not produce conidial heads until the culture becomes several weeks old; its conidial heads exhibit light to dull buff shades and fail to show any blue-green color (Raper and Fennell 1965). Emericella astellata has a characteristic ability to produce aflatoxin B1 and B2 (Frisvad et al. 2004). In addition to ascospore ornamentation, the morphological characteristics of anamorph and mycotoxin production are important characteristics to identify Emericella species. Moreover, the maximum growth temperatures of E. spectabilis, E. foeniculicola, and E. aurantiobrunnea were less than 40 °C, whereas E. nidulans grew in temperatures up to 48 °C. These differences were reflected in the analysis of molecular phylogenetics. Other species that have smooth-walled ascospores with two equatorial crests also differed from E. nidulans with respect to morphological and physiological characteristics other than ascospore ornamentation (Table 2).
https://static-content.springer.com/image/art%3A10.1007%2Fs10267-012-0188-x/MediaObjects/10267_2012_188_Fig6_HTML.jpg
Fig. 6

Comparison of the species of Emericella that have smooth-walled ascospores with two equatorial crests. aE. sublata IFM42029. bE. fruticulosa CBS 650.73. cE. falconensis CBM-FA-82. dE. aurantiobrunnea IMI 74897. eE. foeniculicola IFM 42201. fE. spectabilis CBS 429.77. Barsaf 5 µm

Table 2

Comparison of properties of the species that are related to Emericella pachycristata

Species

Convex wall

Colony color

Conidial head color

Conidiophorous size

Maximal growth temperature (°C)

E. nidulans

Smooth

Deep dull yellow green

Dark green

75–100 × 2.5–3 μm

48

E. sublata

Smooth

Dull brown to grayish olive

Grayish olive green to dull green

Up to 210 × 4–6 μm

48

E. rugulosa

Ribbed

Purple-gray to purple-brown

Green to dark green

50–150 × 4.5–7.5 μm

48

E. pachycristata

Smooth

Pale orange to greyish green or deep green

Grayish olive

Up to 175 × 3.5–6 μm

48

E. fruticulosa

Smooth

Grey green near pale lumiere green

Greyish green to olive yellow

40–60 × 2.2–4.4 μm

45

E. falconensis

Smooth

Orange to bright brown

Dull green

75–240 × 3–6 μm

45

E. spectabilis

Smooth

Vinaceous gray

Dark green

190–364 × 5.7–10.3 μm

<40

E. foeniculicola

Smooth

Pale vinaceous to purplish gray

Grayish green

20–160 × 3–5 μm

<40

E. aurantiobrunnea

Smooth

Cream to buff

Light to dull buff

Up to 250 × up to 8 μm

<40

Emericella pachycristata also has smooth-walled ascospores with two equatorial crests, and the maximum growth temperature is 48 °C. However, the phylogenetic position of E. pachycristata was close to that of E. rugulosa. Klich et al. (2001) reported a new sterigmatocystin-producing variant of Emericella (strain SSRC1398) that exhibits a growth rate on standardized media and Southern blots of genomic DNA similar to E. rugulosa; however, it produces smooth-walled ascospores. According to this report, this variant is likely to be E. pachycristata. The molecular phylogenetic relationship between E. rugulosa and E. pachycristata apparently supports these morphological and physiological characteristics (Figs. 1, 2, 3; clades IV and V).

We conducted multilocus sequencing analysis based on recent Aspergillus taxonomy and indicated the correlations among molecular phylogenetics, morphological data, and growth temperature. However, several species, particularly species belonging to clades I–III, were undistinguishable by phylogenetic analysis alone. Emericella dentata produces smooth-walled ascospores with two dentate equatorial crests (Raper and Fennell 1965). Although it belongs to clade I, with E. nidulans, these species have very different ascospore crests. In the phylogenetic trees based on the β-tubulin and actin genes, species that have ascospores with two broad equatorial crests (clade II) and four equatorial crests (clade III) formed the same clade. However, the species that belong to clades II and III exhibit substantially different morphological characteristics. Thus, it is very difficult to identify Emericella species by phylogenetic analysis alone.

Zalar et al. (2008) reported four new Emericella species. Among them is E. filifera, which forms ascospores with appendaged threads. They also discuss the similarity between the ascospores of E. filifera and E. appendiculata. In addition, E. appendiculata produces ascospores with appendaged threads. We reevaluated E. filifera according to phylogenetic analysis of sequence data and morphological characteristics, and found that E. filifera is identical to E. appendiculata. Therefore, we conclude that E. filifera is a synonym of E. appendiculata based on sequence data and ascospore ornamentation. Moreover, E. appendiculata (synonym: E. filifera) and E. qinqixianii are unique species that produce ascospores with appendaged threads.

In the morphological taxonomy of the genus Aspergillus and its teleomorphs, species are distinguished by phenotypic characteristics such as colony and microscopic characteristics of the conidia and ascospores. Colony diameter at 7 days after inoculation on standard media is another important characteristic (Klich et al. 2001). Emericella species have been mainly classified on the basis of ascospore ornamentation since the reevaluation performed by Horie. However, the results of the present study indicate that morphological characteristics other than ascospore ornamentation and physiological characteristics are important as well. Thus, to identify species in this genus, it is necessary to evaluate both phylogenetic analysis and morphological and physiological characteristics.

Acknowledgments

This work was supported in part by the National Bioresource Project “Pathogenic microbes in Japan” (http://www.nbrp.jp/) and a Grant-in-Aid for Scientific Research (B-18405005) from the Japan Society for the Promotion of Science.

Copyright information

© The Mycological Society of Japan and Springer 2012