Intra- and interspecific diversity of ultrastructural markers in Scedosporium

f u n g a l b i o l o g y 1 2 0 ( 2 0 1 6 ) 1 4 7 e1 5 4

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Intra- and interspecific diversity of ultrastructural markers in Scedosporium Amaliya A. STEPANOVAa,*, G. Sybren DE HOOGb, Nataliya V. VASILYEVAa a

Kashkin Research Institute of Medical Mycology, I.I. Mechnikov North-Western State Medical University, Saint Petersburg 194291, Russia b CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

article info

abstract

Article history:

Ultrastructural features of conidia, lateral walls of aerial and submerged hyphal cells, and

Received 11 June 2015

of septal pore apparatus of Scedosporium apiospermum, S. boydii, Pseudallescheria angusta and

Received in revised form

Scedosporium aurantiacum were studied. Submerged hyphal cells possessed a thick extracel-

29 July 2015

lular matrix. Crystalline satellites accessory to the septal pore apparatus were revealed.

Accepted 4 August 2015

Fundamental ultrastructural features appeared to be heterogeneous at low taxonomic

Available online 12 August 2015

levels. The closely interrelated members of the S. apiospermum complex showed quantita-

Corresponding Editor:

tive ultrastructural variability, but the unambiguously different species S. aurantiacum de-

Sybren De Hoog

viated qualitatively by markers of conidial wall structure, Woronin bodies morphology and presence/absence of crystalline satellites of the septal pore apparatus.

Keywords:

ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Conidial wall Crystalline satellites Pseudallescheria Scedosporium Septal pore apparatus Woronin bodies

Introduction Ultrastructure of conidial wall relations and septal pore apparatus hyphal cells differ significantly between major groups of fungi and have extensively been used as primary characters to define higher taxa (Suh & Sugiyama 1993; Ho & Hyde 2004; Healy et al. 2013). In recent decades the role of ultrastructural markers has largely been taken over by molecular phylogeny. Quite significantly, however and in contrast to phenotypic characters such as microscopic morphology or physiology, the taxonomic value of Transmission Electron Microscopy (TEM) studies has never been falsified by more recent approaches. Although neglected, they are still useful as markers

of diversity, supporting main traits in phylogeny (Moore 1996). On the other hand, molecular methods have led to more precision at all taxonomic levels, and corrected the taxonomy e.g. of groups that appeared to be dramatically polyphyletic, such as Sporothrix (de Beer et al. 2006). Therefore it may be useful to reconsider the validity and diagnostic power of TEM. In the present study we will focus on lower taxonomic levels, to establish variance and reproducibility of selected TEM markers. This is done with members of Scedosporium, a genus of common opportunists causing a wide spectrum of diseases in humans (Cortez et al. 2008; de Hoog et al. 2014). The name Pseudallescheria has been in use for the sexual state of this group of fungi, but this has been replaced by Scedosporium for the

* Corresponding author. E-mail address: [email protected] (A. A. Stepanova). http://dx.doi.org/10.1016/j.funbio.2015.08.004 1878-6146/ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

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Fig 1 e Scanning (a, b, leq) and transmission (cek, rew) electron microscopy of S. apiospermum (aek), S. aurantiacum (leq, ret), S. boydii (u, w) and P. angusta (v). aei. RCPFF 1491/1057; j, k. RCPFF 1490/712; m. CBS 116910; l, n, p, q. CBS 136046; ret. CBS 136047; u. CBS 117432; v e CBS 254.72; w e CBS 301.79. Scale bars: a, b, leq [ 1 mm; c, f, g, iek [ 0.5 mm; d, e, h, ret, v, w [ 0.3 mm; u [ 1.5 mm. Abbreviations used: C [ conidium, CC [ conidiogenous cell, CS [ crystalline satellite, CW [ cell wall, ec [ ectosporium, EM [ extracellular matrix, en [ endosporium, ep [ episporium, ex [ exosporium, GP [ germ pore, S [ septum; WB [ Woronin body(ies), ze [ zone(s) of exclusion.

holomorph (Lackner et al. 2014) for most species except for those of doubtful identity, such as Pseudallescheria angusta. Limited data on TEM of Scedosporium and related fungi in Microascaceae are as yet available in the literature. Certain aspects of conidiogenesis have been studied in Scedosporium apiospermum, Scedosporium boydii, Lomentospora inflata and

Scopulariopsis brumptii (Campbell & Smith 1982; Dykstra et al. 1989; Huang et al. 1990). The aim of the present work was to compare more fundamental criteria which have been in use at higher taxonomic levels, i.e. mature conidial walls, lateral cell walls, septa and septal pore apparatus. As a model set we used strains of the closely interrelated taxa of the S.

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Table 1 e Mean values of mature conidial wall and its layers in S. apiospermum and S. aurantiacum, in mm. Strains

Endosporium

Episporium

Ectosporium

Ectosporium þ episporium

Total

0.28 0.27

0.06 0.08

0.03 0.05

0.09 0.13

0.37 0.40

0.31 0.29 0.11 0.17

0.07 0.04 0.03 0.03

0.07 0.01 0.03 0.01

e e e e

0.45 0.34 0.17 0.21

S. apiospermum RCPFF-1491/1057 RCPFF-1490/712 S. auranticum CBS 116910 CBS 136046 CBS 136047 CBS 136049

apiospermum complex: S. boydii, S. apiospermum and the intermediate cluster of P. angusta (Chen et al. 2016), plus the unambiguously deviating species S. aurantiacum. In the S. apiospermum complex sexual states are known, while S. aurantiacum as yet is treated as being asexual.

Materials and methods Two strains of Scedosporium apiospermum (RCPFF 1491/1057, RCPFF 490/712), four strains of Scedosporium boydii (CBS 117410, CBS 117432, CBS 120157, CBS 301.79), one of Pseudallescheria angusta (CBS 254.72), and four of Scedosporium aurantiacum (CBS 116910, CBS 136046, CBS 136047, CBS 136049). Strains were taken from the Russian Collection of Pathogenic Fungi (RCPFF: St. Petersburg, Russia) and the Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre (CBS: Utrecht, The Netherlands) and were verified by rDNA ITS sequencing. Isolates were cultured on Potato Dextrose Agar (PDA) at 28
C during 7 and 20 d. For Scanning Electron Microscopy (SEM) samples were fixed in 3 % glutaraldehyde, then dehydrated by an ethanol series (30
/70
), critical-point dried (HCP-2) for 15 min, coated with gold and observed under a JSM 35 instrument (Jeol, Tokyo, Japan). For Transmission Electron Microscopy (TEM) blocks of nutrient medium with parts of fungal colonies were fixed during the 3 h in 3 % glutaraldehyde and post-

fixed for 10 h in 1 % osmium tetroxide. Subsequently samples were dehydrated through an ethanol and acetone series and embedded in epon-araldite epoxy resin. Prior to TEM observation, light microscopic investigations of semithin epoxy sections (3e5 mm) were performed, cut by Pyramitome 1180 (LKB, Bromma, Sweden) using glass knives and stained with toluidine blue. Ultrathin sections were cut with an Ultratome 2088 (LKB, Bromma, Sweden), stained with uranyl acetate and lead citrate and examined under a JEM-100 CX II transmission electron microscope (Jeol, Tokyo, Japan). For each strain 40e50 median sections of the septal pore apparatus were observed. Numbers of Woronin bodies and crystalline satellites in the septal pore apparatus of each strain were calculated from serial median ultrathin sections.

Results Mature conidia of Scedosporium apiospermum under SEM were clavate in shape, with narrow base and smooth surface (Fig 1a) in all strains studied. Conidiogenous cells occasionally showed annellidic rings remaining after conidium production (Fig 1b, arrow). Under TEM the conidial initial possessed a thin (0.03e0.05 mm dependent in the strain; Table 1) wall (primary cell wall or ectosporium, Figs 1c, arrow, 4a). During conidial maturation a secondary cell wall (endosporium) developed under the ectosporium (Figs 1d, 4b). Later the upper part of

Fig 2 e Diagram of lateral wall structure in mature cells of aerial (A) and submerged (B) hyphae. a, b. RCPFF 1491/1057, RCPFF 490/712, CBS 117410, CBS 117432, CBS 120157, CBS 301.79, CBS 254.72, CBS 116910, CBS 136046, CBS 136047, CBS 136049. Abbreviations usedLCW [ lateral cell wall, EM [ extracellular matrix, PL [ plasmalemma.

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Fig 3 e Ultrastructure of septal pore apparatus of hyphal cells of S. apiospermum (a), S. aurantiacum (b, c, g), S. boydii (d, e, h, i, j, k, l, n), P. angusta (f, m). a. RCPFF 1490/1057; b, c, g. CBS 136046; d. CBS 120157; e, n. CBS 117432; f. CBS 301.79; h, m. CBS 254.72; i, j. CBS 117432; k, l. CBS 301.79. Scale bars: aem [ 0.2 mm. Abbreviations used: CI [ crystalline inclusion; CS [ crystalline satellite, D [ diaphragm, P [ plug, S [ septum; SP [ septal pore; WB [ Woronin body(ies).

the endosporium darkened leading to the development of an electron-dense layer (episporium) (Figs 1e, 4c, d). Thus two layers were present in the mature conidial wall of this species (Fig 4e): a thin, dark upper layer (ecto- þ episporium) and a thicker, electron-transparent lower layer (endosporium) with thin microfibrils, which were more densely compacted in the upper half of the endosporium. The dark upper layer

was hypothesized to contain melanin, determining the brown colour of mature conidia. No differences were revealed between mean values of dimensions of mature conidial walls among strains of S. apiospermum (Table 1). Several consecutive stages of germ pore formation were demonstrated near the base of mature conidia (Fig 1feh). After separation of mature conidia from senescent conidiogenous cells, remains of the

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151

(Fig 1a, arrow) and in Scedosporium aurantiacum (Fig 1l, arrows), demonstrating that they represent folds (Fig 11, k) in the cell wall. Very often conidiogenous cells without annellidic rings were revealed in both species (Fig 1i, j). Mature conidia of S. aurantiacum under SEM were obovoidal with a narrow base (Fig 1m, n) and initially had smooth surfaces (Fig 1l). During maturation and desiccation, a finely granular structure appeared (Fig 1meq). Lateral (Fig 1p) and apical (Fig 1q) sides of conidial walls of strain CBS 136046 showed circular ‘zones of exclusion’, i.e. without elements of ornamentation (Fig 4h). Under TEM the early stages of conidial development were similar with S. apiospermum (Fig 4aed). In young, smooth-walled conidia, two wall layers were visible: (a) an upper, thin electron-dense, homogenous dark layer (ectosporium), and (b) a lower, thick, electron-transparent layer (endosporium, Fig 1r). During and after desiccation the thin (0.01e0.07 mm dependence on the strain, Table 1) ectosporium locally separated from the lower layer (Fig 1s), then disintegrated (Fig 4f) and fully disappeared in mature conidia by a process of stripping the conidial wall (Figs 1t, 4g, h). An early stage of ectosporium separation from its supporting layer is visible in Fig 1s. An exosporium developed between this layer and the episporium (Fig 1s). The mature conidial wall thus was composed of three layers (Figs 1t, 4g, h): (1) a thin, dark exosporium with longitudinally oriented short microfibrils determining conidial surface finely granular ornamentation; (2) a median layer (episporium), which was similar in thickness in strains CBS 116910, CBS 136047, and with a thicker, dark exosporium in strains CBS 136046 and CBS 136049; (3) a light endosporium with low contrast and randomly distributed microfibrils. Variability in these parameters among S. aurantiacum strains is shown in Table 1. Thickness of lateral walls of aerial and submerged hyphal cells (which exceptionally were conidiogenous cells) was similar in all investigated species (Table 2) and varied between 0.04 and 0.06 mm. Slightly more variability was observed in

Fig 4 e Diagram of conidial wall morphogenesis in S. apiospermum (aee) and S. aurantiacum (aed, f, g, h). aee. RCPFF 1491/1057, 1490/712; aef, g. CBS 116910, 136047, 136049; aef, h. CBS 136046. Abbreviations used: ec [ ectosporium, en [ endosporium, ep [ episporium, ex [ exosporium, s [ septum, ze [ zone of exclusion.

ectosporium were visible near the germ pore (Fig 1g, h). Conidia were formed sessile alongside hyphal cells (Fig 1i) or on short protrusions (Fig 1j). Annellidic rings along the apical part of conidiogenous cells were observed in S. apiospermum

Table 2 e The quantitative and qualitative parameters of lateral cell walls, septa and components of septal pore apparatus in the cells of vegetative mycelium of S. apiospermum, S. boydii, P. angusta and S. aurantiacum. Strains

S. apiospermum RCPFF-1491/1057 RCPFF-1490/712 S. boydii CBS 117410 CBS 117432 CBS 120157 CBS 301.79 P. angusta CBS 254.72 S. aurantiacum CBS 116910 CBS 136046 CBS 136047 CBS 136049

Lateral Extra-cellular Septum, Septal Woronin Number of Crystalline Number of Presence/absence cell matrix, mm mm pore, bodies, Woronin bodies satellites, crystalline of shapeless walls, mm mm mm (min/max) mm satellites crystallic (min/max) components in septal pore 0.05 0.05

0.20 0.18

0.05 0.05

0.15 0.16

0.17 0.17

1e3 1e3

0.13 0.14

1e2 1e2

Absent Absent

0.04 0.06 0.06 0.05

0.30 0.33 0.30 0.31

0.04 0.04 0.05 0.06

0.12 0.16 0.15 0.13

0.17 0.20 0.22 0.22  0.19

2e3 2e3 2e3 2e4

0.13 0.15 0.16 0.15

1e2 1e2 1e2 1e5

þ þ Absent Absent

0.05

0.32

0.05

0.11

0.22  0.19

2e4

0.13

1e4

Absent

0.05 0.06 0.05 0.05

0.30 0.33 0.32 0.34

0.06 0.06 0.06 0.05

0.14 0.17 0.15 0.16

0.22 0.20 0.20 0.21

   

2e4 3e7 2e4 2e4

Absent Absent Absent Absent

Absent Absent Absent Absent

Absent Absent Absent Absent

0.16 0.18 0.19 0.18

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this parameter among stains of S. boydii. Lateral cell walls were thin, electron-transparent (Fig 1u), composed of randomly distributed microfibrils (Fig 2A, B). Cells of the submerged mycelium were covered on the outside with an extracellular matrix (Figs 1u, 2B), which was composed of tightly and randomly compacted, dark microfibrils. Significant variation in thickness was noted (Table 2): thickness of the extracellular layer was four times more than the cell wall in cells of S. apiospermum, but 5e7.7 times thicker in Scedosporium boydii, 6.4 times in Pseudallescheria angusta, and 6e7 times in S. aurantiacum.

Fig 5 e Diagrammatic overview of septal pore components in hyphal cells of S. apiospermum (a), S. boydii (bed), P. angusta (e), and S. aurantiacum (f, g). a. RCPFF 1491/1057, 1490/ 712; b. CBS 120157, 301.79; c. CBS 117410, 117432; d. CBS 301.79; e. CBS 254.72; f. CBS 116910, 136047, 136049; g. CBS 136046. Abbreviations: CI [ crystalline inclusion; CS [ crystalline satellite, LCW [ lateral cell wall, D [ diaphragm, S [ septum; SP [ septal pore, WB [ Woronin body.

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Hyphal cells practically showed no difference in thickness of septa (Table 2). The diameter of the central septal pore was relatively small in S. boydii and P. angusta. Septa were light and straight (Figs 1v, w, 3a, b, d, f, h, k, l, 5a‒g). The septal pore apparatus in hyphal cells of S. apiospermum (Figs 3a, 5a), S. boydii (Figs 3d, e, i, j, 5b‒d) and P. angusta (Figs 3f, m, 5e) was composed of three elements: Woronin bodies, crystalline satellites, and pore plugs. A distinctive peculiarity in the septal pore apparatus of S. aurantiacum was the absence crystalline satellites (Figs 3a, c, 5f, g). The septal pore apparatus of two S. boydii strains (CBS 117410, CBS 117432; Figs 3i, j, 5c) compared to the remaining strains (CBS 120157, CBS 301.79, 5b) possessed larger, irregular crystalline inclusions, which were localized near septa or inside the septal pore. A distinct crystalline structure of the latter type was visible in several sections. Woronin bodies are membrane-bound components of the septal pore apparatus and are distinct from crystalline satellites in having an outer membrane. The septal pore apparatus of S. apiospermum typically had spherical Woronin bodies (Figs 3a, 5a) with a mean diameter 0.17 mm (Table 2). In contrast, those of S. aurantiacum were larger (Table 2) and were ellipsoidal in shape (Figs 3b, c, g, 5f, g). No significant size differences we revealed within the species. The number of Woronin bodies varied from 1 to 3 in S. apiospermum, from 2 to 4 in S. boydii and P. angusta, and from 2 to 7 in S. aurantiacum. The latter high value was particularly caused by strain CBS 136046, where the number of Woronin bodies varied from 3 to 7. Ellipsoidal and relatively large Woronin bodies were further typically present in P. angusta, in one strain (CBS 301.79) of S. boydii (Table 2, Fig 5d). The diameter of Woronin bodies in all hyphal cells was larger than that of the septal pore (Table 2) and thus the bodies effectively close the septal pore between adjacent cells of growing hyphae at onset of senescence and they are also involved in the regulation of transport of organelles and cytosol in growing and mature cells. In intact cells, mostly they are situated at one (Figs 1v, 3n) but sometimes also at both sides of the septal pore (Fig 3l) allowing cytoplasmic flow between adjacent cells. In addition to Woronin bodies, the presence of the spherical, median-electron dense crystalline satellites (Figs 3a, d, e, f, h, i, m, 5aee) was revealed in S. apiospermum, S. boydii, and P. angusta, with diameters close to that of Woronin bodies (Table 2). The number of these elements varied from 1 to 2 in S. apiospermum (Figs 3a, 5a, Table 2) as well as in three strains (CBS 117410, CBS 117432, CBS 120157) of S. boydii. In one strain (CBS 301.79) of the latter species up to five satellites were seen (Fig 5d, Table 2). In P. angusta the number varied from 1 to 4 (Figs 3f, m, 5e, Table 2). Woronin bodies and crystalline satellites proved to be very dynamic. On median sections of a single septal pore apparatus, Woronin bodies were typically located at identical distance to septa, to other Woronin bodies, and to crystalline satellites (Figs 1w, 3aed, f, h, k, m, 5aeg). Septal plugs were frequent components of the pore apparatus of mature hyphal cells. They were dark, homogeneous (Fig 3f, h), variable in size and shape (spherical, irregular, rectangular, or dumbbell-shaped) on median sections, symmetrical or asymmetrical, and might partially or completely fill the septal pore. A thin, dark, hemispherical diaphragm was

Ultrastructural markers in Scedosporium

observed in only a single strain (CBS 136046) of S. aurantiacum (Figs 3b, 5g).

Discussion Mature conidia of Scedosporium apiospermum have smoothwalled conidia, as found earlier by Campbell & Smith (1982) using SEM of mature conidia of the same species. Conidia of Scedosporium aurantiacum differ significantly differ by the presence of a finely granular structure. One strain (CBS 136046) of S. aurantiacum differed under SEM from remaining strains by the occasional presence of rare circular, smooth areas in the conidial outer surface texture. Additional differences in the mature conidial structure between these two species were observed. The mature conidial wall in S. apiospermum consisted of three layers: an ectosporium, an episporium, and an endosporium. Differences between ectosporium and episporium were very difficult to recognize in TEM images, because they had similar electron-density and were positioned in tight contact. Early stages of conidium development in S. aurantiacum were comparable to those of S. apiospermum, leading to the development of ectosporium, episporium, and endosporium. However, later the ectosporium was stripped off was locally loosened, revealing a fourth, very thin layer, an exosporium, formed in upper part of the episporium. After loss of the ectosporium the mature conidial wall consisted of three layers as in S. apiospermum, but these now are exosporium, episporium and endosporium. Thus, the investigated species differ in the patterns of conidial wall morphogenesis, in the fate of the ectosporium, and in the presence of an exosporium. Conidiogenesis in Scedosporium is annellidic, but our data demonstrate holoblastic conidia, as noted by Hironaga & Watanabe (1980). In early stages of development conidia of S. aurantiacum are also holoblastic, but wall relations of mature conidia a more complex, with an ornamented exosporium and with loss of ectosporium. The annellations visible in the apical part of conidiogenous cells are interpreted as folds of the cell walls. They are also demonstrated in TEM images of Scedosporium boydii by Campbell & Smith (1982) and in S. apiospermum by Dykstra et al. (1989). These authors noted that conidiogenesis in Scedosporium did not clearly fit in the bipartition annellidic vs. sympodial. The absence of remains of disruptive scars in Scedosporium suggests a high degree of cell wall plasticity at the moment of inflation of the conidial bud. Thickness and structure of hyphal cell walls and extracellular matrix, and structure of septa were comparable in all species investigated. Presence of a thick, outer extracellular matrix is also known in vegetative cells of Aspergillus fumigatus (Stepanova et al. 2004, 2013; Loussert et al. 2009; Muller et al. 2011; Stepanova & Sinitskaya 2012; Muszkieta et al. 2013) and other Aspergillus species. The layer enhances its adhesive capacity and biofilm formation (Muller et al. 2011). The matrix may also protect the fungus against host defence reactions or antifungal drugs (Muszkieta et al. 2013). Similar functions may be hypothesized for Scedosporium. An additional function might be a compartment for concentration and localization of metabolites and enzymes. The layer contains galactomannans, a-1,3-glucans, monosaccharides, polyols, melanin

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and proteins, including major antigens and hydrophobins (Muller et al. 2011). Melanin is of significance in host tissue invasion. In A. fumigatus, this layer starts concomitant with early stages of conidial growth (Stepanova & Sinitskaya 2012) and increases with maturation (Stepanova et al. 2004) and in infected host tissue (Stepanova et al. 2013). Strains within the S. apiospermum complex differed in size, shape and number of Woronin bodies and crystalline satellites. Strain CBS 301.79 contained significantly more of both septal pore components. Hyphal cells of S. apiospermum, S. boydii and Pseudallescheria angusta had a more complex septal pore apparatus than S. aurantiacum, and contained additional crystalline satellites. In contrast, the septal pore apparatus of S. aurantiacum differed from that of S. apiospermum by the presence more numerous (up to 4e7 on median section), large, ellipsoidal Woronin bodies. In one strain of S. aurantiacum, CBS 136046, they were much more numerous (Fig 5g); in this strain also a diaphragm was visible in the septal pore apparatus, and it also had circular smooth areas (‘zones of exclusion’) on the conidial walls. Despite these differences, its rDNA ITS genotype was identical. A peculiarity found in the septal pore apparatus of two S. boydii strains (CBS 117410, 117432) was the presence of large, irregular crystalline inclusions are these the same as the crystalline satellites in the septal pore. In ascomycetous fungi, e.g. Ascodemis sphaerospora (Bremmer & Carroll 1968), Fusarium oxysporum f. sp. lycopersici (Wergin 1973), Peziza badia and Sarcoscypha coccinea (Kamaletdinova & Vasilyev 1982), Aspergillus nidulans (Momamy et al. 2002), A. fumigatus (Stepanova & Sinitskaya 2012), Woronin bodies are known to originate from microbodies via exocytosis-like activity. In contrast, crystalline satellites, which are topographically similar to Woronin bodies, were stripped from outer membranes, so that we suppose that they originate from directly in the cytosol. Perhaps, crystalline satellites present the ‘primitive’ Woronin bodies and, in S. apiospermum, S. boydii and P. angusta present ‘primitive’ and advanced types of taxonomically important components of septal pore apparatus. Investigated strains of S. boydii (CBS 301.79) and P. angusta (CBS 254.72) contained Woronin bodies that were comparable in number, size and shape and they possessed crystalline satellites that were identical in morphology. P. angusta (CBS 254.72) is possibly a self-sporulating strain of S. boydii or S. apiospermum (Lackner et al. 2014). The ultrastructure of septal pore apparatus is generally taken to represent be very stable and highly conserved; the variation noted between these members of the S. apiospermum complex is quantitative. In contrast, one strain of S. aurantiacum (CBS 136046) differed qualitatively in characters of conidial wall and septal pore apparatus (compare Table 2). Plugs were often revealed in the septal pore of mature and senescent hyphal cells of all investigated species. They are widely distributed components of septal pore apparatus in filamentous fungi (Kamaletdinova & Vasilyev 1982, Kimbrough 1994, Stepanova & Vasilyev 1994, Stepanova et al. 2004, 2013). They may also participate in regulation of flow and distribution of cytosol and cellular organelles flow between the adjacent cells, having similar functions as Woronin bodies. Our data demonstrate that significant variation can be observed in ultrastructural characters which usually are regarded as showing heterogeneity at higher taxonomic levels

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only. Most variation in TEM characteristics within species or species complexes was of quantitative nature, while most qualitative differences are found between S. aurantiacum on one hand and the S. apiospermum complex on the other. While S. apiospermum, S. boydii and P. angusta form a closely interrelated species complex e or perhaps even a single, variable species e S. aurantiacum is unambiguously different (Chen et al. 2016). Strain CBS 301.79 is the type strain of P. ellipsoidea but this species is regarded as synonym of S. boydii. Pseudallescheria angusta is intermediate between S. apiospermum and S. boydii (Lackner et al. 2014) and forms occasional recombinants (Chen et al. 2016). The difference between this complex and the next, unambiguously different species S. aurantiacum is underlined by the absence of crystalline satellites in the latter species. In main traits, ultrastructural data of mature conidial wall and septal pore apparatus structure summarized in Table 2 correspond with molecular-genetic data (Lackner et al. 2014; Chen et al. 2016). The consistent absence of crystalline satellites in S. aurantiacum is a significant difference between S. aurantiatum and the S. apiospermum complex, underlining the need for barcodes at the level of recognized species, while the separation of members of the S. apiospermum complex seems redundant.

Acknowledgement We are indebted to Somayeh Sharifynia for re-establishing the molecular identity of strains analyzed and to the Head of Russian Collection of Pathogenic Fungi Chilina, Galina Anastasievna, for providing Scedosporium apiospermum cultures.

references

Bremmer DM, Carroll GC, 1968. Fine-structural correlates of growth in hyphae of Ascodesmis sphaerospora. Journal of Bacteriology 95: 658e671. Campbell CK, Smith MD, 1982. Conidiogenesis in Petriellidium boydii (Pseudallescheria boydii). Mycopathologia 78: 145e150. Chen M, Zeng JS, de Hoog GS, Stielow B, Gerrits van den Ende AHG, Liao W, Lackner M, 2016. The concept of ‘species complex’ illustrated by the Scedosporium apiospermum complex. Fungal Biology 120 (2): 137e146. Cortez K, Roilides E, Quiroz-Telles F, Meletiadis J, Antachopoulos C, Knudsen T, Buchanan W, Milanovich J, Sutton DA, Fothergill A, Rinaldi MG, Shea YR, Zaoutis T, Kottilil S, Walsh J, 2008. Infections caused by Scedosporium spp. Clinical Microbiological Reviews 21: 157e197. de Beer ZW, Begerow D, Bauer R, Pegg GS, Crous PW, Wingfield MJ, 2006. Phylogeny of the Quambalariaceae fam. nov., including important Eucalyptus pathogens in South Africa and Australia. Studies in Mycology 55: 289e298.
J, Figueras MJ, 2014. Atlas of Clinical de Hoog GS, Guarro J, Gene Fungi (online version). CBS-KNAW Fungal Biodiversity Centre, Utrecht.

A. A. Stepanova et al.

Dykstra M, Salkin IR, McGinnis MR, 1989. Ultrastructural comparison of conidiogenesis in Scedosporium apiospermum, Scedosporium inflatum and Scopulariopsis brumptii. Mycologia 86: 896e904. Healy RA, Kumar TK, Hewitt DA, McLaughlin DJ, 2013. Functional and phylogenetic implications of septal pore ultrastructure in the ascoma of Neolecta vitellina. Mycologia 105: 802e813. Hironaga M, Watanabe S, 1980. Annellated conidiogenous cells in Petriellidium boydii (Scedosporium apiospermum). Sabouraudia 18: 261e268 Mycologia 105: 802813. Ho WH, Hyde KD, 2004. A new type of conidial septal pore in fungi. Fungal Diversity 15: 171e186. Huang HJ, Zhu JY, Zhang YH, 1990. The first case of Pseudallescheria boydii meningitis in China e electron microscopic study and antigenicity of the agent. Journal of the Tongii Medical University 10: 218e221. Kamaletdinova FI, Vasilyev AE, 1982. Cytology of Discomycetes. Alma-Ata: Publishing House “Nauka” of Kazakh SSR. Kimbrough JW, 1994. Septal ultrastructure and ascomycete systematics. In: Hawksworth DL (ed.), Ascomycete Systematics: problems and perspectives in the nineties. Plenum Press, New York, NY, pp. 127e141. Lackner M, Hagen F, Meis JF, Gerrits van den Ende AHG, Vu D, Robert V, Fritz J, Moussa TAA, de Hoog GS, 2014. Susceptibility and diversity in the therapy-refractory genus Scedosporium. Antimicrobial Agents and Chemotherapy 58: 5877e5885. Loussert S, Schmitt S, Prevost M-C, Balloy V, Fadel E, Philippe B,
JP, Beauvais A, 2009. In vivo bioKauffmann-Lacroix C, Latge film composition of Aspergillus fumigatus. Cellular Microbiology 12: 405e410. Momamy M, Richardson EA, van Sickle C, Jedd C, 2002. Mapping Woronon body position in Aspergillus nidulans. Mycologia 94: 260e266. Moore RT, 1996. The dolipore/parenthesome septum in modern taxonomy. In: Sneh B, Jabaji-Hare S, Neate SM, Dijst G (eds), Rhizoctonia Species: taxonomy, molecular biology, ecology, pathology and disease control. Springer, Heidelberg, pp. 13e35. Muller F-M, Seidler M, Beauvais A, 2011. Aspergillus fumigatus biofilms in the clinical setting. Medical Mycology 49: 96e100. € htz V, Gibbons JG, Leberre VA, Beau R, Muszkieta L, Beauvais A, Pa Shibuya K, Rokas A, Francois JM, Kniemeyer O, Brakhage AA,
JP, 2013. Investigation of Aspergillus fumigatus formation Latge by various “omics” approaches. Frontiers in Microbiology 4: 1e15. Stepanova AA, Bosak IA, Sinitskaya IA, 2013. Cytological investigations of Aspergillus fumigatus Fres. in murine lung. Problems in Medical Mycology 15: 52e58. Stepanova AA, Sinitskaya IA, Avdeenko YL, 2004. Submicroscopic study of the cells of vegetative mycelium of Aspergillus fumigatus Fres., Problems in Medical Mycology 6: 34e40. Stepanova AA, Sinitskaya IA, 2012. Cytological investigations of Aspergillus fumigatus Fres. germinating conidia. Problems in Medical Mycology 14: 43e53. Stepanova AA, Vasilyev AE, 1994. Ultrastructural Bases of Mushroom Morphogenesis. Ylym Publishing House, Ashghabat. Suh SO, Sugiyama J, 1993. Septal pore ultrastructure of Leucosporidium larimarini, a basidiomycetous yeast, and its taxonomic implications. Journal of General and Applied Microbiology 39: 257e260. Wergin WP, 1973. Development of Woronin bodies from microbodies in Fusarium oxysporum f. sp. lycopersici. Protoplasma 76: 249e260.