Cladobotryum fungicola ATHUM 6855. (a) colony obverse; (b) colony reverse; (c,d) conidiophores and conidiogenous cells with the characteristic zig-zag apical part (arrows), (c) in phloxine B, (d) in Melzer's; (e-h) diverse conidia, (e,f) typical conidia, and (g,h) conidia with central constriction in Melzer's. Scale bars: 50 µm = (c); 10 µm = (d-h).

... sample preparation step for the 1 H NMR spectroscopy required the re-dissolution of the extracts in deuterated methanol (MeOD) containing tetramethylsilane (TMS) as an internal standard for the calibration of the chemical shift axis. The protocol is depicted in Figure S1 (Supplementary Materials). ...

... The micromorphological characters of our strain fit perfectly with the original description of the anamorphic state [11], where the species is described as Sibirina fungicola. The long, zigzag-shaped apical part of conidiogenous cells is prominent and characteristic (Figure 1c,d). They are vaguely illustrated by Põldmaa and Samuels [31], though they are not mentioned in the text or in the original description. ...

... Qm 3.2; mainly two-celled, rarely three-or four-celled; in slide culture narrowly ellipsoidal to cylindrical or bacilliform; (23)26-33(41) × (4.5)6-7(8.5) µm, Qm 4.8; mainly two-celled, rarely three-celled; hyaline, smooth; basal apiculi central or slightly eccentric (Figure 9f-i Mycelium: cottony to compactly felt-like; white at first, then buff, progressing towards pinkish hues; sporadically white aerial loose tufts; symmetrically spreading, with irregular margin (Figure 10a). Growth rate: moderate. ...

... rate: moderate. Colony reverse: buff, progressively turning from terracotta to various red hues with brownish and purplish undertones (Figure 10b). Conidiophores: very long, unbranched to sparsely branched in a unilateral manner at conidiophore apices (Figure 10c-f). ...

... reverse: buff, progressively turning from terracotta to various red hues with brownish and purplish undertones (Figure 10b). Conidiophores: very long, unbranched to sparsely branched in a unilateral manner at conidiophore apices (Figure 10c-f). Conidiogenous cells: elongated, narrow-cylindrical, subulate, with obtuse apices; arranged in verticils 1-4(5) per verticil, mainly 3-4 at the apex; aseptate, rarely septate; hyaline (Figure 10c-f). ...

... very long, unbranched to sparsely branched in a unilateral manner at conidiophore apices (Figure 10c-f). Conidiogenous cells: elongated, narrow-cylindrical, subulate, with obtuse apices; arranged in verticils 1-4(5) per verticil, mainly 3-4 at the apex; aseptate, rarely septate; hyaline (Figure 10c-f). Conidiogenesis: blastic; a single to multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 6 conidia, mainly 3, in a wreath-shaped arrangement (Figure 10c-f). ...

... cells: elongated, narrow-cylindrical, subulate, with obtuse apices; arranged in verticils 1-4(5) per verticil, mainly 3-4 at the apex; aseptate, rarely septate; hyaline (Figure 10c-f). Conidiogenesis: blastic; a single to multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 6 conidia, mainly 3, in a wreath-shaped arrangement (Figure 10c-f). Conidia: in petri dish culture ovoid, mainly ellipsoidal to cylindrical; (15.5)19-26(28) × 6-10 µm Qm 2.8; sometimes slightly swollen at base and constricted in septal area; mainly two-celled, also three-celled, but rarely fourcelled; hyaline, smooth; many germinated; quite often in maturity disintegration of one cell (out of the two) or separation of the two cells; many variably connected in twos or threes at their base or side, rarely forming short chains; possible secondary production of conidia from germinated cell of conidium; in slide culture fusiform to narrowly ellipsoidal to cylindrical; (14)17-23(30) × 5-8 µm, Qm 3.2; mainly two-celled. ...

... in petri dish culture ovoid, mainly ellipsoidal to cylindrical; (15.5)19-26(28) × 6-10 µm Qm 2.8; sometimes slightly swollen at base and constricted in septal area; mainly two-celled, also three-celled, but rarely fourcelled; hyaline, smooth; many germinated; quite often in maturity disintegration of one cell (out of the two) or separation of the two cells; many variably connected in twos or threes at their base or side, rarely forming short chains; possible secondary production of conidia from germinated cell of conidium; in slide culture fusiform to narrowly ellipsoidal to cylindrical; (14)17-23(30) × 5-8 µm, Qm 3.2; mainly two-celled. (Figure 10f-k). Sclerotioid structures: present, mainly as chains of globose to ellipsoidal cells, thick-walled. ...

... cottony to loosely felt-like; whitish, buff, progressing towards pale ochre to pinkish hues; when mature, secondary whitish cottony at centre (Figure 11a). KOH reaction: positive. ...

... rate: relatively high. Colony reverse: buff, progressively turning from ochre to orange and red hues with pinkish undertones (Figure 11b). Conidiophores: very long, delicate, unbranched or sparsely branched in a unilateral manner at conidiophore apices or solitary, shorter, emerging orthogonally from very long hyphae; septate; hyaline (Figure 11c-e). ...

... reverse: buff, progressively turning from ochre to orange and red hues with pinkish undertones (Figure 11b). Conidiophores: very long, delicate, unbranched or sparsely branched in a unilateral manner at conidiophore apices or solitary, shorter, emerging orthogonally from very long hyphae; septate; hyaline (Figure 11c-e). Conidiogenous cells: elongated, delicate, narrow-cylindrical to almost filiform, subulate, with obtuse apices; arranged in verticils, 1-4(5) per verticil, mainly 3-4 at the apex, sometimes emerging orthogonally to conidiophore axis or in a dichotomous manner at the apex; aseptate, sometimes septate; hyaline (Figure 11c-e). ...

... very long, delicate, unbranched or sparsely branched in a unilateral manner at conidiophore apices or solitary, shorter, emerging orthogonally from very long hyphae; septate; hyaline (Figure 11c-e). Conidiogenous cells: elongated, delicate, narrow-cylindrical to almost filiform, subulate, with obtuse apices; arranged in verticils, 1-4(5) per verticil, mainly 3-4 at the apex, sometimes emerging orthogonally to conidiophore axis or in a dichotomous manner at the apex; aseptate, sometimes septate; hyaline (Figure 11c-e). Conidiogenesis: blastic; a single to multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 3 conidia in a wreath-shaped arrangement (Figure 11c-e). ...

... cells: elongated, delicate, narrow-cylindrical to almost filiform, subulate, with obtuse apices; arranged in verticils, 1-4(5) per verticil, mainly 3-4 at the apex, sometimes emerging orthogonally to conidiophore axis or in a dichotomous manner at the apex; aseptate, sometimes septate; hyaline (Figure 11c-e). Conidiogenesis: blastic; a single to multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 3 conidia in a wreath-shaped arrangement (Figure 11c-e). Conidia: in petri dish culture ellipsoidal to cylindrical or fusiform; (15)18-29(32) × (5.5)6.5-9(10.5) ...

... (5)5.5-6.5(7) µm, Qm 3.9; mainly two-celled, very rarely three-to four-celled; hyaline, smooth (Figure 11f-h). Sclerotioid structures: present, cells globose to elongated, slightly thick-walled, forming monilioid chains, usually branched (Figure 11i). ...

... Qm 3.9; mainly two-celled, very rarely three-to four-celled; hyaline, smooth (Figure 11f-h). Sclerotioid structures: present, cells globose to elongated, slightly thick-walled, forming monilioid chains, usually branched (Figure 11i). Cladobotryum sp. ...

... compactly felt-like to cottony, with white tufts; buff, progressing towards pale ochre and pinkish hues; asymmetrically spreading, with irregular margin (Figure 12a). KOH reaction: negative or faintly positive. ...

... rate: slow to moderate. Colony reverse: buff, progressively turning from ochre to red hues with purplish undertones (Figure 12b). Conidiophores: very long, delicate, unbranched or very sparsely branched in a unilateral manner at conidiophore apices or solitary, shorter, emerging from very long hyphae; septate; hyaline (Figure 12c-e). ...

... reverse: buff, progressively turning from ochre to red hues with purplish undertones (Figure 12b). Conidiophores: very long, delicate, unbranched or very sparsely branched in a unilateral manner at conidiophore apices or solitary, shorter, emerging from very long hyphae; septate; hyaline (Figure 12c-e). Conidiogenous cells: elongated, sometimes very elongated, delicate, narrow-cylindrical to almost filiform, subulate; arranged in verticils, 1-3(4) per verticil, mainly 2-3 at the apex, sometimes emerging orthogonally to conidiophore axis or in a dichotomous manner at the apex; aseptate; hyaline; (Figure 12c-e). ...

... very long, delicate, unbranched or very sparsely branched in a unilateral manner at conidiophore apices or solitary, shorter, emerging from very long hyphae; septate; hyaline (Figure 12c-e). Conidiogenous cells: elongated, sometimes very elongated, delicate, narrow-cylindrical to almost filiform, subulate; arranged in verticils, 1-3(4) per verticil, mainly 2-3 at the apex, sometimes emerging orthogonally to conidiophore axis or in a dichotomous manner at the apex; aseptate; hyaline; (Figure 12c-e). Conidiogenesis: blastic; a single to multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 2 conidia in a V-shaped arrangement (Figure 12c-e). ...

... cells: elongated, sometimes very elongated, delicate, narrow-cylindrical to almost filiform, subulate; arranged in verticils, 1-3(4) per verticil, mainly 2-3 at the apex, sometimes emerging orthogonally to conidiophore axis or in a dichotomous manner at the apex; aseptate; hyaline; (Figure 12c-e). Conidiogenesis: blastic; a single to multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 2 conidia in a V-shaped arrangement (Figure 12c-e). Conidia: in petri dish culture ellipsoidal to cylindrical; (14.5)17.5-25(31) ...

... (3.5)4.5-5.5(6) µm, Qm 4.7, mainly two-celled, rarely one-to four-celled; hyaline, smooth; (Figure 12f-j). Sclerotioid structures: not observed. ...

... structures: not observed. Mycelium: felt-like to compactly cottony; whitish, buff, progressing towards pale ochre to brownish with pinkish hues; symmetrically spreading, with regular margin (Figure 13a). KOH reaction: negative or faintly positive. ...

... rate: relatively slow to moderate. Colony reverse: buff, progressively turning from ochre to bright and dark orangered hues (Figure 13b). Conidiophores: very long, unbranched, sometimes shorter, emerging orthogonally from very long hyphae; septate; hyaline (Figure 13c,d). ...

... reverse: buff, progressively turning from ochre to bright and dark orangered hues (Figure 13b). Conidiophores: very long, unbranched, sometimes shorter, emerging orthogonally from very long hyphae; septate; hyaline (Figure 13c,d). Conidiogenous cells: very elongated, narrow-cylindrical to filiform, subulate, with obtuse apices; in verticils at conidiophores apices, 1-3 per verticil; aseptate, hyaline (Figure 13c,d). ...

... very long, unbranched, sometimes shorter, emerging orthogonally from very long hyphae; septate; hyaline (Figure 13c,d). Conidiogenous cells: very elongated, narrow-cylindrical to filiform, subulate, with obtuse apices; in verticils at conidiophores apices, 1-3 per verticil; aseptate, hyaline (Figure 13c,d). Conidiogenesis: blastic; a single to multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 3 conidia (Figure 13c,d). ...

... cells: very elongated, narrow-cylindrical to filiform, subulate, with obtuse apices; in verticils at conidiophores apices, 1-3 per verticil; aseptate, hyaline (Figure 13c,d). Conidiogenesis: blastic; a single to multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 3 conidia (Figure 13c,d). Conidia: in petri dish culture fusiform to cylindrical or bacilliform, rarely ellipsoidal; (17.5)20-24.5(28) ...

... Qm 3.8; in slide culture cylindrical to filiform; (21)24-38.5(44.5) × 3.5-5 µm, Qm 7.1; one-celled or two-celled; hyaline, smooth; (Figure 13e-g). Sclerotioid structures: not observed. ...

... structures: not observed. Mycelium: uneven in terms of texture and pigmentation, with cottony and compactly felt-like areas irregularly distributed, ranging from white to buff, pale ochre, pink; asymmetrically spreading, with irregular margin (Figure 14a). KOH reaction: negative. ...

... rate: relatively slow. Colony reverse: buff, progressively turning from ochre to terracotta and various red hues with brownish undertones (Figure 14b). Conidiophores: very long, unbranched or sparsely branched in a very irregular and somewhat unilateral or dichotomous manner at conidiophore apices, sometimes shorter, emerging almost orthogonally from very long hyphae; septate; hyaline (Figure 14c). ...

... reverse: buff, progressively turning from ochre to terracotta and various red hues with brownish undertones (Figure 14b). Conidiophores: very long, unbranched or sparsely branched in a very irregular and somewhat unilateral or dichotomous manner at conidiophore apices, sometimes shorter, emerging almost orthogonally from very long hyphae; septate; hyaline (Figure 14c). Conidiogenous cells: very narrow-cylindrical to almost filiform, subulate, with obtuse apices; arranged in verticils at conidiophores apices, 1-4 per verticil; aseptate; hyaline (Figure 14c). ...

... very long, unbranched or sparsely branched in a very irregular and somewhat unilateral or dichotomous manner at conidiophore apices, sometimes shorter, emerging almost orthogonally from very long hyphae; septate; hyaline (Figure 14c). Conidiogenous cells: very narrow-cylindrical to almost filiform, subulate, with obtuse apices; arranged in verticils at conidiophores apices, 1-4 per verticil; aseptate; hyaline (Figure 14c). Conidiogenesis: blastic; a single or multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 2 conidia in a V-shaped arrangement (Figure 14c). ...

... cells: very narrow-cylindrical to almost filiform, subulate, with obtuse apices; arranged in verticils at conidiophores apices, 1-4 per verticil; aseptate; hyaline (Figure 14c). Conidiogenesis: blastic; a single or multiple conidiogenous loci per conidiogenous cell and a single conidium or up to 2 conidia in a V-shaped arrangement (Figure 14c). Conidia: in petri dish culture ellipsoidal to cylindrical, fusiform to bacilliform; (17.5)24-34.5(43.5) ...

... (3)4.5-5.5(6.5) µm, Qm 6.4; mainly two-celled, but also one-celled; hyaline, smooth; (Figure 14d Mycelium: uneven in terms of texture and pigmentation, with compactly felt-like and floccose to cottony areas irregularly distributed, ranging from whitish, buff, pale ochre to red hues with pinkish undertones; asymmetrically spreading, with irregular margin (Figure 15a). KOH reaction: positive. ...

... implemented to clarify the relationships among the 29 examined Greek strains and the 2 ex-type strains (C. rubrobrunnescens and C. tenue) (Figure 17). In order to resolve any phylogenetic ambiguity, 63 publicly available sequences were also included, representing all known ITS sequences of the Cladobotryum species to date. ...

... ITS-based phylogeny shows the cluster of a large group that contains C. tenue, C. rubrobrunnescens, and the Greek Cladobotryum spp. strains (Figure 17). ) was used as an outgroup. ...

... score plot of the unsupervised principal component analysis (PCA) is presented in Figure 18a. The generated model with 15 components and with R2X(cum) = 0.78 and Q2(cum) = 0.54 satisfactorily described the data variation. ...

... analysis was performed using the species information as a Y variable with the aim to reveal species-specific spectral characteristics responsible for the clustering. The score plot of Projection to Latent Structures (PLS-DA) is shown in Figure 18b. The created model had 7 components with R2X(cum) = 0.57 and Q2(cum) = 0.48, while the R2Y(cum) = 0.68. ...

... model was validated through a permutation test (100 permutations; Figure S2, Supplementary Materials). The supervised analysis based on species corroborates the observations of the PCA, suggesting an existing intraspecies chemical similarity, thereby further separating C. fungicola strains from those corresponding to C. varium (Figure 18b). It is interesting to note that on the second axis, macroscopically-observed pigmentation differences of the species/isolates are projected, with the red-pigmented ones being located above and the non-red being located below x-axis. ...

... more thorough investigation was performed for the red-pigmented species, which aimed at a more detailed description of the spectroscopic profile of the URPs, and tried to possibly unravel the similarities and differences hidden within a wider context. The PCA and PLS-DA score plots are shown in Figure 19a,b, respectively. The model that resulted from PCA had 10 principal components and described 76% of the variability, while predicting a lower share (51%). ...

... in the centre of the T 2 Hotteling ellipse, samples from all strains are overlapped and only C. rubrobrunnescens is situated in the 2nd axis, at the margins of the ellipse. The supervised analysis with 6 components and R2X(cum) = 0.58, R2Y(cum) = 0.87, and Q2(cum) = 0.66 distinguishes C. mycophilum from C. dendroides species in the 2nd component, while in the 1st component of the model, the difference of URPs with the rest of the samples is projected (Figure 19b). Cladobotryum rubrobrunnescens and C. tenue ex-type strains are located at two edges of C. dendroides strains, exhibiting a more distant spectroscopic profile when analysed within the red-pigmented strains. ...

... Variable Importance in Projection scores (VIPs) of the 1st component-the axis separating URPs from the other examined red-pigmented species-reveals sugar moieties as the major characteristic of URPs. The 1 H NMR spectra of the hydroalcoholic extracts of the isolates are presented in Figure 20 and grouped according to the results obtained from the PCA analysis (Figure 18a) to facilitate visual inspection. More precisely, panel a shows the 1 H NMR spectra of the isolates corresponding to the species C. mycophilum, C. verticillatum, C. apiculatum, and C. fungicola; panel b shows the isolates of the species C. varium; and panel c shows the isolates of the species C. dendroides, C. tenue, C. rubrobrunnescens, and the URPs. ...

... phylogenetic analysis shows that the examined strains isolated from Greek habitats are spread across the phylogenetic tree among strains that originated from tropical or other climates. Both sequence and metabolic data suggest that the cluster including the species, C. fungicola, diverged early phylogenetically and presents an "ancestral"-like status ( Figure 17). The appearance of red pigmentation, presumably due to aurofusarin and related pigment production [7], possibly occurred as one major evolutionary event at the divergence of C. apiculatum against the earlier diverging species (Figure 17). ...

... sequence and metabolic data suggest that the cluster including the species, C. fungicola, diverged early phylogenetically and presents an "ancestral"-like status ( Figure 17). The appearance of red pigmentation, presumably due to aurofusarin and related pigment production [7], possibly occurred as one major evolutionary event at the divergence of C. apiculatum against the earlier diverging species (Figure 17). However, the exclusion of the possible reappearance of these metabolites in many different evolutionary events cannot be overruled. ...

... Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8080877/s1, Text S1: Analytical Species Descriptions; Figure S1: Simplified diagram of the extraction protocol followed in the present study; Figure S2: Permutation tests, applying 100 random changes, of the PLS-DA models of (a) all the examined Cladobotryum species presented in Figure 18b and (b) the red-pigmented species presented in Figure 19b; Figure S3: (a) 1 H NMR, (b) COSY, (c) HSQC, (d) HMBC spectra of phomalactone in MeOD. File S1: Phylogenetic tree matrix. ...

... Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8080877/s1, Text S1: Analytical Species Descriptions; Figure S1: Simplified diagram of the extraction protocol followed in the present study; Figure S2: Permutation tests, applying 100 random changes, of the PLS-DA models of (a) all the examined Cladobotryum species presented in Figure 18b and (b) the red-pigmented species presented in Figure 19b; Figure S3: (a) 1 H NMR, (b) COSY, (c) HSQC, (d) HMBC spectra of phomalactone in MeOD. File S1: Phylogenetic tree matrix. ...

... Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8080877/s1, Text S1: Analytical Species Descriptions; Figure S1: Simplified diagram of the extraction protocol followed in the present study; Figure S2: Permutation tests, applying 100 random changes, of the PLS-DA models of (a) all the examined Cladobotryum species presented in Figure 18b and (b) the red-pigmented species presented in Figure 19b; Figure S3: (a) 1 H NMR, (b) COSY, (c) HSQC, (d) HMBC spectra of phomalactone in MeOD. File S1: Phylogenetic tree matrix. ...

... [2,3,[6][7][8][9][10][11][12]22,23,[29][30][31]35,36,46,81,82,[101][102][103] are cited in the Supplementary Materials. Figure 1c ...