Morphological studies

Morphological observations were made on herbarium specimens and living cultures. Colonies and ascomata were examined with an Olympus SZX12 dissecting microscope (Olympus America, Inc., Melville, NY, USA) and rehydrated with tap water. Microscopic preparations were mounted in 90 % lactic acid, tap water, or Melzer’s reagent; measurements were made from material mounted in Melzer’s reagent, and means ± standard deviation (SD) based on a minimum of 20–25 measurements were calculated for sizes of asci, ascospores, and conidia. Observations were made using an Olympus BX51 light microscope. Microphotographs were captured using an Olympus DP70 camera with Imaging Software Cell^D (Olympus). Colony macrophotographs were documented with a Canon EOS 77D digital camera with Canon EF 100mm f/2.8L Macro IS USM objective (Canon Europe Ltd., Middlesex, UK) with daylight spectrum 5500K 16W LED lights. Images were processed with Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).

We used slide cultures to observe the ontogeny of chlamydospores, arrangement of conidiophores, conidiogenesis, and early conidial cluster formation. Each strain was inoculated on several small blocks of potato-carrot agar (PCA) (Crous et al. 2019), a nutrient-deficient agar medium, covered with a coverslip, incubated in the dark at 20–23 °C and checked daily for two weeks. Fungi were cultured on several different nutrient media to obtain colony characters and information on diffusible pigments and growth: cornmeal dextrose agar (CMD) (Oxoid Limited; 2 % dextrose), MLA, oatmeal agar (OA) and PCA. Characteristics of colonies were based on 4-wk-old cultures incubated in the dark at 20–23 °C. Strains were also inoculated on cornmeal agar (CMA) with sterile stems of Urtica dioica and synthetic nutrient agar (SNA) with pine (Pinus sylvestris) needles and banana (Musa sp.) leaves (Crous et al. 2019) to induce sporulation.

The effect of temperature on mycelial growth was studied on 31 strains that represent 22 accepted species and six varieties. The strains were pre-grown on malt extract agar (MEA, malt extract 20 g, peptone 1 g, glucose 20 g, agar 20 g, distilled water 1 L, pH 6) for 14 d at 24 °C. An agar disk (5 mm diam) was then excised from the growing front of the fungus and used as an inoculum. Growth rates were measured in triplicate at seven different temperatures ranging from 5–37 °C after 14 d of cultivation at 24 °C in the dark.

Gene markers, DNA extraction, PCR amplification, and sequencing

We analysed sequences of four gene markers. The internal transcribed spacer (ITS1-5.8S-ITS2) (ITS) of the nuclear rRNA cistron is the primary barcode for fungi (Schoch et al. 2012). The nuclear large subunit LSU rDNA gene (LSU) (D1−D3 domains, approximately 5′ 1 800 base pairs) is the most commonly used marker in Chaetosphaeriaceae. It has also been used to explore relationships in the Ascomycota, providing good phylogenetic resolution at the generic and higher taxonomic levels in fungi (e.g. Zhang et al. 2007, Schoch et al. 2009). Two coding markers, i.e. the intermediate section of the coding region of the translation elongation factor 1-α (tef1-α) and β-tubulin (tub2) marked by exons 2−6, were selected. They are known for their ability to distinguish between interspecific relationships (Robert et al. 2011, Stielow et al. 2015, Meyer et al. 2019) and were successfully applied as dual barcodes in Chaetosphaeriaceae in genera Codinaea, Dictyochaeta, and Zanclospora (Réblová et al. 2021a–c). Accession numbers of sequences retrieved from GenBank and published in other studies (Réblová & Winka 2000, Huhdorf & Fernández 2005, Fernández et al. 2006, Somrithipol et al. 2008, Prabhugaonkar & Bhat 2009, Magyar et al. 2011, Crous et al. 2012, 2013, 2014, 2015, 2020, Hashimoto et al. 2015, Ma et al. 2016, Hernández-Restrepo et al. 2017, Wang et al. 2017, Wei et al. 2018, 2022 Yang et al. 2018, Lin et al. 2019, Luo et al. 2019, Vu et al. 2019, Li et al. 2021, Réblová et al. 2021a–e, Manawasinghe et al. 2022, Wu & Diao 2022) are listed in Table 2.

Total genomic DNA was extracted from 3–4-wk-old cultures grown on MLA using the DNeasy^® UltraClean^® Microbial Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s protocol for filamentous fungi. PCR amplifications were carried out in 25 μL volume reactions using a Q5 High Fidelity DNA polymerase kit (New England Biolabs Inc., Hitchin, UK) according to the manufacturer’s protocol.

The ITS and LSU markers were amplified with the V9G/LR8 primer pair (de Hoog & Gerrits van den Ende 1998, Vilgalys unpubl.). The tef1-α region was amplified with the EF1-983F/EF1-2218R primers (Rehner & Buckley 2005) and tub2 with the T1/Bt2b primer pair (O’Donnell & Cigelnik 1997, Glass & Donaldson 1995). PCR of ITS, LSU, and tef1-α was carried out as described in Réblová et al. (2020). PCR of tub2 was carried out in a BioRad C1000 thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) as follows: 98 °C for 1 min; 3 cycles of touchdown PCR denaturation (98 °C for 10 s), annealing (from 62 °C to 58 °C (temperature reduced every cycle for 2 °C) and elongation 72 °C for 90 s; 34 cycles of denaturation (98 °C for 10 s), annealing (57 °C for 30 s) and elongation (72 °C for 90 s) and a final extension step at 72 °C for 5 min.

Amplicons were sequenced in both directions using the PCR and nested primers ITS5, ITS4, JS1, JS7, JS8, and LR7 (Vilgalys & Hester 1990, White et al. 1990, Landvik 1996). Automated sequencing was carried out by Eurofins GATC Biotech Sequencing Service (Cologne, Germany), Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands), and Ottawa Research and Development Centre, Agriculture and Agri-Food Canada (Ottawa, Ontario, Canada). Analysis of raw sequence data and construction of sequence contigs were performed using Sequencher v. 5.4.6 (Gene Codes Corp., Ann Arbor, MI, USA).

For ITS, LSU, tef1-α, and tub2 markers, histograms of intraspecific and interspecies distances of Chloridium spp. were generated to illustrate the degree of overlap (i.e. barcoding gap). Species from which only rDNA markers were available were not included in the analysis. The matrix of pairwise distances was computed with MEGA v. 11.0.13 using the Kimura two parameters (K2P) model (Tamura et al. 2021). The histogram was plotted in GraphPrism software v. 7.03 (Graphpad Software Inc., LaJolla, CA, USA) using a bin size of 0.002 (ITS), 0.005 (tub2) and 0.0005 (tef1-α, LSU). The estimates of evolutionary divergence are provided in the Supplementary Table S1.

Phylogenetic analyses and species delimitation

The gene sequences, newly generated in this study and those obtained from GenBank, were aligned in MAFFT v. 7.487 (Katoh & Standley 2013) implemented in the CIPRES Science Gateway v. 3.3 (Miller et al. 2010) and manually corrected in BioEdit v. 7.1.8 (Hall 1999), if necessary. Consensus secondary structure (2D) models acquired for the ITS1 and ITS2 of Chloridium spp., and also Codinaea and related taxa (Réblová et al. 2021b), were used to improve the alignment by comparing nucleotides at homologous positions (in helices and loops). The LSU alignment was enhanced using a predicted 2D model of this gene of Saccharomyces cerevisiae (Gutell et al. 1993). The single gene data sets were concatenated and subjected to several phylogenetic analyses. The multilocus sequence alignment was deposited in TreeBASE (29771). The relationships of 11 species of Chloridium described by Wu & Diao (2022) that contained only ITS and several LSU rDNA sequences were included in separate reduced analyses.

We performed species delimitation analyses to provide hypotheses about the species boundaries in the genus Chloridium. They comprised one multilocus method, STACEY (Jones 2017), and three single-locus methods, each based on a different approach, namely, GMYC (Bayesian Inference) (Fujisawa & Barraclough 2013), PTP (Maximum Likelihood) (Zhang et al. 2013) and ABGD (pairwise phylogenetic distance) (Puillandre et al. 2012).

The data set was reduced to strains with unique sequences using the haplotype function from R v. 4.2.1 (R. Core Team 2015) package PEGAS (Paradis 2010). STACEY analysis was performed on a data set of four loci: ITS, LSU, tef1-α, and tub2. In the case of the single-locus methods, LSU alignment was omitted due to a low number of informative sites. The best-fitting model of DNA evolution for each locus was determined using Bayesian information criterion (BIC) in jModelTest v. 2.1.7 (Posada 2008) with the following results: a selected model for ITS was TrNef+I+G, for LSU TrN+I+G, for tef1-α TrN+I+G, and for tub2 TrN+G.

STACEY analysis was calculated in BEAST v. 2.6.7 (Bouckaert et al. 2019) using the add-on STACEY v. 1.2.5 with the following parameters: four separate runs (later combined using LogCombiner software from BEAST package) with chain length of 1 × 10⁸ generations; species tree prior set to Yule model; growth rate prior set to lognormal distribution (M = 5, S = 2); PopPriorScale prior set to lognormal distribution (M = -7, S = 2); relativeDeathRate prior set to beta distribution (α = 1, β = 1 000). The convergence of mcmc was checked in Tracer v. 1.7.1 (Rambaut et al. 2018). The output was processed with Species Delimitation Analyzer (Jones et al. 2015) with the varying value of the collapseheight parameter (0.001–0.02). Out of these values, we chose to present two (0.0067 and 0.102), which resulted in highly supported scenarios.

The GMYC method was performed in R v. 4.2.1 using the package SPLITS (Fujisawa & Barraclough 2013) with input trees calculated in BEAST v. 2.6.7. The PTP method was performed in Python v. 3 (van Rossum & Drake 2019) package PTP (Zhang et al. 2013) with 100 standard bootstrap trees calculated in IQ-TREE v. 2.1.2 (Minh et al. 2020) used as an input. The ABGD (Automatic Barcode Gap Discovery) (Puillandre et al. 2012) method was performed on the ABGD web server (wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html). For results interpretation, we followed the recommendation of Puillandre et al. (2012) and Kekkonen & Herbert (2014), so we present the results of the initial partition closest to P = 0.01. The graphical representation of the species delimitation results was created in iTOL (Interactive Tree Of Life) (Letunic & Bork 2016).

To further inspect the boundaries between species, we classified the strains from our data set into hypothetical populations and performed the species tree inference by starBEAST v. 2.0 (Drummond et al. 2012) implemented in BEAST v. 2.6.7. The hypothetical populations consisted of smallest possible and highly supported clades or individual strains. The species tree was calculated in four separate runs with chain length of 1 × 10⁹ generations, strict molecular clock and coalescent constant population model.

Additional phylogenetic reconstructions of members of Chaetosphaeriaceae and Chloridium s. str. were based on partitioned analyses of three- (ITS, LSU, and tef1-α) and four (ITS, LSU, tef1-α and tub2) loci, respectively. They were calculated using Bayesian Inference (BI) and Maximum Likelihood (ML) analyses through the CIPRES Science Gateway v. 3.3. The best-fit models of nucleotide evolution for each partition (ITS, LSU, tef1-α and tub2) were selected in jModelTest v. 2.1.7. as specified above. The ML analysis was performed with RAxML-HPC v. 8.2.12 (Stamatakis (2014) with a GTRCAT approximation. Statistical support for the nodes was determined by non-parametric bootstrapping (BS) with 1 000 replicates. The BI analysis was performed with MrBayes v. 3.2.7 (Ronquist et al. 2012). Two Bayesian searches were conducted using default parameters. The B-MCMCMC (Bayesian-Metropolis-coupled Markov chain Monte Carlo) analyses lasted until the average standard deviation of split frequencies was below 0.01, with trees saved every 1 000 generations with burn-in 25 %. The BI and ML phylogenetic trees were compared visually in terms of topological conflicts between the supported clades.

To assess the relationships of representatives of Chloridium, Gonytrichum, and Pseudophialocephala with other members of Chaetosphaeriaceae, we performed a phylogenetic analysis based on ITS-LSU-tef1-α concatenated data set. The alignment consisted of 118 strains representing 114 species; it has 3 469 characters including gaps and 1 572 unique character sites (RAxML). The first 90 nucleotides (nt) at the 5′-end of LSU were excluded because most sequences retrieved from GenBank were incomplete. Tracylla aristata and T. eucalypti (Tracyllales) were selected as the outgroup (Réblová et al. 2021a–e, Wu & Diao 2022).

Phylogenetic analysis investigating inter- and intraspecific relationships of Chloridium was performed using ITS-LSU-tef1-α-tub2 sequence data set. The sequences of the tub2 gene were added to improve the node resolution and help explore interspecific relationships. The four-gene analysis was based on the alignment of 124 strains, of which 119 were ingroup taxa representing 29 species and varieties of the genus Chloridium; it has 3 690 characters, including gaps and 587 unique character sites (RAxML). The non-coding regions of tub2 were excluded from the analysis. Codinaeella minuta, Codinaea gonytrichodes, Menispora caesia, M. ciliata, and M. tortuosa were selected to root the tree (these species were selected from close relatives of Chloridium with available genes based on the results of previous analysis).

Relationships of 11 species of Chloridium sensu Wu & Diao (2022), for which only rDNA sequences were available, were investigated separately. The original three- and four-gene data sets of Chaetosphaeriaceae and Chloridium were reduced to the two-gene data sets and combined with 11 Chloridium sensu Wu & Diao (2022). Phylogenetic analysis of the genus Chloridium was based on a reduced ITS-LSU sequence data set, including 53 Chloridium strains representing 37 species and varieties. The alignment had 2 384 characters, including gaps and 492 unique character sites (RAxML). The same outgroup as in the previous analysis was selected. Because some of the sequences of new Chloridium species (Wu & Diao 2022) were difficult to align using 2D structures, we applied Gblocks (Castresana 2000) using default options to delimit and remove putative ambiguous regions. We performed three following phylogenetic analyses. The phylogenetic analysis of Chloridium based on ITS-LSU data set initially had 2 384 characters, and 1 264 characters after applying Gblocks, i.e. 53 % of the characters were preserved. To test the phylogenetic hypothesis proposed by Wu & Diao (2022), showing Chloridium divided into two clades by a nested Adautomilanezia-Sporoschisma clade, we performed two ITS-LSU analyses. The Chaetosphaeriaceae data set initially had 2 487 characters, 1 143 characters after applying Gblocks, i.e. 46 % of the characters were preserved. The third reduced ITS-LSU data set comprised only Chloridium and Adautomilanezia-Sporoschisma clades, it had 2 422 characters, and 1 237 characters after application of Gblocks, i.e. 51 % of the characters were preserved.

Relationships among members of the Chl. chlamydosporum species complex were investigated in the phylogenetic analysis of ITS-LSU-tef1-α-tub2 loci. The alignment consisted of 14 sequences representing three species and two varieties. It had 4 142 characters including gaps and 304 unique character sites (RAxML); both coding and non-coding regions of tub2 were included. For this small data set, no outgroup was selected.

Ancestral state reconstruction

We analysed ancestral states of five morphological features, which are used as taxonomic characters, and the ancestral areas of distribution. The data set involving morphological characters, the areas of distribution (gathered from the published distribution of each species from the GlobalFungi database (Větrovský et al. 2020)) and the DNA sequence data spanned across the entire Chloridium genus.

The alignment comprised representative sequences of each species (ex-type strains when possible) from four loci (ITS, LSU, tef1-α, and tub2) with the best-fit models of nucleotide evolution selected in jModelTest v. 2.1.7. The phylogenetic tree for the reconstruction was calculated in MrBayes v. 3.2.7 using partitioned analysis of the four loci.

The reconstruction of the ancestral states and areas was performed in the software package RASP v. 4.2 (Yu et al. 2020) using Bayesian Binary MCMC (Markov chain Monte Carlo) (BBM) method with the following settings: number of cycles 1 000 000; number of chains 10; frequency of samples 100; discard samples 100; temperature: 0.1; state frequencies: estimated (F81).

We split the morphological characters into the following states: I. Conidiophores: (a) unbranched, apex fertile, no collar-like hyphae; (b) unbranched, apex sterile or fertile, with collar-like hyphae; (c) unbranched, apex always sterile, setiform, with collar-like hyphae; (d) branched penicillately, apex fertile, no collar-like hyphae. II. Phialides: (a) terminal, discrete phialides absent; (b) discrete in whorls along the entire conidiophore length, sometimes terminal; (c) discrete in whorls or solitary in the lower part of the conidiophore. III. Conidial colour (a) hyaline; (b) hyaline may become pale brown upon aging; (c) subhyaline to olivaceous or pale brown. IV. Conidial shape: (a) ellipsoidal to obovate, oblong, subglobose; (b) obovate to cuneiform; (c) T- or U-shaped. V. Chlamydospores: (a) absent; (b) present, solitary; (c) present, solitary and in chains; (d) present, in clusters.

The areas of distribution were split into four climate types: (a) all climates; (b) all climates except boreal; (c) all climates except tropics; (d) temperate, subtropics and xeric.

Prediction and identification of base-pair changes in 2D structures of ITS of Chloridium

We used PPfold (Sükösd et al. 2012) and UNAfold (Markham & Zuker 2008) programs to improve multiple sequence alignment of studied sequences of members of Chloridium. The PPfold program uses an explicit evolutionary model and a probabilistic model of structures and provides consensus 2D structures of ITS1 or ITS2 for input multiple sequence alignment. Obtained 2D structure was then refined with the UNAfold web server (www.unafold.org, accessed on 1 July 2022), which employs the Mfold program (Zuker 2003). Final models were visualised with VARNA: Visualisation Applet for RNA program (Darty et al. 2009). In addition, we employed the CBC analyser program (Wolf et al. 2005) to capture CBC (Compensatory Base Change) changes for input sequences. These changes represent the replacement of one standard WC (Watson-Crick) base pair (A-T or G=C) for another WC base pair, thus preserving the RNA secondary structure. In addition to CBC, we can identify two other types of substitutions in helices: hemi-compensatory base change (hCBC), where standard WC base pair is replaced with near-isosteric “wobble pairs” (G/U or A+/C) and non-compensatory base change (non-CBC), where standard base pair is replaced with any non-canonical base pair.

Barcode analyses

In all markers, species of the Pseudophialocephala section recognised in our study differed little or not at all. This divergence falls within the range of intraspecific variation observed in the other species in this study (Fig. 9). Therefore, we do not consider species of that section in the barcoding gap analysis. Other genetically very closely related species, Chl. bellum and Chl. guttiferum, Chl. gamsii and Chl. biforme, were responsible for the overlapping intraspecific and interspecific variability and the absence of a clear barcoding gap in all markers. For the ITS marker, the similarity between some Chl. fuscum strains and between Chl. bellum and Chl. guttiferum species was the same (0.011). In other species, similarities of 0.000–0.011 were intraspecific, values of 0.011–0.015 represented the barcoding gap, and higher similarities represented interspecific variability. Similarly, in the case of tef1-α, no barcoding gap was observed. Still, we could trace a zone starting at the distance of 0.013, where intraspecific variation transitions into the variation observed between related species (e.g. distance 0.014, Chl. guttiferum vs Chl. peruense) or varieties of the same species. Distances above 0.015 (Chl. luteum var. luteum vs var. bellum) represented exclusively interspecific variability. No clear barcoding gap was found for the marker tub2, and values around 0.04 represented both intraspecific (Chl. chlamydosporum) and interspecific (Chl. gamsii vs Chl. biforme) variation. Values higher than 0.05 belonged exclusively to interspecific variability. In the case of LSU, the pure intraspecific distance was observed only at values of 0.0–0.0027. Between the values 0.0027 and 0.004, there is a mixture of intraspecific and interspecific variability; higher values belong to pure interspecific variability.

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Fig. 9.

Barcoding gap. The frequency distribution graphs of the Kimura 2-parameter distances of ITS, LSU, tef1-α and tub2 DNA sequences (i.e. barcoding gaps). The intraspecific distances are shown as blue bars and interspecific distances as grey bars. The asterisk (*) indicates values where intra and interspecies variability overlap.

Ancestral state reconstruction

The reconstruction of ancestral states and climatic regions is shown in Fig. 10. The ancestral state of the conidiophore type was an unbranched conidiophore with a fertile apex and without collar-like hyphae. The unbranched setiform conidiophores with a sterile apex and collar-like hyphae are derived and evolved independently in at least two lineages. Unbranched conidiophores, with a sterile or fertile apex and collar-like hyphae, evolved in one lineage only. Penicillately branched conidiophores without collar-like hyphae are highly derived and evolved only in one lineage. The conidiophore types correlate with the arrangement of phialides. Terminal integrated phialide is the ancestral state that occurs in all major lineages. The presence of a discrete phialide formed in whorls along the entire length of the conidiophore is a derived character that evolved in only one lineage. Discrete phialide formed in whorls or solitary in the lower part of the conidiophore is also derived and originated at least two times independently. The ancestral and most prevalent shape of conidia was ellipsoidal, oblong, and subglobose, whereas other types were rare, derived, and evolved independently twice.

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Fig. 10.

Reconstruction of ancestral states and areas using Bayesian Binary MCMC (BBM) method in Rasp v. 4.2 using phylogenetic trees calculated in MrBayes v. 3.2.7. A–E. Reconstruction of ancestral states of morphological characters. F. Ancestral areas reconstruction of species distribution.

Similarly, the dominant and ancestral conidial colour was hyaline, and darker shades were derived and evolved in five independent lineages. The chlamydospore types are randomly distributed across the phylogenetic tree. In the common ancestors of Chloridium, Gonytrichum, and Volubilia sections, the presence of chlamydospores is an ancestral state. The presence of chlamydospores in chains or clusters was derived and evolved independently twice in Chl. chloroconium (sect. Gonytrichopsis) and Chl. elongatum (sect. Metachloridium). Ancestral reconstruction of the geographical distribution showed that the occurrence in temperate, subtropical or xeric climatic regions together with all climates except boreal and tundra, is ancestral. Interestingly, the ability to colonise colder areas (i.e. all climates, all climates accept boreal and tundra, and all climates except tropics) is derived.

Consensus 2D structure of ITS1 and ITS2

2D models of the ITS1 and ITS2 consensus structures for the genus Chloridium mapped to Chl. virescens were generated. For ITS1, we identified only two helices (not shown). The H1 helix has a variable length of 28–71 nt and represents a region of high variability. In Chl. virescens, the H1 helix is 61 nt long; it contains two nt bulge (TT), a bulge loop with five nt (GCTAA), one non-WC pair C/C and four nt hairpin loop. The second helix consists of only three WC base pairs and four nt hairpin loops and is highly conserved in all species.

The H1 helix of ITS1 was exploited in the classification of members of the Chl. chlamydosporum species complex, where it represented the main variability of the whole ITS region. In particular, the H1 helix exhibited changes in its second part, which can be divided into three zones (Fig. 8). In Chl. chlamydosporum, this upper part is the longest. It consists of 18 bp, including two non-canonical G/A and G/G bp and 2–3 G/U wobble bp, and contains all three zones. Other species, such as Chl. peruense, Chl. detricola var. detricola and var. effusum, have this part reduced to 12, 13 and 7 bp. In comparison, two zones are present in Chl. detriticola var. detriticola and Chl. peruense, only one zone is typical of Chl. detriticola var. effusum. There is also a reduction in the number of non-WC and wobble pairs. In the case of Chl. peruense, the duplex contains only one non-WC U/C pair and one wobble pair. Chloridium detriticola var. detriticola has only two wobble pairs, while Chl. detriticola var. effusum consists of only standard WC pairs.

The consensus ITS2 structure of members of Chloridium consisted of three helices (H1–H3). Helix H4 was not detected due to large sequence variability. In the ITS2 molecule, we identified all existing substitutions among species of Chloridium, which were mapped onto the predicted 2D structure model of ITS2 of Chl. virescens (Fig. 11). In Chl. virescens, H1 is the shortest helix consisting of five WC base pairs with three nt hairpin loops. H2 helix contains nine WC base pairs, one bulge base (A) in the middle of the helix, and four nt hairpin loops. The H3 is the longest duplex (69 nt). It contains five-bulge bases, one non-WC C/U base pair, a short internal loop with three base pairs (A/C, A-U, A/G), and a four nt hairpin loop.

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Fig. 11.

ITS2 secondary structure of Chloridium virescens (GenBank accession no. OP455441) and 5.8S-28S rRNA gene hybridisation (proximal stem region). ITS2 helices are numbered I–III. All substitutions recorded among members of Chloridium are mapped on the 2D model. Identified substitutions are colour coded: CBC (green), hCBC (blue) and non-CBC (red). Hairpin loops, junction regions and an area of the putative IV helix are highlighted with grey colour to represent regions with a variable number of nucleotides or sequence variation.

Helix H3 of ITS2 is a site of high interspecific variability. In Chl. virescens, the H3 helix consists of 26 base pairs. Among them, there are two tandem wobble pairs G/U. The first one changes into canonical G=C pair in Chl. crousii and Chl. xishuangbannaense (sect. Chloridium), Chl. cylindrosporellum and Chl. elongatum (Metachloridium), Chl. subglobosum (Gonytrichum) and in all species of section Pseudophialocephala, while the second G/U pair is substituted by entirely different non-WC pairs G/G for the sect. Pseudophialocephala and U/U for Chl. crousii and Chl. xishuangbannaense (Chloridium). The H3 duplex shows only one CBC change A-U → G=C in the central part, which is seen in the Chl. chlamydosporum species complex and several other species of its sister clade. Interestingly, in several species of the section Chloridium, the standard A-U changes to wobble pair G/U (Chl. caudigerum, Chl. jilinense) or non-canonical C/C (Chl. crousii and Chl. xishuangbannaense), while in Chl. caesium (sect. Gonytrichum), it is replaced by A/G. In the upper part of the H3 helix occur three near-isosteric substitutions between standard and wobble pairs (G=C → G/U), two exclusively exhibit Chl. chloridioides (Gonytrichum), and the third one is shared by all members of the Mesobotrys section and selected species of the sections Chloridium and Cryptogonytrichum. The fourth near-isosteric substitution between standard and wobble pair (A-U → A/C) occurs in Chl. chloroconium, Chl. tropicale, and Chl. chloridioides representing three different sections. The last pair adjacent to the hairpin loop is highly variable; we observed that this position tolerates standard, wobble, or non-WC pairs.

Biogeography, ecology, and diversity analysis of Chloridium in environmental samples

Data mining in the GlobalFungi database, based on the Exact hit similarity rule, resulted in 8 806 021 reads that matched the ITS1 or ITS2 sequences of the studied taxa. This means that reads assigned to Chloridium account for 0.3 % of all reads in the database. Chloridium tropicale is the only species in the genus that was not found in metabarcoding data. On average, each species was found in 1 485 samples (minimum: 1–2 samples for Chl. xishuangbannaense and Chl. novae-zelandiae, maximum: 7 903 samples for Chl. detriticola var. detriticola). Most samples came from bulk, top or rhizosphere soil (87 %), while other substrates such as roots (6 %), litter (3 %), and deadwood (3 %) were less frequent. Each species shows a preference for soil type, especially for bulk soil, i.e. soil outside the rhizosphere where nutrients are scarce (e.g. Chl. caudigerum, Chl. guttiferum, Chl. gamsii, Chl. mirabile) over the topsoil. In contrast, the topsoil, the upper layer of soil where organic nutrients are abundant, is strongly preferred by other species (e.g. Chl. cylindrosporellum, Chl. fuscum, Chl. moratum, Chl. volubile). Several species are relatively more common on organic residues, such as litter (Chl. caudigerum, Chl. chloridioides, Chl. chloroconium, Chl. subglobosum) and deadwood (Chl. caudigerum, Chl. jilinense, Chl. subglobosum).

Most of the samples were taken in the forest (94 %), followed by grasslands (2 %), croplands (1 %), and other biomes. In addition to the general abundance in forests, several species are fairly common in arable land (Chl. bellum, Chl. elongatum, Chl. fuscum, Chl. mirabile, Chl. humicola species complex, Chl. proliferatum) or grasslands (Chl. bellum, Chl. caesium, Chl. chlamydosporum, Chl. chloroconium, Chl. fuscum, Chl. gamsii, Chl. guttiferum, Chl. humicola complex, Chl. setosum). The most abundant species were Chl. detriticola var. detriticola (7 903 samples, 132 studies), Chl. fuscum (7 246 samples, 186 studies), Chl. moratum (4 405 samples, 57 studies), Chl. volubile (3 559 samples, 81 studies), Chl. shangsiense (3 314 samples, 71 studies), Chl. humicola species complex (3 287 samples, 174 studies), Chl. bellum var. bellum (2 549 samples, 97 studies) and Chl. cylindrosporellum (2 529 samples, 47 studies). Given the density of sampling in different regions, we can draw some well-founded conclusions (Fig. 12, Supplementary Fig. S4, Suplemmentary Tables S2, S3). Most species are widely distributed and only some species have a more or less restricted geographical distribution, i.e. China or Vietnam: Chl. crousii, Chl. proliferatum, Chl. xishuangbannaense; Australasia: Chl. biforme, Chl. novae-zelandiae; Northern Hemisphere: Chl. chloridioides, Chl. jilinense, Chl. kirkii, Chl. subglobosum.

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Fig. 12.

Biogeography of Chloridium inferred from the GlobalFungi database data. The distribution types are demonstrated on example species. A. Global distribution across all climatic zones (Chl. detriticola var. detriticola). B. Global distribution across multiple climatic zones, but not in boreal (Chl. bellum var. bellum). C. As in the previous case, but with a high preference for warmer and humid areas and South America. D. Distributed in temperate and colder climatic zones (Chl. caudigerum). E. Global distribution in temperate, subtropical and xeric sites (Chl. caesium). F. Australasian distribution on temperate and xeric sites (Chl. biforme in green, Chl. novae-zelandiae in red).

In general, the distribution of species is primarily governed by climatic zones and secondarily by geography. The species can be divided into six types based on climatic conditions and geography preferences. Group 1: Species distributed globally (i.e. with exceptions of little sampled Africa and Antarctica, in some species) in all climatic zones (Chl. chloroconium, Chl. culmicola, Chl. detriticola var. detriticola, Chl. detriticola var. effusum, Chl. shangsiense, Chl. volubile). The average Mean Annual Temperature (MAT) was 8.4–13.4 °C, and the Mean Annual Precipitation (MAP) was 974–1 349 mm. Group 2: Distributed in temperate, continental humid, subtropical, tropical, and drier climates but with absence or rare occurrence in the colder zones (boreal, tundra), either globally (Chl. bellum var. bellum, Chl. chlamydosporum, Chl. cylindrosporellum, Chl. fuscum, Chl. guttiferum, Chl. humicola species complex, Chl. mirabile, Chl. moratum, Chl. peruense, Chl. setosum, Chl. ypsilosporum) or with geographically limited distribution (Chl. gamsii, Europe, N. America, Australasia) (MAT 7.8–20 °C, MAP 860–1 801 mm). Group 3: Distributed in subtropical, temperate, continental humid, tundra, or boreal climatic zone in Northern Hemisphere (Chl. chloridioides var. chloridioides) or also in Australasia (Chl. caudigerum) (MAT 6.9–7.5 °C, 657–743 mm). Group 4: Occurring in temperate and subtropical climatic regions, and sometimes also on xeric sites either globally (Chl. caesium), almost globally (Chl. virescens mainly in Europe, absent in Northern America), limited to Northern Hemisphere (Chl. chloridioides var. convolutum, Chl. culmicola, Chl. elongatum, Chl. jilinense, Chl. kirkiii, Chl. subglobosum), Australasia (Chl. biforme, Chl. novae-zelandiae) or China (Chl. crousii) (MAT 7.6–19.75 °C, MAP 645–1 550 mm). Group 5: Species such as Chl. proliferatum and Chl. xishuangbannaense, both very rare and known from subtropical/tropical regions in China and Vietnam.

Using GlobalFungi database, we identified several diversity hotspots. As the temperate and relatively humid climate suits all studied species, 23 species (except Chl. culmicola, Chl. crousii, Chl. mirabile, Chl. biforme, Chl. novae-zelandiae, Chl. xishuangbannaense, Chl. proliferatum) are found in the forests of Central Europe. Another hotspot was a humid subtropical forest on the South China-Laos border, where species preferring temperate, subtropical, and tropical climate meet (Chl. bellum, Chl. caudigerum, Chl. chlamydosporum, Chl. chloridioides var. convolutum, Chl. chloroconium, Chl. culmicola, Chl. cylindrosporellum, Chl. detriticola var. detriticola and var. effusum, Chl. elongatum, Chl. mirabile, Chl. fuscum, Chl. humicola species complex, Chl. kirkii, Chl. moratum, Chl. peruense, Chl. shangsiense, Chl. sinense, Chl. volubile, Chl. ypsilosporum). This is also due to the large sampling depth of the study of Sun et al. (2021) focusing on this area.

Taxonomy

Chloridium Link, Mag. Gesell. naturf. Freunde, Berlin 3: 13. 1809. Emend. Réblová & Hern.-Restr.

Synonyms: Gonytrichum Nees & T. Nees, Nova Acta Phys.-Med. Acad. Caes. Leop.-Carol. Nat. Cur. 9: 244. 1818.

Mesobotrys Sacc., Michelia 2: 27. 1880.

Cirrhomyces Höhn., Ann. Mycol. 1: 529. 1903.

Melanopsammella Höhn., Ann. Mycol. 17: 121. 1920.

Bisporomyces van Beyma, Antonie van Leeuwenhoek 6: 276. 1940.

Pseudophialocephala M.S. Calabon et al., Mycosphere 13: 502. 2022.

Monostachys Arnaud, Bull. Trimestriel Soc. Mycol. France 69: 278. 1953. (Nom. inval. Art. 36).

Sphaeromycetella Arnaud, Bull. Trimestriel Soc. Mycol. France 69: 279. 1953. (Nom. inval. Art. 36.)

? Piminella Arnaud, Bull. Trimestriel Soc. Mycol. France 69: 281. 1953. (Nom. inval. Art. 36.)

Type species: Chloridium virescens (Pers.) W. Gams & Hol.-Jech.

Emended description: Colonies effuse, hairy or finely bristle-like, or pulvinate, brown, becoming whitish, white grey, yellow, yellow-green, green, beige, olivaceous brown, olivaceous grey or amber when sporulating; composed of conidiophores and ascomata. Asexual morph. The chloridium-type conidiophores absent or present; they are formed in one layer (all conidiophores of the same average length) or two layers (as shorter and longer setiform but always fertile conidiophores), solitary, scattered, crowded or caespitose, unbranched, sometimes branched (in culture), often with percurrent proliferations, brown, cylindrical or subulate. Conidiogenous cells phialidic, terminal, integrated, extending percurrently, occasionally sympodially (in culture), conidiogenous locus with multiple loci, sometimes protruding above the collarette, with annellations; collarettes flaring, shortly funnel-shaped, often inconspicuous. Conidia ellipsoidal, ellipsoidal to oblong, subglobose, suballantoid, obovate, sometimes cuneiform, hyaline, occasionally turn pale brown (in aged material), or pale brown at maturity, aseptate, smooth, adhering in heads or cirrhi. Chlamydospores absent or present; lateral, terminal, intercalary, sessile or on a stipe, solitary, or in chains, aseptate, brown, thick-walled, smooth. The gonytrichum-type conidiophores absent or present; solitary, scattered, crowded or caespitose, the main stalk is unbranched, brown, cylindrical or subulate, apex setiform, sterile, straight, flexuous, uncinate or circinate, sometimes terminating into a phialide. In the nodes grow collar-like hyphae from which arise phialides and setae. Conidiogenous cells phialidic, discrete, borne on a collar-like hypha in whorls or directly on the conidiophore, or integrated terminal, conidiogenous locus with multiple loci, sometimes protruding above the collarette; collarettes flaring, inconspicuous. Conidia ellipsoidal, ellipsoidal-oblong, subglobose, occasionally T- or U-shaped to almost deltoid, mostly hyaline, sometimes subhyaline to pale olivaceous, aseptate, adhering in heads or cirrhi. Chlamydospores absent or present; lateral, terminal, intercalary, sessile or on a stipe, solitary, in short chains or clusters, aseptate, brown, thick-walled, smooth. The phialocephala-type conidiophores absent or present; macronematous, mononematous, solitary or fasciculate, erect, composed of a penicillately branched head and a stipe that is cylindrical to subulate, stout, straight or slightly flexuous, brown, proliferating, base slightly inflated, bulbous or composed of a true foot cell. Conidiogenous apparatus compact, with dense penicillate branching, consisting of several levels of hyaline or pale brown branches and conidiogenous cells arising in groups from the tip of metulae. Conidiogenous cells monophialidic, cylindrical to lageniform, integrated, terminal, hyaline to subhyaline, extending percurrently, conidiogenous locus with multiple loci sometimes protruding above the collarette, with annellations; collarettes short, flaring or inconspicuous. Conidia ellipsoidal to obovate, cuneate to rhomboid, subhyaline, becoming pale brown after detachment, aseptate, smooth, accumulating in slimy glossy heads. Chlamydospores absent. The pseudogliomastix-type conidiophores absent or present; semimacronematous or micronematous with a loosely penicillately branched head or reduced to scattered single phialides on undifferentiated hyphae, solitary, erect or slightly decumbent, subhyaline, later elongated and pigmented. Conidiogenous cells monophialidic, cylindrical, integrated, terminal, hyaline or subhyaline, conidiogenous locus with multiple loci; collarettes short, flaring or indistinct. Conidia mostly obovate, sometimes with a basal scar, hyaline to subhyaline becoming olivaceous brown, smooth, accumulating in heads or irregularly shaped slime. Chlamydospores absent. Sexual morph. Ascomata perithecial, non-stromatic, superficial, glabrous or setose, setae always sterile, acute. Ostiole periphysate. Paraphyses present, disintegrating with age. Asci unitunicate, stipitate, arising from croziers, with a non-amyloid, refractive apical annulus. Ascospores fragmenting within the ascus into part spores, fusiform, hyaline, 1-septate, finely verrucose, or smooth.

Habitat and geographical distribution: Members of Chloridium are saprobes on decaying plant material in terrestrial and freshwater habitats, especially on wood in advanced stages of decay, buried in soil or leaf litter; many species are soil dwellers. Members of Chloridium occur worldwide, mostly in temperate, subtropical, or tropical regions. In contrast, only a few species inhabit the boreal and tundra biomes. Typical habitats are moist temperate, and subtropical forests.

Notes: The present phylogenetic analyses confirmed that the genera Chloridium, Gonytrichum, and Pseudophialocephala are closely related and form a monophyletic, well-supported clade with eight subclades that share some morphological characteristics. These subclades are introduced as sections: Chloridium, Cryptogonytrichum, Gonytrichopsis, Gonytrichum, Mesobotrys, Metachloridium, Pseudophialocephala, and Volubilia (Figs 5, ​,6).6). The generic concept has been emended. Chloridium is presented as a polythetic taxon with a new infrageneric classification. The disposition of species of genera Bisporomyces, Cirrhomyces, Gonytrichum, Mesobotrys, and Pseudophialocephala, accepted as synonyms of the genus Chloridium, is given in Table 3.

Epitypification was considered for all known species which lack the type strain. In the end, only strains of Chl. fuscum and Chl. subglobosum were suitable for epitype selection. Strains of other species come from different countries and even continents than the holotypes and cannot be considered suitable candidates.

A specific mode of phialidic conidiogenesis occurs in all members of the genus Chloridium. The formation of conidia on multiple loci within the collarette represents a modification of phialidic conidiogenesis (Hammill 1972, Gams & Holubová-Jechová 1976, Onofri et al. 1994). Two, usually unequally sized, successively formed conidia are often seen at the tip of the conidiogenous locus (Fig. 13A–D). The phialides extend percurrently, which is more pronounced in older cultures, giving the conidiophores a slightly geniculate appearance. The conidiogenous locus often protrudes beyond the collarette, even to several micrometres, and may also extend percurrently after each conidium bearing several minute annellations. The annellations are scarcely visible with a light microscope, preferably using phase contrast, as “closely-spaced progressive series of ring-like scars encircling the cell” (Seifert et al. 2011). The meristem remains active, and as the phialide repeatedly percurrently extends, clusters of conidia remain attached in place at the site of the original collarette and conidiogenous locus (Fig. 13E, M). The phialides are either terminal at the tip of the conidiophore or arise from metulae, or are discrete in whorls or solitary, lateral, producing conidia in heads or cirrhi (Fig. 13).

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Fig. 13.

Conidiogenesis, phialides and conidial formations in the genus Chloridium. A–D. Phialides with conidia borne on multiple conidiogenous loci. E. Conidiophores with conidial heads attached after each percurrent elongation of the phialide. F. Conidiophores with conidial heads and cirrhi. G, H. Phialides arranged in whorls, growing from collar-like hyphae. I. Phialides solitary or in pairs, with and without collar-like hyphae. J. Phialides growing from metulae at the tip of a penicillately branched conidiophore. K, L. Conidia adhering in cirrhi. M–O. Conidia adhering in heads. Scale bars: A–D = 5 μm; E = 100 μm; F = 200 μm; G–N = 10 μm; O = 50 μm.

Sections of Chloridium are characterised by a particular conidiophore morphotype (Fig. 14) that corresponds to the morphology of four hyphomycetous genera and includes simple and more complex forms named the chloridium-, gonytrichum-, phialocephala- and pseudogliomastix-types. Sometimes pairs of the conidiophore types occur together and form identical or nearly identical conidia. In each pair, a simple form (chloridium- or pseudogliomastix-type) of its more complex counterpart (gonytrichum- and phialocephala-type) is present. Because of their ontogeny, co-occurrence in nature, loss of the ability to form complex conidiophores in favour of simple ones, and identical conidia borne on these morphotypes (explained below, also see Discussion), we do not consider them synasexual morphs and prefer to use the term “conidiophore type”.

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Fig. 14.

Conidiophore types distinguished in the genus Chloridium. A, B. Chloridium-type. C–I. Gonytrichum-type. J. Phialocephala-type. K. Pseudogliomastix-type. Scale bars: A = 25 μm; B, J, K = 20 μm; C–I = 50 μm.

The chloridium-type conidiophores occur in all sections except Pseudophialocephala. Members of sections Chloridium, Cryptogonytrichum, and Metachloridium produce only this type of conidiophore. Whether the conidiophores form in one or two layers is considered a species-specific trait that can be observed especially on natural substrate. Conidiophores of the chloridium-type occur together with the gonytrichum-type in some species. In the section Mesobotrys, the chloridium-type conidiophores occur in culture as an early developmental stage, and soon, first collar-like hyphae are formed in the nodes. Similar in vitro observations were made in Chl. chloroconium (section Gonytrichopsis). Such ontogeny is challenging to observe on the natural material; we usually see solitary or caespitose chloridium-type conidiophores at the margin of the colony. After repeated in vitro transfers, the ability to form collar-like hyphae decreases and may even be absent, but it varies among species. The loss of this ability and formation of the chloridium-type conidiophores instead of the gonytrichum-type occurs in both varieties of Chl. chloridioides and was regularly observed in Chl. volubile and Chl. chloroconium (see also Barron & Bhatt 1967).

Species of the Gonytrichopsis, Gonytrichum, Mesobotrys, and Volubilia sections produce gonytrichum-type conidiophores. The collar-like (encircling) hyphae are borne below the septa of the central stalk and setae. At first, they appear as a hyaline to subhyaline outgrowth that enlarges, grows forward, and closely encircles the conidiophore as a collar. The septa develop later, and the collar-like hypha becomes pigmented. The phialides and setae are borne on the collar-like hyphae and not directly on the conidiophore. Thus, the conidiophore and setae are essentially unbranched. This concept was introduced by Hughes (1951) on the example of Chl. caesium, and is accepted in this study. However, there are a few exceptions. Chloridium mirabile is unique in that it lacks collar-like hyphae, or they occur infrequently. The discrete phialides and lateral setae are borne directly on the conidiophore; they have a broadly lobate base that encircles the conidiophore in the same way as a collar-like hypha. The conidiophores of the section Gonytrichum are one of the most complex structures in the genus Chloridium. Conidiophores and lateral setae are geniculate in the nodes; they grow in all directions, the collar-like hyphae form along their entire length, and the conidiophores assume an arborescent appearance (Fig. 14C–E). In other sections with the gonytrichum-type conidiophores, the geniculation at the nodes is absent or less pronounced, collar-like hyphae and setae form in the lower fertile part of the conidiophore, and the conidiophores are subulately tapering and point upwards in one direction (Fig. 14F–I).

Chlamydospores (Fig. 15) are found only in culture and can be used as a diagnostic marker to distinguish between sections and, to some extent, at the species level. They are solitary, in chains or irregular clusters on vegetative mycelium, and consistently present or absent in individual species.

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Fig. 15.

Chlamydospores in the genus Chloridium. A–C. Lateral, on stipe or sessile, sometimes intercalary. D. Intercalary in long chains. E, F. Intercalary, solitary or in short chains. G. Intercalary, in series or clusters. Scale bars: A–F = 10 μm; G = 5 μm.

Regarding growth at different temperatures, cardinal temperature values vary from species to species and represent a good tool for distinguishing the taxa of the genus Chloridium. The ability to distinguish all varieties within a species is remarkable. Optimum growth is between 20–30 °C, with frequent ability to grow also at 5 °C. Only two species show good growth at 37 °C (Chl. guttiferum and some isolates of Chl. bellum). Eight species were unable to grow at 30 °C. In general, Chloridium is mesophilic, with a good ability to tolerate lower temperatures (5 °C, 10 °C) and is less able to grow at temperatures above 37 °C. (Fig. 16, Table S4).

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Fig. 16.

Growth of Chloridium spp. at different temperatures (5–37 °C) on MEA after 14 d and their phylogenetic relationships.

Key to the sections

1a. Conidiophores unbranched ....................................................................................................................................................................... 2

1b. Conidiophores with a condensed branching system ............................................................................................................................................................... Pseudophialocephala

2a. Conidiophores lacking collar-like hyphae; phialides integrated, terminal ................................................................................................ 3

2b. Conidiophores with collar-like hyphae; phialides integrated, terminal and discrete, lateral ............................................................................................................................................................... 5

3a. Chlamydospores absent ............................................................................................................................................................... Cryptogonytrichum

3b. Chlamydospores present ............................................................................................................................................................... 4

4a. Conidia hyaline, sometimes pale brown in old material; conidiophores cylindrical; chlamydospores solitary, intercalary, lateral, terminal, rarely in short chains ............................................................................................................................................................... Chloridium

4b. Conidia pale brown; conidiophores subulately tapering; chlamydospores solitary, intercalary, sometimes lateral, with age arranged in long chains or clusters ............................................................................................................................................................... Metachloridium

5a. Collar-like hyphae formed along the entire length of the conidiophore and setae, which become geniculate; conidiophores cylindrical, assuming an arborescent appearance; chlamydospores absent ............................................................................................................................................................... Gonytrichum

5b. Collar-like hyphae formed in the lower fertile part of the conidiophore; conidiophores are subulately tapering and point upwards in one direction ............................................................................................................................................................... 6

6a. Chlamydospores absent ............................................................................................................................................................... Mesobotrys

6b. Chlamydospores present ............................................................................................................................................................... 7

7a. Chlamydospores abundant, in conspicuous series and clusters ............................................................................................................................................................... Gonytrichopsis

7b. Chlamydospores rare, solitary ............................................................................................................................................................... Volubilia

Chloridium section Chloridium Link

Type species: Chloridium virescens (Pers.) W. Gams & Hol.-Jech.

Description: Colonies effuse, hairy, brown, becoming whitish, green to yellow-green, yellow, beige, olivaceous grey or amber when sporulating; composed of conidiophores and ascomata. Asexual morph. The chloridium-type conidiophores produced in one or two layers, solitary, scattered, crowded or caespitose, unbranched, but sometimes branched in culture, often with percurrent proliferations, brown, cylindrical, slightly tapering apically. Conidiogenous cells phialidic, terminal, integrated, extending percurrently, occasionally sympodially in culture, paler than the conidiophore; collarettes flaring, shortly funnel-shaped, inconspicuous. Conidia ellipsoidal, ellipsoidal-oblong, subglobose, obovate, suballantoid, hyaline, occasionally turn pale brown in older cultures, aseptate, smooth, accumulating in heads or cirrhi. The gonytrichum-type conidiophores absent. Chlamydospores present, they are lateral, terminal or intercalary, aseptate, brown, thick-walled, smooth. Sexual morph. Ascomata perithecial, non-stromatic, superficial, glabrous or setose; setae always sterile, acute. Ostiole periphysate. Paraphyses present, disintegrating with age. Asci unitunicate, stipitate, with a non-amyloid apical annulus. Ascospores fragmenting within the ascus, fusiform, hyaline, 1-septate, finely verrucose.

Notes: The section Chloridium comprises 19 species and varieties. Because of their extensive morphological homoplasy, especially in culture, and lack of data from nature, a synoptic table with salient features of each species is provided (Table 4).

Chloridium bellum var. bellum Réblová & Hern.-Restr., sp. nov. MycoBank MB 846081. Fig. 17, ​,1818.

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Fig. 17.

Chloridium bellum var. bellum. A–C. Colonies. D–G. Conidiophores. H. Conidia. I. Chlamydospores. A–I. On CMA with Urtica stems. Images: A, C, D, E. CBS 127310; B, F–I. CBS 709.73A. Scale bars: A = 500 μm; B, C = 250 μm; D, E, I = 20 μm; F–H = 10 μm.

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Fig. 18.

Diversity of colony morphology in Chl. bellum on CMD, MLA, OA, PCA after 4 wk. A–C. Chl. bellum var. bellum (A. CBS 709.73A; B. CBS 709.73B; C. CBS 127310). D. Chl. bellum var. luteum CBS 141.54. Scale bar = 1 cm.

Etymology: Bellus (Latin) beautiful, pretty, referring to the lovely long conidial cirrhi.

Typus: Germany, Schleswig-Holstein, Kiel-Kitzeberg, isolated from wheat-field soil, date unknown, W. Gams No. C 339 (holotype CBS H-25016, culture ex-type CBS 709.73A).

Culture characteristics: On CMD: colonies 62–64 mm diam, circular, flat, entire, velvety becoming mucoid, cobwebby at the margin, beige-brown, darker at the margin, with white zones of aerial mycelium, reverse of the same colour. On MLA: colonies 75–77 mm diam, circular, slightly raised centrally, flat margin, margin entire, velvety to cobwebby, locally mucoid, deeply furrowed, zonate, grey to beige grey centrally, olivaceous brown at the mucoid spots, cinnamon-brown towards the margin, reverse olivaceous grey to brown. On OA: colonies 84–85 mm diam, circular, flat, margin entire, cobwebby, sometimes with irregular mucoid zones, dark grey brown with white grey conidial masses, olivaceous grey to brown at the margin, reverse black. On PCA: colonies 77–80 mm diam, circular, flat, margin entire, velvety, cobwebby to mucoid towards the margin, olivaceous black, locally whitish beige due to conidial masses, reverse of the same colour. Sporulation was absent on CMD, abundant on MLA, OA and PCA. Growth at 5–37 °C with an optimum at 30 °C.

Description in culture: On MLA, vegetative hyphae 1.5–3.5 μm diam, branched, septate, hyaline and smooth, becoming pale brown and encrusted. Asexual morph. The chloridium-type conidiophores 67–278(–330) × 2.5–3.5 μm, macronematous, solitary, erect, unbranched, commonly with 3–6 percurrent proliferations, and with up to 15 proliferations in older cultures (>8 wk) assuming a conspicuously geniculate appearance, cylindrical, straight or flexuous, brown. Conidiogenous cells 12–29 × 2.5–3 μm, tapering to 1.5–2 μm below the collarette, monophialidic, subcylindrical to lageniform, integrated, terminal, pale brown to subhyaline, paler towards the apex, extending percurrently, collarettes 2.5–4 μm wide, 1.5–2 μm deep, flaring, pale brown to subhyaline. Conidia 3.5–5 × 2–3 μm (mean ± SD = 4.3 ± 0.4 × 2.4 ± 0.2 μm), ellipsoidal to slightly obovate, hyaline, aseptate, smooth, accumulating in slimy whitish heads and cirrhi. Chlamydospores 4.5–7.5 × 3.5–4.5 μm, intercalary or lateral, sessile or on a short stalk, sometimes terminal, subglobose, ellipsoidal or pyriform, truncate at the base, brown, thick-walled, smooth, with age becoming verrucose. Sexual morph. Unknown.

Additional specimens examined: Germany, Schleswig-Holstein, Kiel-Kitzeberg, isolated from wheat-field soil, date unknown, W. Gams No. C 773 (CBS 709.73B). USA, Kansas, isolated from soil, date unknown, collector unknown (CBS 127310).

Habitat and geographical distribution: Saprobe in soil, known from Germany and USA. According to GlobalFungi, identical sequences were found in 2 549 samples from 97 studies, collected primarily in forest soil (topsoil, bulk soil, and rhizosphere soil), croplands and grasslands on all continents. The species is typical of temperate, subtropical, or tropical climatic regions but is almost absent in the boreal and tundra regions of Europe and North America (MAT 13.1 °C, MAP 1 114 mm).

Notes: The Chl. bellum clade (79/1.0) comprises four strains; three strains isolated from soil and identified initially as Chl. virescens var. caudigerum (CBS 141.54, CBS 709.73A, CBS 709.73B) (Gams & Holubová-Jechová 1976), and Chl. virescens var. virescens (CBS 127310). Chloridium bellum has two incarnations, var. bellum and var. luteum, whose identity inside the single species, Chl. bellum, was demonstrated by STACEY analysis. The subclade containing three isolates (CBS 709.73A, CBS 709.73B, CBS 127310) that originate from the northern temperate zone (Germany, USA) represents the type variety. The conidia of var. bellum adhere predominantly in heads, occasionally in cirrhi that form only in older cultures (>8 wk). Conidial clusters are whitish to white grey in mass on all four media and have longer conidiophores that are typically geniculate due to frequent percurrent elongation of the phialides. The chlamydospores are slightly narrower. The lineage represented by the strain CBS 141.54 from Central Africa (Democratic Republic of the Congo) is described as var. luteum. It differs from var. bellum in shorter conidiophores and conidia that are dark yellow in the mass on OA and change to amber in older cultures (>12 wk) and produce a pale yellow soluble pigment on MLA.

Although the two groups of strains have similar colony morphology and may be difficult to distinguish in culture, traits such as the cardinal temperatures and growth optima, the colour of the conidia in the mass and their preferred mode of clustering, the conspicuous elongation of the conidiophores in vitro and genetic divergence in the two coding loci justify their distinction as varieties.

Chloridium bellum is characterised by simple conidiophores with ellipsoidal to slightly obovate conidia adhering in heads or cirrhi and mostly lateral or terminal chlamydospores. Chloridium setosum is similar to Chl. bellum but differs in shorter, ellipsoidal to subglobose conidia, branched conidiophores (in culture), and colony characteristics with mostly dark cinnamon or chestnut brown tones.

Chloridium bellum var. luteum Réblová & Hern.-Restr., var. nov. MycoBank MB 846706. Fig. 18, ​,1919.

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Fig. 19.

Chloridium bellum var. luteum (CBS 141.54). A, B. Colonies. C–H. Conidiophores. I–K. Conidia arranged in cirrhus (I). L–N. Chlamydospores. A, B. On MLA. C–N. On PCA. Scale bars: A = 250 μm; B = 100 μm; C, L–N = 20 μm; D–K = 10 μm.

Etymology: Luteus (Latin) yellow, referring to the yellow pigment diffusing in the agar and the yellow colour of the conidia in the mass.

Typus: Democratic Republic of the Congo, Yangambi, isolated from soil, date unknown, J.A. Meyer No. 4747 (holotype CBS H-10170, culture ex-type CBS 141.54 = MUCL 15746).

Culture characteristics: On CMD: colonies 56–64 mm diam, circular, flat, margin fimbriate, smooth, mucoid, locally cobwebby, colony centre yellowish-beige, pale brown towards the margin, reverse pale brown. On MLA: colonies 76–77 mm diam, circular, flat, margin fimbriate, mucoid centrally, cobwebby to velvety-lanose towards the periphery, beige grey, yellowish grey at the centre, cinnamon brown to grey-brown towards the margin, pale yellow pigment diffusing into the surrounding agar, reverse olivaceous brown. On OA: colonies 84–86 mm diam, circular, flat, margin fimbriate, cobwebby, farinose when sporulating, olivaceous yellow due to conidial masses, dark grey to almost black at the margin, reverse dark grey. On PCA: colonies 80–82 mm diam, circular, flat, margin fimbriate, farinose when sporulating, beige due to conidial masses, dark brown towards the periphery, reverse dark chocolate brown. Sporulation was abundant on all media, delayed after 12 wk on CMD. Growth at 10–30 °C with an optimum at 25 °C.

Description in culture: On PCA, vegetative hyphae 1.5–3.5 μm diam, branched, septate, often adhering in aerial strands, hyaline, smooth, later pale brown and encrusted. Asexual morph. The chloridium-type conidiophores 60–182 × 2.5–4.5 μm, macronematous, solitary, erect, unbranched, commonly with 1–4 percurrent proliferations, cylindrical to somewhat subulately tapering upwards, straight or flexuous, brown, darker near the base, gradually paler towards the apex. Conidiogenous cells 15–30(–37) × 2.5–3 μm, tapering to 1.5–2 μm below the collarette, monophialidic, subcylindrical to lageniform, integrated, terminal, pale brown to subhyaline, paler towards the apex, extending percurrently; collarettes 2–3 μm wide, ca. 1.5 μm deep, subhyaline, flaring, shortly funnel-shaped. Conidia 3.5–5 × (2–)2.5–3 μm (mean ± SD = 4.3 ± 0.4 × 2.5 ± 0.2 μm), ellipsoidal to obovate, hyaline becoming pale brown upon ageing, aseptate, smooth, accumulating in slimy heads and cirrhi, conidia in mass whitish to white grey (CMD, MLA, and PCA), becoming olivaceous grey (MLA), or yellow to dark yellow turning to amber (OA), especially in older cultures (>12 wk). Chlamydospores 5–8 × 4–5 μm, lateral, sessile or on a stalk, or terminal, seldom intercalary, ellipsoidal, subglobose or pyriform, truncate at the base, brown, thick-walled, smooth becoming verrucose with age. Sexual morph. Unknown.

Habitat and geographical distribution: Saprobe in soil from the Democratic Republic of the Congo. According to GlobalFungi, identical sequences were found in 842 samples from 33 studies, collected mainly in forest soil (topsoil, bulk soil, and rhizosphere soil), croplands and grasslands on all continents except Antarctica. It typically occurs in temperate, subtropical, or tropical climatic regions but is almost absent in the boreal and tundra regions of Europe and North America (MAT 16.5 °C, MAP 1 161). Its distribution overlaps with Chl. bellum var. bellum.

Notes: Chloridium bellum var. luteum produces conidia accumulating mainly in the cirrhi in vitro. A distinctive feature is yellow to the dark yellow colour of conidia in the mass on OA. For comparison with the var. bellum, see notes in the latter taxon. Among other taxa, Chl. bellum var. luteum, together with Chl. guttiferum, is unique in its ability to grow at 37 °C.

Chloridium biforme Réblová & Hern.-Restr., sp. nov. MycoBank MB 846707. Fig 20.

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Fig. 20.

Chloridium biforme (ICMP 23429). A. Ascomata with conidiophores. B. Colony. C, D. Asci. E. Ascospores. F, G, N. Conidiophores. H–K. Conidia. L. Tip of the phialide. M. Chlamydospores. O. Colonies on CMD, MLA, OA, PCA (from left to right) after 4 wk. A–E, G. From nature. F, H–J, L. On MLA. K. On CMA with Urtica stems. M, N. On PCA. Scale bars: A, B = 250 μm; C–E, H–K = 10 μm; F, G, M, N = 20 μm; L = 5 μm; O = 1 cm.

Etymology: Bifôrmis (Latin) having two forms, referring to the conidiophores and ascomata that occur together on a natural substrate.

Typus: New Zealand, Southland Region, Fiordland National Park, Lake Monowai 40 km S of Manapouri, Borland Nature Walk, on decaying wood of Nothofagus sp., 9 Mar. 2005, M. Réblová M.R. 3173/ NZ 422 (holotype PDD 120397, culture ex-type ICMP 23429).

Description on the natural substrate: Colonies effuse, hairy, dark brown, white grey when sporulating; composed of conidiophores and ascomata. Asexual morph. The chloridium-type conidiophores 67–134 × 2.5–5 μm, macronematous, solitary, growing on ascomata or nearby wood, erect, straight, unbranched, with 1–3 percurrent proliferations, cylindrical, slightly tapering upwards, septate, brown, paler towards the apex. Conidiogenous cells 10.5–20(–24) × 2.5–3.5(–4) μm, tapering to 2–2.5 μm below the collarette, monophialidic, integrated, terminal, extending percurrently, subcylindrical to lageniform, subhyaline, pale brown at the base; collarettes 3.5–4 μm wide and 1.5–2 μm deep, hyaline, flaring, shortly funnel-shaped. Conidia 4.5–5.5 × 2.5–3.5 μm (mean ± SD = 4.9 ± 0.5 × 3.0 ± 0.2 μm), ellipsoidal, hyaline, aseptate, smooth, accumulating in whitish heads and cirrhi. Sexual morph. Ascomata 186–220 μm diam, 200–250 μm high, non-stromatic, superficial, solitary or aggregated, subglobose to conical, papillate, dark brown to black, glabrous, glossy. Ostiole periphysate. Ascomatal wall 29–36 μm thick, fragile, carbonaceous, two-layered. An outer layer consisting of brown, more or less polyhedral cells with opaque walls. An inner layer composed of several rows of elongated, thin-walled, hyaline cells. Paraphyses 2.5–4 μm, septate, branched, longer than the asci, disintegrating with age. Asci 74.5–88.5 × (4.5–)5.5–6.5 μm (mean ± SD = 80.7 ± 5 × 66.9 ± 4.3 μm), cylindrical-clavate, stipitate, ascal apex obtuse to broadly rounded with a non-amyloid apical annulus ca. 2 μm wide, 1.5 μm high. Ascospores easily fragmenting within the ascus, part spores 4–5.5 × 2.5–3.5 μm (mean ± SD = 4.7 ± 0.4 × 2.8 ± 0.2 μm), fusiform, slightly asymmetrical, the upper half is slightly shorter and wider, hyaline, 1-septate, finely verrucose, obliquely uniseriate or 1–2-seriate in the ascus.

Culture characteristics: On CMD: colonies 46–48 mm diam, circular, slightly raised, margin fimbriate, cobwebby, floccose, somewhat zonate, creamy, with grey-brown centre and irregular pigmented zones due to chlamydospore production, reverse beige. On MLA: colonies 36–40 mm diam, circular, raised, margin entire to weakly fimbriate, velvety-lanose, deeply furrowed, brown grey, whitish at the margin with a beige-ochre outer zone of submerged growth, reverse olivaceous beige. On OA: colonies 48–55 mm diam, flat, margin entire, cobwebby to mucoid, zonate, grey-isabelline, centre brown, with circular olivaceous zones, with colourless droplets of the exudate, reverse olivaceous grey. On PCA: colonies 52–53 mm diam, circular, slightly convex, margin lobate, lanose, cobwebby at the margin, cinnamon brown, olivaceous brown at the margin, with an outer milky zone of submerged growth, reverse brown. Sporulation was abundant on all media. Growth at 5–25 °C with an optimum at 25 °C.

Description in culture: On MLA, vegetative hyphae 1.5–2.5 μm diam, branched, septate, hyaline to subhyaline, smooth, later pale brown and encrusted. Asexual morph. Conidiophores, conidiogenous cells, and conidia are similar to those from nature. The chloridium-type conidiophores 43–141(–260) × 3–4.5 μm, with up to eight percurrent proliferations. Conidiogenous cells 20.5–26.5 × 2.5–3.5 μm, tapering to 1.5–2 μm below the collarette, conidiogenous locus protruding 3–4.5 μm above the collarette; collarettes 3–3.5 μm wide and 1–1.5 μm deep, subhyaline, inconspicuous. Conidia (3–)3.5–4.5 × 2–2.5 μm (mean ± SD = 3.9 ± 0.3 × 2.3 ± 0.1 μm), broadly ellipsoidal to suballantoid, hyaline, accumulating in whitish or white-yellow slimy heads that turn olivaceous grey in older cultures (>12 wk). Chlamydospores 4–6 × 3.5–5 μm, usually lateral on stipe or terminal, subglobose, ellipsoidal, brown, smooth, thick-walled. Sexual morph. Not observed.

Habitat and geographical distribution: Saprobe on decaying wood of Nothofagus sp., known from New Zealand. According to GlobalFungi, identical sequences were found in 58 samples from three studies, originating from soil and roots from forest, cropland, shrubland, and grassland biomes in Australasia. It occurs in locations of temperate or xeric climate (MAT 11.6 °C, MAP 1 051 mm). Chloridium biforme is one of the few species with a limited distribution range.

Notes: The shape of conidia in culture can vary from broadly ellipsoidal to suballantoidal; suballantoidal shapes predominating on CMA. Chloridium biforme is similar to Chl. virescens in characters of ascomata, asci, and ascospores, but is well distinguished by a single layer of conidiophores and conidia arranged in short whitish cirrhi and sometimes heads.

Chloridium caudigerum (Höhn.) S. Hughes, Canad. J. Bot. 36: 748. 1958. Fig. 21, ​,2222.

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Fig. 21.

Chloridium caudigerum. A, B. Colonies composed of ascomata and conidiophores. C. Asci. D. Ascospores. E. Seta. F, G. Conidia. H–J, P. Conidiophores. K, L. Upper parts of conidiophores with percurrently elongating phialides (arrows indicate elongated parts of phialides with active meristem and visible annellations). M–O. Chlamydospores. A–F, H, I. From nature. G, J–L, P. On MLA. M–O. On CMD. Images: A, C–E, J–O. ICMP 22547; B, F. CBS 138691. G, P. CBS 248.75; H, I. CBS 145490. Scale bars: A, B = 250 μm; C–G, M–O = 10 μm; H–L, P = 20 μm.

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Fig. 22.

Diversity of colony morphology in Chl. caudigerum on CMD, MLA, OA, PCA after 4 wk. A. CBS 248.75. B. CBS 675.74. C. CBS 146196. D. CBS 145489. E. ICMP 22547. Scale bar = 1 cm.

Basionym: Cirrhomyces caudiger Höhn. [as ‘caudigerus’], Ann. Mycol. 1: 529. 1903.

Synonyms: Chloridium virescens var. caudigerum (Höhn.) W. Gams & Hol.-Jech., Stud. Mycol. 13: 19. 1976.

Botryonipha capillamentosa (Preuss) Kuntze, Revis. Gen. Pl. 2: 845. 1891.

Stilbella capillamentosa (Preuss) Lindau, Rabenh. Krypt.-Fl. 1(9): 292. 1908.

? Stilbum capillamentosum Preuss, Linnaea 24: 132. 1851.

Sphaeromycetella leucocephala Arnaud, Bull. Trimestriel Soc. Mycol. France 69: 274. 1953. (Nom. inval. Art. 36).

Synonymy was adopted after Gams & Holubová-Jechová (1976).

Description on the natural substrate: Colonies effuse, brown to black, white grey when sporulating; composed of conidiophores and ascomata. Asexual morph. The chloridium-type conidiophores (60–)108–170(–235) × 3.5–4.5 μm, macronematous, solitary, crowded or in groups, erect, straight, occasionally slightly bent, septate, unbranched, with 1–3 percurrent proliferations, cylindrical, base bulbous, brown, paler towards the apex. Conidiogenous cells 18–28 × 3–4 μm, tapering to 2–2.5 μm below the collarette, monophialidic, integrated, terminal, extending percurrently, subcylindrical to lageniform, pale brown to subhyaline; collarettes 3.5–4 μm wide and 1.5(–2) μm deep, subhyaline, flaring, short funnel-shaped. Conidia 3.5–4.5(–5) × 2.5–3 μm (mean ± SD = 4.0 ± 0.4 × 2.3 ± 0.1 μm), ellipsoidal to oblong, hyaline, ageing conidia turn pale brown, aseptate, smooth, accumulating in slimy whitish to white-yellow heads or cirrhi. Sexual morph. Ascomata 150–190 μm diam, 165–200 μm high, non-stromatic, superficial, subglobose, papillate, dark brown, setose, often covered with conidiophores; setae 24–66 × 2–2.5 μm, brown, septate, acute, always sterile. Ostiole periphysate. Ascomatal wall 25–30 μm thick, fragile, carbonaceous, two-layered. An outer layer consisting of brown, more or less polyhedral cells with opaque walls. An inner layer composed of several rows of elongated, thin-walled, hyaline cells. Paraphyses 1.5–3.5 μm wide, septate, branched, longer than the asci, disintegrating with age. Asci (70–)74.5–84.5 × (5–)5.5–6.5 μm (mean ± SD = 77.3 ± 4.8 × 5.9 ± 0.5 μm), cylindrical-clavate, short-stipitate, ascal apex obtuse to broadly rounded with a non-amyloid apical annulus ca. 1.5–2 μm wide, 1–1.5 μm high. Ascospores fragmenting within the ascus (but somewhat less easily than in other Chloridium species), part spores 4–5.5 × 2.5–3.5 μm (mean ± SD = 4.7 ± 0.4 × 3.1 ± 0.2 μm), fusiform, slightly asymmetrical with the upper part slightly shorter or almost symmetrical, hyaline, 1-septate, finely verrucose, obliquely uniseriate in the ascus.

Culture characteristics: On CMD: colonies 48–50 mm diam, circular, flat, margin entire to weakly fimbriate, velvety to cobwebby, mucoid towards the margin, brown, beige centrally, sometimes with whitish outer zone, reverse brown. On MLA: colonies 36–38 mm diam, circular, convex centrally, flat margin, margin entire, velvety-lanose, somewhat floccose, furrowed, cobwebby to mucoid at the margin, ash grey to beige-brown at the centre, brown towards the periphery, olivaceous brown at the margin, reverse olivaceous brown. On OA: colonies 65–70 mm diam, circular, flat, margin entire to undulate, cobwebby to sparsely velvety with irregular smooth and mucoid spots, beige, colony centre olivaceous yellow when sporulating, becoming brown, dark brown to dark grey towards the periphery, reverse brown. On PCA: colonies 52–66 mm diam, circular, flat, margin entire to fimbriate, velvety, seldom smooth and mucoid towards the periphery, beige-brown, dark brown towards the margin, with a beige outer zone of submerged growth, reverse brown. Sporulation was absent on CMD, abundant on MLA, OA, and PCA. Growth at 5–30 °C with an optimum at 25 °C.

Description in culture: On MLA, vegetative hyphae 1.5–3 μm diam, branched, septate, hyaline, smooth, becoming pale brown, encrusted. Asexual morph. Conidiophores, conidiogenous cells and conidia are similar to those from nature. The chloridium-type conidiophores 118–384(–430) × 3–3.5 μm, with up to 10 percurrent proliferations. Conidiogenous cells 20–26.5 × 2.5–3 μm, tapering to 1.5–2 μm below the collarette, conidiogenous locus protruding 1.5–2.5 μm above the collarette, in places where the meristem remains active after regeneration of the phialide additional minute annellations are formed intercalary; collarettes 3.5–4 μm wide and 1–1.5 μm deep, subhyaline to pale brown, flaring, inconspicuous. Conidia 3.5–4.5(–5) × 2.5–3 μm (mean ± SD = 4.2 ± 0.4 × 2.3 ± 0.2 μm), ellipsoidal to oblong, occasionally some conidia slightly truncate at the base, hyaline, smooth, accumulating in slimy heads, whitish in mass (PCA, CMD) or becoming yellowish to olivaceous yellow (MLA, OA), especially in older cultures (>12 wk). Chlamydospores 5–6.5 × 4–4.5(–5) μm, terminal, lateral and intercalary, globose, subglobose or ellipsoidal, brown, thick-walled, smooth-walled, solitary, rarely in a chain of two or three. Sexual morph. Not observed.

Specimens examined: Austria, Wiener Wald, Gelber Berg, on decaying wood of Quercus sp., 29 May 1904, F. von Höhnel (neotype of Cirrhomyces caudiger FH 1552). Belgium, Ardennes, Le Roptai near Ave-et-Auffe, on decaying wood, Sep. 1975, W. Gams (CBS 601.75A); Ardennes, Grande Tinémont near Han-sur-Lesse, on decaying wood, Sep. 1975, W. Gams (CBS 621.75). Czech Republic, Central Bohemian Region, Úpor Forest near Mělník, on decaying wood of Populus nigra, 24 Jun. 1976, V. Holubová-Jechová (CBS 421.76); Pardubice Region, Železné hory Mts. Protected Landscape Area, Horní Bradlo, Malá Střítež settlement, Polom National Nature Reserve, alt. 600 m, on decaying wood of Acer pseudoplatanus, 10 Nov. 2018, M. Réblová M.R. 4069 (PRA-21331, CBS 145488); ibid., M.R. 4074 (PRA-21332, CBS 145489); ibid., on decaying wood of Fagus sylvatica, M.R. 4077 (PRA-21333, CBS 145490); South Moravian Region, Lanžhot, Ranšpurk National Nature Reserve, on decaying wood of Carpinus betulus, 26 Oct. 2018, M. Réblová M.R. 4010 (PRA-21334, CBS 145430); South Moravian Region, Valtice, Rendez-vous National Nature Monument, on decaying wood of Quercus cerris, 28 Oct. 2018, M. Réblová M.R. 4029 (PRA-21335, CBS 145432); ibid., M.R. 4035 (PRA-21336, CBS 145433); ibid., M.R. 4040A (CBS 146196); South Bohemian Region, Novohradské hory Mts., Dobrá voda, Hojná voda National Nature Reserve, on decaying wood of Fagus sylvatica, 13 Oct. 2003, M. Réblová M.R. 3819 (PRA-21337, CBS 138691). France, Plateau du Lubéron, on decaying wood of Cedrus atlantica, Oct. 1975, W. Gams (CBS 248.75); Arboretum Champenoux-Nancy, on decaying wood of Carya alba, Sep. 1974, W. Gams (CBS 675.74). New Zealand, Canterbury Region, Rangitata Orari Bridge Highway 79, Peel Forest ca. 23 km N of Geraldine, on decaying wood of Nothofagus sp., 6 Mar. 2005, M. Réblová M.R. 3165/NZ 412 (PDD 120398, ICMP 22547). Spain, Cantabria, San Pedro de Romeral, Collados de Ason Natural Park, on twig, 27 Sep. 2010, M. Hernández-Restrepo, J. Mena Portales & J. Guarro (FMR 11338 = IMI 500655); Castilla y Leon, Cerro de la Dehesa, isolated from soil, 9 Nov. 2010, M. Hernández-Restrepo & J. Gené (FMR 12411). The Netherlands, Utrecht Province, Baarn, Pijnenburg Forest, on decaying wood, Aug. 1972, W. Gams (CBS 263.76B).

Habitat and geographical distribution: Saprobe on decaying wood of Acer pseudoplatanus, Carpinus betulus, Carya alba, Cedrus atlantica, Fagus sylvatica, Nothofagus sp., Populus nigra, Quercus cerris, Quercus sp. and other unknown hosts. Chloridium caudigerum is a common species, it is known from Austria, Belgium, Czech Republic, France, New Zealand, Spain, and the Netherlands (von Höhnel 1903, Gams & Holubová-Jechová 1976, this study). According to GlobalFungi, identical sequences were found in 2 164 samples from 107 studies, collected mainly in soil (topsoil, bulk soil, and rhizosphere soil) but also commonly in the litter, deadwood, and roots. It is frequently found in forests, but unlike other species, also in cropland, shrubland, grassland, and wet habitats (wetland, freshwater, and marine habitats) in subtropical, temperate, cold humid, and boreal zones in Asia, Australasia, Europe and North America (Canada, USA) (MAT 6.9 °C, MAP 743 mm). It is absent in Central and South America and tropical parts of Asia.

Notes: Von Höhnel (1903) introduced Cirrhomyces caudiger as a fungus with effuse colonies and pigmented conidiophores in tufts bearing oblong conidia 3–4 × 1.5–2 μm in whitish cirrhi. Von Höhnel stated that the fungus is very common and may have already been described in the genera Acrotheca or Chloridium. Still, the short published diagnoses do not allow a closer comparison. Hughes (1958) noticed its resemblance to Chloridium and transferred C. caudiger to this genus. Gams & Holubová-Jechová (1976) revised the type material of C. caudiger, designated a neotype, and proposed a new combination Chl. virescens var. caudigerum for von Höhnel’s species, separating it from the type variety and var. chlamydosporum by whitish cirrhi and ellipsoidal, somewhat shorter conidia.

Our phylogenetic and morphological analyses of 10 of the 13 strains identified by Gams & Holubová-Jechová (1976) as Chl. virescens var. caudigerum showed a rich complex of several species; only one isolate represents Chl. caudigerum, while the others belong to five species and two varieties (Table 5). Revision of the neotype of C. caudiger FH 1552 revealed a fungus with conidiophores solitary or in groups 110–150 × 3–3.5 μm forming a single layer and ellipsoidal to oblong conidia accumulating in whitish heads, 3.5–4.5 × 2.5 μm, slightly larger than those given in the protologue (von Höhnel 1903).

Eighteen isolates that matched the description of C. caudiger formed a strongly supported monophyletic clade unrelated to Chl. virescens. Therefore, we propose to reinstate the species status of Chl. caudigerum (Hughes 1958). Chloridium caudigerum is characterised by simple, solitary, or caespitose conidiophores in a single layer and ellipsoidal to oblong conidia in slimy whitish to white-yellow heads or cirrhi on the natural substrate. In culture, conidia in mass on MLA and OA become yellowish to olivaceous yellow with age. Conidiophores contain many percurrent proliferations, making them much longer than on the natural substrate. The meristematic tip of the phialide remains active after the percurrent extension of the original phialide; the meristem becomes intercalary, and several minute annellations are formed (Fig. 21J–L). Our observations of fresh material revealed that conidial cirrhi are common only in some parts of the colony, while in other parts, conidia arranged in heads predominate. Similar observations were made in Chl. virescens; cirrhi formation is age-dependent and preceded by conidial heads (Gams & Holubová-Jechová 1976, this study).

Chloridium caudigerum closely resembles Chl. chlamydosporum and Chl. virescens. Chloridium chlamydosporum differs in having obovate conidia, pale brown mycelium, and mostly lateral chlamydospores. Chloridium virescens differs from Chl. caudigerum in slightly shorter ellipsoidal to subglobose conidia that accumulate in grass-green to yellow-green cirrhi at maturity and conidiophores that form two distinct layers on the natural substrate, glabrous ascomata, and mostly intercalary chlamydospores and hyaline mycelium in culture.

The life cycle of Chl. caudigerum was experimentally verified in vitro in this study; the isolates derived from ascospores (CBS 138691, ICMP 22547) yielded conidiophores with conidia identical to those on the natural substrate. All collections of Chl. caudigerum are well comparable on the natural substrate. In culture, the isolates differed slightly in colony appearance, particularly in the intensity of the chlamydospore production (Fig. 22). Abundant aerial mycelium formed only on MLA; on other media, the aerial mycelium was often reduced, and colonies appeared sparsely velvety to cobwebby.

Chloridium chlamydosporum (J.F.H. Beyma) S. Hughes, Canad. J. Bot. 36: 748. 1958. Figs 23–25.

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Fig. 23.

Chloridium chlamydosporum. A–C. Colonies. D–I. Conidiophores. J, K. Tip of the phialide. L–Q. Chlamydospores. R–U. Conidia. A, F–M, R–T. On PCA. B, C, U. On CMA with Urtica stems. D, E, N–Q. On MLA. Images: A, L, M. CBS 149052; B, C, U. CBS 149053; D, E, N–Q. ICMP 16193; F, G, J, T. CBS 149054; H, I, K. CBS 916.73; R. CBS 114.41; S. CBS 239.75C. Scale bars: A–C = 250 μm; D–I, L–S = 10 μm; J, K, T, U = 5 μm.

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Fig. 25.

Diversity of colony morphology in Chl. chlamydosporum on CMD, MLA, OA, PCA after 4 wk. A. CBS 423.76. B. CBS 916.73. C. CBS 239.75C. D. CBS 15193. Scale bar = 1 cm.

Basionym: Bisporomyces chlamydosporus J.F.H. Beyma [as ‘chlamydosporis’], Antonie van Leeuwenhoek 6: 277. 1940 [1939–40].

Synonym: Chloridium virescens var. chlamydosporum (J.F.H. Beyma) W. Gams & Hol.-Jech., Stud. Mycol. 13: 21. 1976.

Culture characteristics: On CMD: colonies 55–56 mm in diam, circular, flat, margin entire, cobwebby to mucoid, whitish beige, pale brown towards the periphery, reverse brown. Rarely are colonies mucoid, smooth, and milky with reverse of the same colour. On MLA: colonies 82–83 mm in diam, circular, slightly raised, margin entire to fimbriate, velvety-lanose, floccose, funiculose, furrowed, zonate, pale olivaceous grey centrally, cinnamon brown to dark chocolate brown towards the periphery, sometimes pale yellow to pale ochre pigment diffusing into agar, reverse brown. On OA: colonies 69–70 mm in diam, circular, flat, margin entire, velvety to cobwebby, olivaceous grey, cinnamon to dark brown towards the margin, rarely ochre pigment diffusing into the agar, reverse brown. On PCA: colonies 64–67 mm in diam, circular, flat, margin fimbriate, velvety, cobwebby at the margin, white grey, cinnamon to dark brown towards the periphery, beige at the margin, reverse brown. Sporulation was abundant on all media. Growth at 5–30 °C with an optimum at 25–30 °C.

Description in culture: On PCA, vegetative hyphae 1.5–3 μm diam, branched, septate, hyaline to pale brown, smooth. Asexual morph. The chloridium-type conidiophores 43–164(–245) × 2.5–3.5(–4.5) μm, macronematous, solitary, straight, unbranched, with none or 1–6 percurrent proliferations, septate, cylindrical or slightly tapering upwards, base somewhat bulbous, brown, paler towards the apex. Conidiogenous cells (6.5–)10–34 × 2.5–3.5 μm, tapering to 1.5–2.5 μm below the collarette, monophialidic, integrated, terminal, extending percurrently, cylindrical to subcylindrical, pale brown to subhyaline, conidiogenous locus protruding 2–6(–8) μm above the collarette; collarettes 2.5–3.5 μm wide and 1–2 μm deep, subhyaline to pale brown, flaring, funnel-shaped. Conidia 3.5–5 × 2–3 μm (mean ± SD = 4.1 ± 0.3 × 2.5 ± 0.2 μm), obovate to ellipsoidal-obovate, hyaline, pale brown upon ageing, aseptate, smooth, accumulating in slimy whitish heads, becoming beige to pale olivaceous in mass, especially in older cultures (>8 wk). Chlamydospores 4–6(–7) × 4–5 μm, mostly lateral, sessile or on a short stalk, sometimes terminal, seldom intercalary, solitary or in short chains of two cells, globose, subglobose, pyriform or ellipsoidal, brown, thick-walled, smooth. Sexual morph. Unknown.

Specimens examined: Chile, locality unknown, volcanic ash soil, date unknown, J. Grinbergs No. 526/73 (CBS 916.73). Czech Republic, Central Bohemian Region, Lánská obora near Rakovník, on the wood of Quercus petraea, 12 Jun. 1976, V. Holubová-Jechová (CBS 423.76). France, Forêt Senart, isolated from forest soil, date unknown, O. Reisinger (CBS 239.75C). New Zealand, Bay of Plenty Region, Waihi Beach, on dead wood in the broadleaved forest, 9 May 2003, J.A. Cooper JAC8645 (PDD 79986, ICMP 15193). The Netherlands, Utrecht Province, Baarn, isolated from soil, date unknown, J.F.H. van Beyma (holotype of Bisporomyces chlamydosporus CBS H-6914, culture ex-type CBS 114.41 = MUCL 10050); Gelderland Province, Meeuwenkampje, isolated from soil, 9 Jun. 2021, F. ten Hoove (CBS 149054 = SPC122.7); ibid., (CBS 149053 = SPC122.24A); ibid., (CBS 149052 = SPC122.18).

Habitat and geographical distribution: Chloridium chlamydosporum dwells mainly in the soil, but it can also inhabit decaying wood of Quercus petraea and other unidentified hosts or needles of Abies firma. It is known from Chile, Czech Republic, Japan, France, New Zealand, and the Netherlands (van Beyma 1940, Matsushima 1975, Gams & Holubová-Jechová 1976, Iwamoto & Tokumasu 2001, this study). According to GlobalFungi, identical sequences were found in 1 666 samples from 72 studies, collected primarily in bulk and topsoil but also commonly in deadwood, litter, shoots, and roots. Most samples came from forest, woodland, cropland, shrubland, grassland, desert, and anthropogenic habitats. It is common across the globe (except Antarctica) but absent in some well-sampled areas (Spain, Scandinavia, and Western USA) and is relatively rare in South America. Interestingly, this species was also absent in France, which is well-sampled, although one of the collections examined by us came from that country. It was usually found in temperate, continental humid, tropical, and drier climates but was absent in the boreal zone (MAT 12.6 °C, MAP 1 241 mm).

Notes: Thirteen morphologically similar strains, some of which were initially identified as Chl. virescens var. chlamydosporum (Gams & Holubová-Jechová 1976) formed a strongly supported monophylum distantly related to Chl. virescens. Based on these results, the species status of Chl. chlamydosporum is reinstated. The variability of the species complex centred on the ex-type strain of Chl. chlamydosporum CBS 114.41 was assessed by STACEY analysis, ML/BI phylogeny of four markers, analysis of the secondary structure of ITS (Figs 3, ​,8,8, ​,11),11), and comparative morphology. Chloridium chlamydosporum species complex was divided into three lineages, whose representatives differ mainly in conidial features and the number of nucleotides and WC base pairs in the first helix of the ITS1 molecule. These lineages are proposed here as three species and two varieties, i.e. Chl. chlamydosporum (conidia 3.5–5 × 2–3 μm; 175–176 nt in ITS1), Chl. detriticola var. detriticola (conidia (4–)4.5–5.5 × (2.5–)3–3.5 μm; 165 nt in ITS1), Chl. detriticola var. effusum (conidia (4–)4.5–5.5 × 2.5–3 μm; 152 nt in ITS1), and Chl. peruense (3–4 × 2–2.5 μm; 164 nt in ITS1). All lineages can also be distinguished by cardinal growth temperatures (Fig. 16, Table S4) and particularly by biogeography. Chloridium detriticola var. detriticola and var. effusum are outstanding by their occurrence in the boreal climatic zone, Chl. peruense by its common distribution in the tropics of Central and South America.

Chloridium chlamydosporum is characterised by solitary, brown, unbranched conidiophores that form a single layer, obovate to ellipsoidal-obovate conidia accumulating in slimy whitish heads and mostly lateral, sessile, globose chlamydospores. The subclade of Chl. chlamydosporum was separated into three lineages A, B, C (Fig. 8). Their representatives are difficult to distinguish based on morphological characters. Thus, more strains are needed to resolve the intraspecific variation of Chl. chlamydosporum. The pale yellow to pale ochre diffusible pigment was observed on MLA in strains CBS 239.75C and CBS 149054.

The ex-type strain of Chl. chlamydosporum, obtained from both CBS (CBS 114.41) and MUCL (MUCL 10050) culture collections, formed mostly smooth, mucoid-waxy colonies with aerial mycelium absent or significantly reduced and sporulation was delayed (>6 months), possibly due to long storage for over 80 years. However, the freshly collected strain CBS 149054 (also isolated from the soil) with identical sequences produced abundant aerial mycelium in vitro and sporulated soon. The appearance of colonies of other studied strains is well-comparable, and the sporulation appeared within a week (Figs 24, ​,2525).

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Fig. 24.

Diversity of colony morphology in Chl. chlamydosporum on CMD, MLA, OA, PCA after 4 wk. A. CBS 114.41. B. CBS 149054. C. CBS 149052. D. CBS 149053. Scale bar = 1 cm.

Phylogenetic analyses and comparative morphology of 10 of 12 strains of Chl. virescens var. chlamydosporum sensu Gams & Holubová-Jechová (1976) revealed a complex comprising four species (Table 5). Chloridium caudigerum closely resembles Chl. chlamydosporum but differs in ellipsoidal conidia accumulating in whitish heads and cirrhi and terminal, lateral and intercalary chlamydospores.

Chloridium crousii W.P. Wu & Y.Z. Diao, Fungal Diversity 116: 235. 2022.

For a description and illustration, see Wu & Diao (2022).

Habitat and geographical distribution: Saprobe on decaying wood from China (Wu & Diao 2022). According to GlobalFungi, identical sequences were found in five samples from two studies collected in forest soils in subtropical and temperate climatic regions in Asia (China) (MAT 19.7 °C, MAP 1 452 mm).

Notes: Chloridium crousii forms conidiophores in a single layer and ellipsoidal-oblong, hyaline conidia 2.8–3.2 × 2.5–2.8 μm aggregated in slimy heads (Wu & Diao 2022). It is closely related to morphologically similar Chl. tropicale and Chl. xishuangbannaense.

Chloridium detriticola var. detriticola Réblová & Hern.-Restr., sp. nov. MycoBank MB 846708. Fig 26.

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Fig. 26.

Chloridium detriticola var. detriticola. A, B. Conidiophores. C, D. Chlamydospores. E, F. Tip of the phialide with conidia. G–J. Conidia. K, L. Colonies on CMD, MLA, OA, PCA (from left to right) after 4 wk. A–J. On MLA. Images: A–F, H–K. CBS 345.67; G, L. CBS 581.73. Scale bars: A, B = 20 μm; C–J = 10 μm; K, L = 1 cm.

Etymology: Detritus (Latin) means plant debris and incolo (Latin) to inhabit, referring to the habitat of the fungus growing on decaying plant material.

Typus: Germany, Bavaria, Bayrischer Wald, Höllbachgspreng, on decaying wood, Jul. 1967, W. Gams (holotype CBS H-10187, culture ex-type CBS 345.67).

Culture characteristics: On CMD: colonies 56–59 mm diam, circular, flat, margin entire, mucoid, smooth, pale brown, beige grey centrally, reverse grey-brown. On MLA: colonies 70–72 mm diam, circular, convex, flat margin, margin fimbriate, velvety, furrowed, cobwebby to mucoid at the margin, dark chocolate brown, whitish grey due to heavily sporulating conidiophores, beige at the margin, reverse dark brown grey. On OA: colonies 83–84 mm diam, circular, flat, margin entire, cobwebby to mucoid, smooth, dark brown to dark grey, interrupted by paler rings of sparse growth, whitish centrally due sporulating conidiophores, reverse dark grey. On PCA: colonies 76–77 mm diam, circular, flat, margin fimbriate, velvety to cobwebby, zonate, farinose when sporulating, at the centre, dark brown to cinnamon brown, whitish grey centrally, with an outer beige zone of submerged growth, reverse of the same colour. Sporulation was abundant on MLA and OA, moderate on PCA, absent on CMD. Growth at 5–25 °C with an optimum at 5 °C.

Description in culture: On MLA, vegetative hyphae 1.5–3.5 μm diam, branched, septate, subhyaline to pale brown. Asexual morph. The chloridium-type conidiophores 72–162 × 3–4 μm, macronematous, solitary, straight, unbranched, with none or 1–4 percurrent proliferations, septate, cylindrical, brown, darker at the base, paler towards the apex. Conidiogenous cells 17.5–38 × 2.5–3.5 μm, tapering to 2–2.5 μm below the collarette, monophialidic, integrated, terminal, extending percurrently, cylindrical to subcylindrical, usually pale brown, conidiogenous locus protruding 2–6.5 μm above the collarette; collarettes 2.5–3 μm wide and ca. 1.5 μm deep, subhyaline to pale brown, flaring, shortly funnel-shaped. Conidia (4–)4.5–5.5 × (2.5–)3–3.5 μm (mean ± SD = 4.6 ± 0.3 × 3.0 ± 0.2 μm), obovate to ellipsoidal-obovate, somewhat truncate at the base, hyaline, aseptate, smooth, ageing conidia turn pale brown (>5 wk), accumulating in slimy whitish heads. Chlamydospores 4.5–6 × 4.5–5 μm, mainly lateral, sessile or on a short stalk, sometimes terminal, rarely intercalary, solitary or in short chains of two cells, globose, subglobose or pyriform, brown, thick-walled, smooth. Sexual morph. Unknown.

Additional specimens examined: Czech Republic, South Bohemian Region, Novohradské hory Mts., Pohoří na Šumavě, Myslivna Mt., on decaying wood of Fagus sylvatica, 6 Oct. 2012, M. Réblová M.R. 3774. Sweden, Stockholm, host unknown, date unknown, J.E. Nylund strain 26 (CBS 581.73).

Habitat and geographical distribution: Chloridium detriticola is a saprobe on decaying wood known from the Czech Republic, Germany, and Sweden. According to GlobalFungi, identical sequences were found in 7 903 samples from 132 studies, making it the most frequently found taxon. Samples were collected mainly from the soil, less regularly from deadwood and litter in forests across the globe (except Africa) in all climatic zones, with a preference for temperate and boreal climates. Of all the taxa studied, it was the most abundant; it is typically found in the boreal zone of the Northern Hemisphere (Alaska, Canada, and all of Scandinavia), where most other species were absent (MAT 8.4 °C, MAP 1 115 mm). The growth capabilities support the observed preference for colder climates. Of all the taxa studied, together with Chl. detriticola var. effusum, it has the fastest growth at 5 °C and 10 °C, low optimum at 20 °C and was not able to grow at 30 °C.

Notes: Chloridium detriticola forms a strongly supported subclade (100/1.0) in the Chl. chlamydosporum species complex, divided into two lineages introduced as two varieties (Fig. 8). The three isolates of var. detriticola from Europe (CBS 345.67, CBS 581.73, M.R. 3774) can be distinguished from the var. effusum ICMP 15144 from New Zealand, by their broader conidia (3–3.5 μm vs 2.5–3 μm) and significantly longer ITS1 region (165 nt vs 152 nt). The decreased number of nucleotides in ICMP 15144 corresponds to a shorter H1 helix of the ITS1 caused by the absence of several canonical pairs (Fig. 8).

Chloridium detriticola var. detriticola overlaps with the less common Chl. detriticola var. effusum in cardinal growth temperatures and biogeography.

Chloridium detriticola var. effusum Réblová & Hern.-Restr., var. nov. MycoBank MB 846709. Fig 27.

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Fig. 27.

Chloridium detriticola var. effusum (ICMP 15144). A, B. Colonies. C–H. Conidiophores. I, J. Tip of the phialide. K–N. Conidia. O. Chlamydospores. P. Colonies on CMD, MLA, OA, PCA (from left to right) after 4 wk. A, C, D, K–M. From nature. B, E–G, I, J, N, O. On PCA. H. On MLA. Scale bars: A, B = 250 μm; C = 25 μm; D–H, O = 20 μm; I–N = 5 μm; P = 1 cm.

Etymology: Effūsus (Latin) vast, sprawling, referring to the effuse colonies.

Typus: New Zealand, West Coast Region, Westland District, Jackson River valley, Lake Ellery track SW of Haast, on decaying wood, 11 Mar. 2003, M. Réblová M.R. 2785/NZ 295 (holotype PDD 120399, culture ex-type ICMP 15144).

Description on the natural substrate: Colonies effuse, dark brown, whitish when sporulating; composed of conidiophores. Asexual morph. The chloridium-type conidiophores 114–218 × 3.5–4.5 μm, macronematous, solitary or in groups, erect, straight or slightly flexuous, unbranched, usually with one percurrent proliferation, cylindrical to somewhat subulate, base bulbous, septate, dark brown, paler towards the apex. Conidiogenous cells 10–29 × 2.5–3.5 μm, tapering to 2–2.5 μm below the collarette, monophialidic, integrated, terminal, extending percurrently, subcylindrical, pale brown; collarettes 3–3.5 μm wide and 1.5–2 μm deep, pale brown to subhyaline, flaring, inconspicuous. Conidia 4–5 × 2.5–3.5 μm (mean ± SD = 4.5 ± 0.3 × 3.0 ± 0.3 μm), obovate to oblong-obovate, hyaline, ageing conidia turn pale brown, aseptate, smooth, accumulating in slimy whitish heads. Sexual morph. Unknown.

Culture characteristics: On CMD: colonies 53–54 mm diam, circular, flat, margin entire, lanose, floccose centrally, cobwebby to mucoid at the margin, pale brown, whitish centrally, reverse brown. On MLA: colonies 70–72 mm diam, circular, convex, flat margin, margin entire, velvety-lanose, cobwebby at the margin, furrowed, dark chocolate brown, whitish grey centrally and star-shaped extending, with an isabelline outer zone of submerged growth, reverse brown. On OA: colonies 74–75 mm diam, circular, flat, margin fimbriate, mucoid, smooth, cobwebby, and somewhat farinose due to sporulating conidiophores at the centre, dark brown, whitish grey centrally, reverse brown. On PCA: colonies 70–73 mm diam, circular, flat, margin entire, lanose, floccose, cobwebby at the margin, dark brown, whitish grey at the centre, with an outer beige zone of submerged growth, reverse dark brown. Sporulation was abundant on all media. Growth at 5–25 °C with an optimum at 20 °C.

Description in culture: On MLA, vegetative hyphae 1.5–3.5 μm diam, branched, septate, subhyaline to pale brown. Asexual morph. Conidiophores, conidiogenous cells, and conidia are similar to those from nature. The chloridium-type conidiophores 58–115 × 3–4.5 μm, with none or 1–3 percurrent proliferations. Conidiogenous cells 16–39 × 3–3.5 μm, tapering to 2–2.5 μm below the collarette, conidiogenous locus protruding 2–4 μm above the collarette; collarettes 2.5–3.5 μm wide and ca. 1.5 μm deep. Conidia (4–)4.5–5.5 × 2.5–3 μm (mean ± SD = 4.5 ± 0.3 × 2.7 ± 0.2 μm), obovate to oblong-obovate, hyaline, in slimy whitish heads. Chlamydospores 4–6.5(–7) × 4–6 μm, mainly lateral, sessile or on a short stalk, sometimes terminal and intercalary, solitary or in short chains, globose, subglobose, ellipsoidal or pyriform, brown, thick-walled, smooth. Sexual morph. Unknown.

Habitat and geographical distribution: Saprobe on decaying wood, known from New Zealand. According to GlobalFungi, identical sequences were found in 524 samples from 32 studies, collected mainly in soil and often in roots, deadwood, and litter in forests, grasslands, shrublands, and woodlands worldwide (except Africa) in all climatic zones. It also occurred in the boreal and tundra zones (Alaska, Canada, Scandinavia), where most other species did not occur (MAT 9.6 °C, MAP 1 318 mm). Its observed preference for colder climates is supported by its growth capabilities, similarly to Chl. detriticola var. detriticola.

Note: Chloridium detriticola var. effusum resembles var. detriticola but differs in having longer conidia, and the H1 helix of the ITS1 region is shorter by 10 canonical pairs (Fig. 8).

Chloridium gamsii Réblová & Hern.-Restr., sp. nov. MycoBank MB 846710. Fig. 28.

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Fig. 28.

Chloridium gamsii (CBS 667.75). A, B. Colonies. C–F. Conidiophores. G. Conidia. H. Tip of the phialide. I, J. Chlamydospores. K. Colonies on CMD, MLA, OA, PCA (from left to right) after 4 wk. A, B, F, G. On CMA with Urtica stems. C–E, H–J. On MLA. Scale bars: A, K = 1 cm; B = 250 μm; C–J = 10 μm.

Etymology: Named after Walter Gams to honour his contribution to mycology and outstanding expertise in the taxonomy and systematics of hyphomycetes and particularly Chloridium.

Typus: Belgium, Ardennes, Le Roptai near Ave-et-Auffe, on decaying wood, Sep. 1975, W. Gams (holotype CBS H-25019, culture ex-type CBS 667.75)

Culture characteristics: On CMD: colonies 28–29 mm diam, circular, flat, margin entire, mucoid, smooth, dark brown, paler towards the margin, reverse of the same colour. On MLA: colonies 29–30 mm diam, circular, convex, margin entire, velvety, deeply furrowed, developing mucoid cracks at the top of the ridges at the centre of the colony, zonate, dark grey to black at the centre, with white-beige and brown rings, reverse dark brown. On OA: colonies 63–65 mm diam, circular, flat, margin entire, mucoid, smooth, cobwebby centrally, zonate, pale brown, beige towards the periphery, reverse of the same colour. On PCA: colonies 46–48 mm diam, circular, slightly raised, margin entire, velvety-lanose, zonate, olivaceous-beige, farinose, grey towards the margin, with a pale cinnamon outer zone of submerged growth, reverse dark brown. Sporulation was absent on CMD, sparse on MLA and OA, abundant on PCA. Growth at 5–25 °C with an optimum at 20 °C.

Description in culture: On MLA, vegetative hyphae 1.5–3.5 μm diam, branched, septate, hyaline, subhyaline or pale brown, smooth. Asexual morph. The chloridium-type conidiophores 34–140 × 2.5–3.5 μm, macronematous, solitary, erect, straight or flexuous, unbranched, with none or 1–3 percurrent proliferations, septate, cylindrical, brown, paler towards the apex. Conidiogenous cells 16–25 × 2–3 μm, tapering to 1.5–2 μm below the collarette, monophialidic, integrated, terminal, extending percurrently, subcylindrical to lageniform, pale brown to subhyaline; collarettes 2.5–3.5 μm wide, 1.5–2 μm deep, subhyaline, flaring, short funnel-shaped. Conidia 3.5–4.5× 2.5–3 μm (mean ± SD = 3.7 ± 0.2 × 2.6 ± 0.2 μm), subglobose to ellipsoidal, to somewhat obovate, hyaline, aseptate, smooth, accumulating in whitish to white-yellow slimy heads and cirrhi. Chlamydospores 5–7.5 × 4.5–6 μm globose, subglobose or clavate, mostly lateral, sessile or on a stipe or in a chain of 2–3 cells, sometimes terminal or intercalary, brown, thick-walled, smooth. Sexual morph. Not observed.

Habitat and geographical distribution: Saprobe on decaying wood, known from Belgium. According to GlobalFungi, identical sequences were found in 753 samples from 48 studies, collected mainly in soil but also commonly in litter, deadwood, and roots. Most samples came from forest, cropland, shrubland, grassland, desert, and anthropogenic habitats. The species is widespread in Europe (but rare in Scandinavia) and Australasia, with a few records in Canada and the USA. It was found in all climate types, mainly in temperate zones, more rarely in colder (Sweden), drier or tropical habitats (Australia) (MAT 8.7 °C, MAP 846 mm).

Notes: The strain CBS 667.75 was derived from ascospores of a fungus identified as Ch. vermicularioides with a conidial state Chl. virescens var. caudigerum (Gams & Holubová-Jechová 1976). The authentic collection on the natural substrate was not available for study. Conidiophores associated with ascomata had conidia 3 × 2 μm arranged in whitish cirrhi. They are slightly smaller than conidia formed in culture 3.5–4.5× 2.5–3 μm (this study).

Based on the analysis of DNA sequences of four markers, we confirm that the strain CBS 667.75 is unrelated to Chl. virescens or Chl. caudigerum and forms a separate lineage. Therefore, a new species Chl. gamsii is proposed for it. Its closest relative is Chl. biforme from New Zealand, also derived from the ascospores. Chloridium biforme differs from Chl. gamsii in ellipsoidal to suballantoid, somewhat narrower conidia ((3–)3.5–4.5 × 2–2.5 μm in vitro) that turn olivaceous grey in mass in older cultures.

Chloridium guttiferum Réblová & Hern.-Restr., sp. nov. MycoBank MB 846711. Fig. 29.

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Fig. 29.

Chloridium guttiferum (CBS 126073). A, B. Colonies. C–G. Conidiophores. H. Chlamydospores. I, J. Conidia. K. Colonies on CMD, MLA, OA, PCA (from left to right) after 4 wk. A–C. On CMA with Urtica stems. D–G. On MLA. H–J. On PCA. Scale bars: A, B = 250 μm; C = 25 μm; D–G = 20 μm; H–J = 10 μm; K = 1 cm.

Etymology: Gutta (Latin) drop, fero (Latin) to bear or carry, referring to the heads of conidia resembling drops attached to the conidiogenous loci.

Typus: Peru, Amazon Center for Environmental Education and Research (ACEER) near Iquitos, Medicinal plants trail, primary rainforest, isolated from soil (horizon A1), Jan. 2001, collector unknown (holotype CBS H-25020, culture ex-type CBS 126073 = RMF 47.01).

Culture characteristics: On CMD: colonies 25–30 mm diam, circular, flat, margin entire, cobwebby to mucoid, zonate, white-beige, colony centre dark brown, with a milky glistening margin surrounded by a beige outer zone of submerged growth, reverse brown. On MLA: colonies 53–56 mm diam, circular, flat, margin entire, velvety, floccose, furrowed centrally, colony centre olivaceous grey, dark olivaceous brown at the margin with a beige zone of submerged growth, reverse dark brown. On OA: colonies 72–73 mm diam, circular, flat, margin fimbriate, cobwebby, partly mucoid, smooth, beige with a dark brown irregular zone, reverse olivaceous brown. On PCA: colonies 50–53 mm diam, circular, flat, margin entire, cobwebby, mucoid and smooth towards the periphery, beige-brown, dark brown at the margin, reverse of the same colour. Sporulation was absent on CMD, abundant on MLA, and sparse on OA and PCA. Growth at 10–37 °C with an optimum at 30 °C.

Description in culture: On CMA with U. dioica stems, vegetative hyphae 1.5–2.5 μm diam, branched, septate, smooth, hyaline, subhyaline to pale brown, sometimes finely encrusted. Asexual morph. The chloridium-type conidiophores 80–314(–377) × 2.5–3.5 μm, macronematous, solitary or in tufts, erect, unbranched or sparsely branched, commonly with 1–5 percurrent proliferations, with up to 15 proliferations in older cultures (>8 wk), cylindrical, straight or slightly bent, septate, brown, paler towards the apex; branches 52–228(–260) μm, primary branches arise below the septa, curved upwards, occasionally secondary branches are formed, always fertile, terminating into a phialide. Conidiogenous cells 15.5–27 × 2.5–3 μm, tapering to 1.5–2 μm below the collarette, monophialidic, subcylindrical to lageniform, integrated, terminal, pale brown, subhyaline towards the apex, extending percurrently, rarely sympodially; collarettes 2–3 μm wide, ca. 1.5 μm deep, subhyaline, flaring, inconspicuous. Conidia (3–)3.5–4.5 × (2–)2.5–3 μm (mean ± SD = 3.9 ± 0.3 × 2.5 ± 0.2 μm), ellipsoidal to obovate, hyaline, aseptate, smooth, accumulating in olivaceous to olivaceous grey slimy heads. Chlamydospores 4.5–7 × 4.5–5.5 μm, intercalary and lateral, sessile or on a stipe, globose, subglobose or pyriform, brown, thick-walled, smooth. Sexual morph. Unknown.

Habitat and geographical distribution: This species has been isolated from the soil and is known from Peru. According to GlobalFungi, identical sequences were found in 237 samples from 36 studies, collected primarily in bulk soil or roots and litter, less often in topsoil or rhizosphere soil, sediment, and water in forest, grassland, cropland, water, wetland, desert and anthropogenic habitats across the globe (except Antarctica). It occurs in temperate, continental humid, tropical, subtropical, and drier zones but not in the boreal zone. It prefers tropical climates and is one of the few species commonly found in South America (MAT 17.9 °C, MAP 1 801 mm). Its preference for warmer climates is consistent with its high growth optimum (30 °C) and temperature tolerance of 37 °C.

Notes: Chloridium guttiferum was resolved as a sister to Chl. bellum in the multigene phylogeny. Strains of both species occur in soil and form ellipsoidal to obovate conidia of nearly identical size. Chloridium guttiferum differs in the caespitose conidiophores, which often branch in older cultures, and the conidia accumulate in olivaceous to olivaceous grey heads on Urtica stems on CMA. Conidiophores of Chl. bellum are solitary, unbranched, and conidia adhere in whitish cirrhi or heads on Urtica stems on CMA. Of all the species, it shares with the phylogenetically related Chl. bellum var. bellum the ability to grow at 37 °C, from which it differs in the absence of growth at 5 °C.

Chloridium jilinense W.P. Wu & Y.Z. Diao, Fungal Diversity 116: 241. 2022.

For a description and illustration, see Wu & Diao (2022).

Habitat and geographical distribution: Saprobe on decaying wood from China (Wu & Diao 2022). According to GlobalFungi, identical sequences were found in 30 samples from 10 studies, collected mainly in bulk soil but quite often also on deadwood and litter in forest biomes. It occurs in Northern Hemisphere only in Northern America (East part only), Central Europe, and Asia (China), exclusively in temperate climatic regions (MAT 7.7 °C, MAP 880 mm).

Notes: In the ITS-LSU phylogeny (Fig. 7), Chl. jilinense is shown as the closest relative to Chl. caudigerum. These species have similar conidiophores formed in a single layer, solitary or in small groups, and ellipsoidal-oblong conidia adhering in slimy heads. Chloridium jilinense has slightly longer conidia, 4–5 × 2.5–3 μm. In culture, conidia are mostly subglobose to slightly obovate (size was not given); chlamydospores were abundant, terminal and lateral, occasionally intercalary (Wu & Diao 2022).

Chloridium kirkii W.P. Wu & Y.Z. Diao, Fungal Diversity 116: 243. 2022.

For a description and illustration, see Wu & Diao (2022).

Habitat and geographical distribution: Saprobe on decaying wood from China (Wu & Diao 2022). According to GlobalFungi, identical sequences were found in 351 samples from 29 studies, collected mainly in bulk or topsoil, less often in deadwood, and predominantly in forest biomes in Asia, Europe, and the western part of Northern America. Locations are usually in the humid temperate climatic region, with a few records in tropical and subtropical areas (Southern China) and the Mediterranean (Southern Europe) (MAT 11.5 °C, MAP 1 550 mm).

Notes: This species is characterised by conidiophores that are solitary or in small groups, somewhat short 22–65 × 2–2.5 μm, the shortest likely reduced to single conidiogenous cells, ellipsoidal-oblong conidia 3–3.5 × 2–2.5 μm in slimy heads and lateral or intercalary chlamydospores, which are sometimes arranged in chains. In the ITS-LSU phylogeny (Fig. 7), Chl. kirkii is shown as sister to a strongly supported clade containing other three species, Chl. crousii, Chl. tropicale and Chl. xishuangbannaense.

Chloridium moratum Réblová & Hern.-Restr., sp. nov. MycoBank MB 846712. Fig. 30.

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Fig. 30.

Chloridium moratum. A. Colony. B, C. Conidiophores. D–F. Conidia. G, H. Maturing pale brown conidia. I. Chlamydospores. J, K. Colonies on CMD, MLA, OA, PCA (from left to right) after 4 wk. A, C–E, H–J. On CMA with Urtica stems. B. On PCA. F, G, K. On MLA. Images: A, F, G, K. FMR 11343; B–E, H–J. CBS 127627. Scale bars: A = 250 μm; B–I = 10 μm; J, K = 1 cm.

Etymology: Morātus (Latin) delayed, referring to the pigmented conidia in aged cultures.

Typus: USA, North Carolina, Coweeta Long-term Ecological Research site near Otto, 40-year-old eastern white pine (Pinus strobus) plantation, conifer forest, isolated from soil (horizon A1), 1988, collector unknown (holotype CBS H-25083, culture ex-type CBS 127627 = RMF 8889 = RMF 8849)

Culture characteristics: On CMD: colonies 46–47 mm diam, circular, flat, margin entire to fimbriate, mucoid, glistening, somewhat funiculose at the centre, milky, with pale olivaceous brown ring, centre sometimes brown due to chlamydospore production, reverse beige. On MLA: colonies 84–86 mm diam, circular, flat, margin entire to fimbriate, mucoid to cobwebby, somewhat funiculose, whitish creamy, cinnamon centrally or with a cinnamon middle ring, with a pale ochre outer zone of submerged growth, golden-ochre pigment diffusing into the agar, reverse dark orange to amber. On OA: colonies 83–85 mm diam, circular, flat, margin fimbriate, mucoid, locally cobwebby, milky, brown centrally with irregular whitish patches of aerial mycelium, with a prominent submerged growth, reverse olivaceous brown. On PCA: colonies 80–81 mm diam, circular, flat, margin fimbriate, mucoid, cobwebby centrally, white-beige, brown at the centre, sometimes pale olivaceous brown at the margin, reverse beige. Sporulation was absent on CMD, sparse on MLA, OA and PCA, though delayed (>6–8 wk). Growth at 5–25 °C with an optimum at 20 °C.

Description in culture: On CMA with U. dioica stems, vegetative hyphae 1.5–3.5 μm diam, branched, septate hyaline, subhyaline to pale brown hyphae, smooth, finely encrusted with age. Asexual morph. The chloridium-type conidiophores 30–120 × 2.5–4 μm, macronematous, solitary, erect, unbranched, with 1–3 percurrent proliferations, straight or slightly bent, brown, paler towards the apex, base bulbous or slightly inflated. Conidiogenous cells 16–35 × 2.5–4 μm tapering to (1.5–)2–2.5 μm below the collarette, monophialidic, subcylindrical, integrated, terminal, pale brown to subhyaline, paler towards the apex, seldom extending percurrently; collarettes 2.5–3.5 μm wide, 1.5–2 μm deep, subhyaline, flaring, inconspicuous. Conidia (3.5–)4–5 × 2.5–3.5 μm (mean ± SD = 4.3 ± 0.2 × 2.9 ± 0.2 μm), irregularly ellipsoidal to subglobose to slightly obovate with a truncate base, hyaline, smooth, some conidia turn brown and become finely verrucose upon ageing, aseptate, accumulating in slimy whitish heads and cirrhi. Chlamydospores 4.5–6.5 × 4–5.5 μm, mostly globose, but also subglobose, ellipsoidal or clavate, solitary or in short chains, often intercalary, sometimes terminal, seldom lateral. Sexual morph. Unknown.

Other specimen examined: Spain, Asturias, La Molina, Picos de Europa National Park, on decaying wood of a twig, 22 Oct. 2010, M. Hernández-Restrepo, J. Mena Portales & J. Guarro (FMR 11343 = MUCL 53625).

Habitat and geographical distribution: Saprobe in soil and decaying wood, known from Spain and the USA. According to GlobalFungi, identical sequences were found in 4 405 samples from 57 studies, collected primarily in topsoil in forests across the globe (except Africa and Antarctica). It typically occurs in temperate, subtropical, or tropical climatic zones, but it is almost absent in the boreal region of Europe (MAT 10.8 °C, MAP 1 355 mm).

Notes: Chloridium moratum has hyaline conidia that vary in shape from ellipsoidal to subglobose to obovate with a basal scar and produces soluble golden-ochre pigment on MLA. Some conidia on PCA and CMA in ageing cultures turned brown and slightly verrucose. The conidiophores are straight or slightly bent with seldom percurrent proliferation. Chloridium gamsii is similar to Chl. moratum but differs in smaller conidia, mostly lateral or terminal chlamydospores, and lacks diffusible pigments.

Chloridium peruense Réblová & Hern.-Restr., sp. nov. MycoBank MB 846713. Fig. 31.

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Fig. 31.

Chloridium peruense (CBS 126074). A, B. Colonies. C–F. Conidiophores. G–I. Chlamydospores. J. Conidia. K. Colonies on CMD, MLA, OA, PCA (from left to right) after 4 wk. A–D. On CMA with Urtica stems. E, F. On MLA. Scale bars: A, B = 250 μm; C–E = 20 μm; F–J = 10 μm; K = 1 cm.

Etymology: Indicating the origin of this species from Peru.

Typus: Peru, Amazon Center for Environmental Education and Research (ACEER) near Iquitos, Medicinal Plants Trail, primary rainforest, isolated from soil (horizon A1), Jan. 2001, collector unknown (holotype CBS H-25017, culture ex-type CBS 126074).

Culture characteristics: On CMD: colonies 82–83 mm diam, circular, flat, margin entire to fimbriate, velvety-lanose, floccose, cobwebby towards the margin, pale brown, whitish beige centrally, reverse brown. On MLA: colonies 76–77 mm diam, circular, flat, margin fimbriate, furrowed, velvety-lanose, locally floccose, cobwebby to mucoid towards the periphery, whitish grey, olivaceous brown towards the margin, reverse brown. On OA: colonies 78–79 mm diam, circular, flat, margin fimbriate, velvety, farinose and beige grey due to sporulating conidiophores, olivaceous brown at the margin, reverse olivaceous brown. On PCA: colonies 86–88 mm diam, circular, flat, margin fimbriate, sparsely lanose, beige-pink to dark brown, reverse dark brown. Sporulation was abundant on MLA, OA and PCA, sparse on CMD. Growth at 10–30 °C with an optimum at 30 °C.

Description in culture: On MLA, vegetative hyphae 1–4 μm diam, branched, septate, subhyaline to pale brown, smooth. Asexual morph. The chloridium-type conidiophores 74–175 × 2.5–3 μm, solitary, in groups or crowded, straight or slightly flexuous, unbranched, with none or 1–2 percurrent proliferations, septate, cylindrical, brown, paler towards the apex. Conidiogenous cells 18–25 × 2.5–3 μm, tapering to (1.5–)2 μm below the collarette, monophialidic, integrated, terminal, extending percurrently, cylindrical to somewhat lageniform, pale brown; collarettes 2.5–3 μm wide and ca. 1 μm deep, subhyaline to pale brown, flaring, shortly funnel-shaped. Conidia 3–4 × 2–2.5 μm (mean ± SD = 3.5 ± 0.3 × 2.2 ± 0.2 μm), obovate to ellipsoidal-obovate, hyaline, aseptate, smooth, accumulating in slimy whitish heads. Chlamydospores 4.5–6 × 4–5.5 μm, mostly lateral, sessile or on a short stalk, sometimes terminal or intercalary, solitary, globose, subglobose or pyriform, brown, thick-walled, smooth. Sexual morph. Unknown.

Habitat and geographical distribution: Chloridium peruense was isolated from soil in Peru. According to GlobalFungi, identical sequences were found in 697 samples from 33 studies, collected mainly in topsoil and soil, less often in rhizosphere soil, roots, deadwood, litter, and shoots in forest, woodland, shrubland, wetland, or aquatic habitats worldwide (except Antarctica) in temperate, continental humid, tropical and drier climates but not in the boreal zone. Unlike most other species, it is found in South America and prefers warm and humid climates (MAT 20 °C, MAP 1 685 mm). The affinity to the warmer regions corresponds to its relatively high growth optimum (30 °C) and inability to grow at 5 °C.

Note: Chloridium peruense is well distinguishable among other species of the Chl. chlamydosporum complex, for it has the smallest conidia.

Chloridium proliferatum W.P. Wu & Y.Z. Diao, Fungal Diversity 116: 249. 2022.

For a description and illustration, see Wu & Diao (2022).

Habitat and geographical distribution: Saprobe on decaying wood from China (Wu & Diao 2022). According to GlobalFungi, identical sequences were found in nine samples from two studies, collected in bulk and rhizosphere soil in forest and grassland biomes. It occurs in Asia (China) in the tropical or subtropical climatic regions (MAT 23.6 °C, MAP 1 848 mm).

Notes: Chloridium proliferatum is characteristic of having ellipsoidal conidia with a conspicuous basal scar adhering in cirrhi and conidiophores in a single layer on the natural substrate. The production of chlamydospores in cultures was omitted from the protologue (Wu & Diao 2022). It was difficult to align the ITS sequences of this species. Analysis that included the ITS sequence generated from the ex-type strain CGMCC 3.20766 (GenBank accession OL628678) resulted in a position in the centre of the Chl. fuscum clade, which is otherwise morphologically and genetically homogeneous. Therefore, only after applying GBlocks and creating a reduced ITS-LSU data set, the position of Chl. proliferatum could be resolved as a separate lineage close to Chl. crousii, Chl. tropical and Chl. xishuangbannaense (Supplementary Fig. S1).

Chloridium setosum W.P. Wu & Y.Z. Diao, Fungal Diversity 116: 251. 2022. Fig. 32.

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Fig. 32.

Chloridium setosum (CBS 263.76A). A, B. Colonies. C–G. Conidiophores. H, I. Conidia. J, K. Chlamydospores. L. Colonies on CMD, MLA, OA, PCA (from left to right) after 4 wk. A–D, G, H. On CMA with Urtica stems. E, J. On PCA. F, I, K. On MLA. Scale bars: A, L = 1 cm; B = 250 μm; C, D = 25 μm; E–G = 20 μm; H–K = 10 μm.

For a description on the natural substrate, see Wu & Diao (2022).

Culture characteristics: On CMD: colonies 50–51 mm diam, circular, flat, margin entire, mucoid, smooth, beige, milky at the margin, reverse of the same colour. On MLA: colonies 70–72 mm diam, circular, convex, flat margin, margin entire, velvety-lanose, sparsely funiculose, somewhat mucoid centrally, zonate, mouse grey, brown at the margin, reverse dark brown. On OA: colonies 49–92 mm diam, circular, flat, margin fimbriate, cobwebby to mucoid, chestnut brown, dark chocolate brown centrally and at the margin, with sparse whitish aerial mycelium at the centre, reverse dark brown. On PCA: colonies 88–90 mm diam, circular, slightly raised, margin fimbriate, velvety, farinose, olivaceous beige to olivaceous yellow due to conidial masses, brown at the margin, reverse dark brown. Sporulation was absent on CMD, abundant on MLA, OA and PCA. Growth at 5–30 °C with an optimum at 25 °C.

Description in culture: On CMA with U. dioica stems, vegetative hyphae 1–3.5 μm, sparsely branched, septate, hyaline to subhyaline, smooth. Asexual morph. The chloridium-type conidiophores 50–206 × 2–3 μm, macronematous, solitary or in bundles, erect, straight or flexuous, unbranched or branched, with 1–10 percurrent proliferation, septate, cylindrical, pale brown, paler near the apex; primary lateral branches 35–105 × 2–3 μm, arise above the septa or between them, directed upwards at a wide (nearly 90 °) or narrower angle, usually with a bulbous base, with several percurrent proliferations, always terminating in a phialide. Conidiogenous cells 10–25 × 2.5–3.5 μm, tapering to 1.5–2 μm, monophialidic, integrated, terminal, extending percurrently, subcylindrical to lageniform, pale brown to subhyaline; collarettes 2–2.5 μm wide, ca. 1.5 μm deep, subhyaline, flaring, inconspicuous. Conidia 3–4.5 × 2–3 μm (mean ± SD = 3.6 ± 0.4 × 2.4 ± 0.2 μm), ellipsoidal to subglobose, hyaline, aseptate, smooth, accumulating in yellow-white to pale olivaceous slimy heads or cirrhi, whitish (MLA, OA) or pale olivaceous to olivaceous yellow (PCA). Chlamydospores 4.5–7 × 3.5–4.5 μm sparse, primarily terminal, or lateral on a stipe, subglobose or clavate, thick-walled, smooth. Sexual morph. Unknown.

Specimen examined: Sri Lanka, Central province, Mt. Pidurutalagala, alt. 2 000 m, on decaying wood, Jan. 1973, W. Gams (CBS H-25022, CBS 263.76A).

Habitat and geographical distribution: Saprobe on decaying wood, pine cones, plant litter, and dead leaves of bamboo, known from China and Sri Lanka (Wu & Diao 2022, this study). According to GlobalFungi, identical sequences were found in 515 samples from 42 studies, collected mainly in various soil types, roots, deadwood, and sediment in forest, grassland, woodland, cropland, shrubland, anthropogenic or aquatic habitats across the globe (except Antarctica) in temperate, continental humid, tropical, subtropical and drier climates, but is absent in the boreal zone (MAT 13.4 °C, MAP 1 420 mm).

Notes: The species was described from a dead cone of Pinus sp. from China (Wu & Diao 2022). On the natural substrate, conidiophores formed two distinct layers of taller setiform, fertile, dark brown conidiophores in fascicles with shorter and paler conidiophores. The conidia from nature are comparable to those in culture (this study); they are ellipsoidal, subglobose, adhering in yellowish cirrhi but somewhat shorter (3–3.8 × 2–2.5 μm) (Wu & Diao 2022). In culture (this study), the morphology of conidiophores varied; however, they always formed a single layer. The conidiophores were solitary and unbranched, with one to several percurrent proliferations on MLA, OA, and PCA. When grown on U. dioica stems on CMA, the conidiophores were solitary or fasciculate, with several primary lateral branches, which were always fertile, terminating into a phialide. The conidia accumulated in cirrhi and heads. Branched conidiophores were rarely observed in other Chloridium species and always in culture, e.g. Chl. guttiferum, Chl. novae-zelandiae and Chl. virescens.

In the phylogenetic tree, Chl. setosum is the closest relative to Chl. virescens. Both species are morphologically highly similar and share similar size and shape of conidia in culture. Chloridium virescens can be distinguished in conidia adhering in yellow-green or green, sometimes whitish cirrhi or heads and the presence of chlamydospores, which are abundant, primarily intercalary, lateral, sometimes aggregated in short chains. In contrast, in Chl. setosum, conidia adhere in yellow white cirrhi, and chlamydospores are rare and usually in a terminal or lateral position. Both species also differ in colony characteristics.

Chloridium tropicale W.P. Wu & Y.Z. Diao, Fungal Diversity 116: 256. 2022.

For a description and illustration, see Wu & Diao (2022).

Habitat and geographical distribution: Saprobe on dead leaves of an unidentified tree from China (Wu & Diao 2022). The species was not found in the GlobalFungi database and seems to be rare.

Notes: Chloridium tropicale is characterised by simple conidiophores and oblong to ellipsoidal, straight or slightly curved conidia adhering in heads or cirrhi (Wu & Diao 2022). It is closely related to Chl. crousii and Chl. xishuangbannaense.

Chloridium virescens (Pers.) W. Gams & Hol.-Jech., Stud. Mycol. 13: 17. 1976. Figs 33–35.

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Fig. 33.

Chloridium virescens. A, B. Colonies composed of conidiophores and ascomata. C. Asci. D. Ascogenous hyphae with croziers. E, F. Fragmenting ascospores. G, H. Conidiophores. I, J. Conidia arranged in cirrhi. K. Phialide. A–K. From nature. Images: A, C–G. CBS 145487; B, I–K. CBS 138683; H. CBS 145480. Scale bars: A, B = 250 μm; C, D, J, K = 10 μm; G, H = 20 μm; E, F, I = 5 μm.

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Fig. 35.

Diversity of colony morphology in Chl. virescens on CMD, MLA, OA, PCA after 4 wk. A. CBS 676.74. B. CBS 145481. C. CBS 102341. D. CBS 144663. E. CBS 601.75B. Scale bar = 1 cm.

Basionym: Dematium virescens Pers., Neues Mag. Bot. 1: 121. 1794.

Synonyms: Sporotrichum virescens (Pers.) Link, Mag. Gesell. naturf. Freunde, Berlin 3: 13. 1809.

Helicosporium virescens (Pers.) Sivan., Bitunicate Ascomycetes and their Asexual morphs, p. 591. 1984.

Chloridium viride Link, Mag. Gesell. naturf. Freunde, Berlin 3: 13. 1809.

Corticium viride (Link) Bres., Oesterr. Bot. Z. 54: 428. 1904.

Cirrhomyces viridis (Link) Höhn., Rabenh. Krypt.-Fl. 1(9): 793. 1910.

Haplaria viride (Link) Rabenh., Klotzschii Herb. Viv. Mycol., Edn Nov, Ser. Sec., Cent. 8: no. 770. 1858.

Dematium asserculorum Pers., Mycol. Europ. 1: 15. 1822.

Chloridium dispersum Nees, Syst. Pilze Schwämme, p. 66. 1816 [1816–17].

Doratomyces viridis Corda, Weitenweber’s Beitr. ges. Nat.-Heilwiss. 1, p. 83. 1837; Icon. Fungorum 1: 19, tab. 5: 262B. 1837.

Haplaria chlorina Ellis & Everh., Bull. Torrey Bot. Club 10: 97. 1883.

Eriosphaeria vermicularioides Sacc. & Roum., Revue Mycol. 5: 235. 1883.

Chaetosphaeria vermicularioides (Sacc. & Roum.) W. Gams & Hol.-Jech., Stud. Mycol. 13: 15. 1976.

Melanopsammella vermicularioides (Sacc. & Roum.) Réblová et al., Sydowia 51: 65. 1999.

Mesobotrys flavovirens Höhn., Sber. Akad. Wiss. Wien, Math.-naturw. Kl., Abt. 1, 111: 1048. 1902.

Cirrhomyces flavovirens (Höhn.) Höhn., Rabenh. Krypt.-Fl. 1(8): 760. 1907.

Synonymy was partially adopted after Gams & Holubová-Jechová (1976).

Description on the natural substrate: Colonies effuse, hairy, brown, green to yellow-green when sporulating; composed of conidiophores and ascomata. Asexual morph. The chloridium-type conidiophores macronematous, solitary, crowded, in tufts or loose groups, erect, straight, unbranched, with one to several percurrent proliferations, cylindrical, septate, dark brown, paler towards the apex, forming two layers; conidiophores of the upper layer 106–180 × 2.5–4 μm, setiform but always fertile, conidiophores of the lower layer 48–73 × 2–3.5 μm. Conidiogenous cells 12–14(–23) × 2.5–3 μm, tapering to ca. 1.5–2 μm below the collarette, monophialidic, extending percurrently, subcylindrical to lageniform, pale brown to subhyaline; collarettes 2.5–3 μm wide, ca. 1.5 μm deep, hyaline to subhyaline, flaring, shortly funnel-shaped to inconspicuous. Conidia 2.5–3.5 × 2–2.5 μm (mean ± SD = 3.1 ± 0.2 × 2.1 ± 0.2 μm), subglobose, occasionally somewhat ellipsoidal, hyaline, aseptate, smooth, accumulating in slimy whitish, yellow-green or grass-green cirrhi, sometimes heads. Sexual morph. Ascomata 150–180(–200) μm diam, 160–200 μm high, non-stromatic, superficial, solitary or aggregated, subglobose, papillate, dark brown to black, glabrous, glossy. Ostiole periphysate. Ascomatal wall 22–33 μm thick, fragile, carbonaceous, two-layered. An outer layer consisting of brown, more or less polyhedral cells with opaque walls. An inner layer consisting of several rows of elongated, thin-walled, hyaline cells. Paraphyses 2–4.5 μm, septate, branched, longer than the asci, disintegrating with age. Asci 70–81.5(–86) × 5.5–6.5(–7) μm (mean ± SD = 73.6 ± 3.1 × 64.5 ± 3.7 μm), cylindrical-clavate, stipitate, ascal apex broadly rounded with a non-amyloid apical annulus ca. 2.5 μm wide, 1.5 μm high. Ascospores easily fragmenting within the ascus, part spores 4–5(–5.5) × 2.5–3 μm (mean ± SD = 4.5 ± 0.4 × 2.8 ± 0.2 μm), of almost equal size and shape, fusiform, hyaline, 1-septate, finely verrucose, obliquely 1(–2)-seriate in the ascus.

Culture characteristics: On CMD: colonies 56–60 mm diam, circular, flat, margin fimbriate to rhizoidal, velvety, whitish to whitish beige, with an ochre outer zone of submerged growth, pale gold pigment diffusing into the agar, reverse brown. On MLA: colonies 56–58 mm diam, circular, slightly raised, margin fimbriate to lobate, velvety-lanose, somewhat floccose, furrowed, whitish to pale ochre with a prominent ochre zone of submerged growth or brown at the margin due to chlamydospore production, pale gold pigment diffusing into the agar, reverse ochre. On OA: colonies 70–73 mm diam, circular, flat, margin entire, cobwebby to mucoid, colony centre gold-beige to cinnamon, sometimes dark olivaceous grey due to production of chlamydospores, sometimes zonate with zones of rich and sparse growth and chlamydospore production, sometimes pale yellow pigment diffusing into the agar, reverse beige to pale olivaceous brown. On PCA: colonies 61–63 mm diam, circular, flat, margin fimbriate to lobate, whitish with a conspicuous yellow to ochre outer zone of submerged growth, or beige centrally becoming brown to dark brown towards the periphery due to production of chlamydospores, pale yellow to ochre pigment occasionally diffusing into the agar, reverse ochre-beige. Sporulation was abundant on all media, delayed after 6–8 wk. Growth at 5–30 °C with an optimum at 25 °C.

Description in culture: On MLA, vegetative hyphae 1–3 μm diam, branched, septate, hyaline, smooth. Asexual morph. Conidiophores, conidiogenous cells and conidia are similar to those from nature. The chloridium-type conidiophores 80–253 × 3–4 μm, sometimes with short rhizoids at the base, growing in a single layer, with up to 11 percurrent proliferations, unbranched or occasionally branched with 1(–2) primary lateral branches. Conidiogenous cells 11–24.5 × 2.5–3.5 μm, tapering to 1.5–2.5 μm below the collarette; collarettes 3–3.5 wide, ca. 1.5 μm deep. Conidia 2.5–4(–4.5) × 2–2.5(–3) μm (mean ± SD = 3.5 ± 0.4 × 2.4 ± 0.2 μm), subglobose to ellipsoidal, occasionally ellipsoidal-oblong, hyaline, accumulating in slimy whitish to white-green heads or cirrhi. Chlamydospores abundant, 4.5–6.5 × 4–5 μm, mostly intercalary, solitary or in short chains, lateral, sessile or on a stipe, or terminal, globose, subglobose, ellipsoidal or pyriform, brown, thick-walled, smooth. Sexual morph. Not observed.

Specimens examined: Belgium, Ardennes, near Neupont, on decaying wood, Sep. 1975, W. Gams (CBS 601.75B); Héverlée, Parc d’Arenberg, on decaying wood of Quercus robur, date unknown, G.L. Hennebert (MUCL 7830-A = CBS 424.76); Malmedy, Fungi Gall. exs. 2693, (isotype of Eriosphaeria vermicularioides L 90.236-665). Czech Republic, Liberec Region, Liberec (Reichenberg), on decaying leaves, twigs and wood, A.C.J. Corda (holotype of Doratomyces viridis PRM 155471, not seen; microscopic slide ex holotype DAOM 40986); South Bohemian Region, Novohradské hory Mts., Èerné údolí, Žofín, the road to National Natural Reserve Žofín, on decaying wood of Acer sp., 6 Oct. 2017, M. Réblová M.R. 3930 (PRA-21338, CBS 144660); South Bohemian Region, Novohradské hory Mts., Horní Stropnice, Bedřichovský potok Natural Monument, on decaying wood of Quercus sp., 5 Oct. 2018, M. Réblová M.R. 3977 (PRA-21339, CBS 145345); ibid., M.R. 3986A (PRA-21340, CBS 145479); ibid., M.R. 3986B (CBS 145480); ibid., M.R. 3987A (PRA-21341, CBS 145481); ibid., M.R. 3987B (CBS 145348); ibid., on decaying wood of Alnus glutinosa, M.R. 3979 (PRA-21342, CBS 145347); ibid., M.R. 3989 (PRA-21343, CBS 145482).; Pardubice Region, Železné hory Mts. Protected Landscape Area, Horní Bradlo, Malá Střítež settlement, Polom National Nature Reserve, alt. 600 m, on decaying wood of Fagus sylvatica, 10 Nov. 2018, M. Réblová M.R. 4062 (PRA-21344, CBS 145487); South Moravian Region, Lanžhot, Ranšpurk National Nature Reserve, on decaying wood, 15 Oct. 1997, M. Réblová M.R. 1148 (PRA-21345); South Moravian Region, Milovice, Milovický les Nature Reserve, on decaying wood of Quercus sp., 18 Nov. 2010, M. Réblová M.R. 3588 (PRA-21346); ibid., M.R. 3599 (PRA-21347); South Moravian Region, Pohansko near Lanžhot, on decaying wood, 16 Oct. 1997, M. Réblová M.R. 1119 (PRA-21348, CBS 102341); South Moravian Region, Skařiny Nature Reserve near Mikulèice, on decaying deciduous wood, 24 Oct. 2004, M. Réblová M.R. 2898 (PRA-21349, CBS 144663). Germany, Rostock, on wood, very probably of Fagus, date unknown, J.H.F. Link (neotype of Chloridium viride B, not seen; microscopic slide ex neotype DAOM 40815). France, Ariège, Pyrénées Mts., Rimont, road D18 ca. 1.5 km from the village, banks of the Le Baup brook, on decaying wood, 3 Oct. 2013, M. Réblová & J. Fournier M.R. 3803 (PRA-21350); Ariège, Pyrénées Mts., Montsegur, road D9 ca. 1 km from the village, banks of the Le Lesset brook, on decaying wood, 1 Oct. 2013, M. Réblová & J. Fournier M.R. 3727 (PRA-21351, CBS 138683); locality unknown, Acer sp., date unknown, F. Mangenot (CBS 152.53). The Netherlands, South Holland Province, Schaesberg near Valkenburg, on decaying angiosperm wood, Jul. 1974, W. Gams (CBS 676.74); South Holland Province, Leiden, Leidse Hout urban park, isolated from soil, 25 Jun. 2021, V. Merckx, M. Stech & S. van Melis (CBS 149051 = SPC87.37). USA, New Jersey, Newfield, on rotten wood of Magnolia, Jul. 1883, J.B. Ellis (holotype of Haplaria chlorina NY, not seen; microscopic slide ex holotype DAOM 35346).

Habitat and geographical distribution: Chloridium virescens is a saprobe on decaying wood of Abies alba, Acer sp., Alnus glutinosa, Fagus sylvatica, Fraxinus excelsior, Quercus petraea, Quercus robur, Quercus sp. and other unidentified hosts and was also isolated from the soil. It is known from Belgium, Czech Republic, France, Germany, Ivory Coast, the Netherlands, and the USA (Link 1809, Corda 1837, Ellis & Everhart 1883, Gams & Holubová-Jechová 1976, Maggi et al. 1990, this study). It has also been reported as an endophyte of Catharanthus roseus (Kharwar et al. 2008) and Terminalia arjuna and Azadirachta indica (Kharwar et al., unpublished) from India. According to GlobalFungi, identical sequences were found in 619 samples from 39 studies, collected mainly in bulk soil, litter and roots in forest and grassland, less often in woodland or anthropogenic habitats. It is abundant in Europe, rare in other regions (Asia, Australasia, South America), and absent in Northern America. All locations are temperate, subtropical, or Mediterranean climates (MAT 9 °C, MAP 743 mm).

Notes: Chloridium virescens is characterised by unbranched conidiophores growing in two distinct layers on the natural substrate, usually in tufts or loose groups, with conidia accumulating into grass-green to yellow-green cirrhi and producing hyaline mycelium and mostly intercalary chlamydospores in culture. Because of its striking appearance, this species has been described under several names listed in the synonymy above.

The remarkable similarity of Chl. virescens with Cirrhomyces caudigerus (von Höhnel 1903) and Bisporomyces chlamydosporus (van Beyma 1940) was reported by Mangenot (1952), Meyer (1959) and Ellis (1971). They attempted to distinguish them by characters of conidia such as size, formation of cirrhi or heads, the colour of conidia in mass, and morphology of chlamydospores. Although Meyer (1959) regarded B. chlamydosporus, C. caudigerus, and Sphaeromycetella leucocephala conspecific, Hughes (1958) considered the first two species separate and transferred them to the genus Chloridium. Gams & Holubová-Jechová (1976) adopted Meyer’s treatment and regarded Chl. caudigerum and Chl. chlamydosporum as synonyms of Chl. virescens for which they proposed varieties. The three taxa were resolved as separate species lineages in the present phylogeny.