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Cadophora species from marine glaciers in the Qinghai-Tibet Plateau: an example of unsuspected hidden biodiversity
IMA Fungus volume 13, Article number: 15 (2022)
Large numbers of marine glaciers in the Qinghai-Tibet Plateau are especially sensitive to changes of climate and surface conditions. They have suffered fast accumulation and melting and retreated quickly in recent years. In 2017, we surveyed the cold-adapted fungi in these unique habitats and obtained 1208 fungal strains. Based on preliminary analysis of ITS sequences, 41 isolates belonging to the genus Cadophora were detected. As one of the most frequently encountered genera, the Cadophora isolates were studied in detail. Two phylogenetic trees were constructed: one was based on the partial large subunit nrDNA (LSU) to infer taxonomic placement of our isolates and the other was based on multi-locus sequences of LSU, ITS, TUB and TEF-1α to investigate more exact phylogenetic relationships between Cadophora and allied genera. Combined with morphological characteristics, nine Cadophora species were determined, including seven new to science. Among the new species, only C. inflata produces holoblastic conidia and all the others express phialidic conidiogenesis. All isolates have optimum growth temperature at 20 °C or 25 °C. With more species involved, the currently circumscribed genus became obviously paraphyletic. All members are clustered into two main clades: one clade mainly includes most of the Cadophora species which have phialidic conidiogenesis and we refer to as ‘Cadophora s. str.’; the remaining Cadophora species have multiform conidiogenesis and are clustered in the second clade, with members of other genera in Ploettnerulaceae interspersed among the subclades. The results show a high diversity of Cadophora from marine glaciers in the Qinghai-Tibet Plateau and most of them are novel species.
The genus Cadophora was first established in 1927, with C. fastigiata as the type species, to accommodate dematiaceous hyphomycetes producing solitary phialides with distinct hyaline collarettes (Lagerberg et al. 1927). Due to subtle differences in morphology, Conant (1937) transferred eight Cadophora species to the genus Phialophora. Later, Gams (2000) proposed that Phialophora species related to discomycete sexual morphs of Mollisia and related genera belonging to Helotiales should be accommodated in Cadophora. This proposal was supported by subsequent rDNA sequence analysis of LSU (Harrington and McNew 2003).
Currently, the genus is included in the family Ploettnerulaceae of Helotiales (Johnston et al. 2019; Ekanayaka et al. 2019) and comprises some species with multiform morphological characters deviated from the original morphological generic concept. For example, C. antarctica and C. fascicularis produce chains of ramoconidia and conidia on holoblastic conidiogenous cells (Crous et al. 2017; Maciá-Vicente et al. 2020); while C. obovata has putatively monoblastic conidiogenous cells that may represent a retrogression of enteroblastic phialidic conidiogenesis and C. fallopiae is only observed as a cladophialophora-like synasexual morph in culture (Maciá-Vicente et al. 2020; Crous et al. 2020). Besides, C. lacrimiformis only found by its sexual morph, is also included in this asexually typified genus (Ekanayaka et al. 2019). Recent studies based on molecular data have shown that Cadophora is apparently paraphyletic and species with distinct morphological variations may share ancestors with other related genera (Maciá-Vicente et al. 2020).
Species of Cadophora normally possess multiple trophic modes. They are commonly considered as plant pathogens, root associates or wood and soil colonizers with cosmopolitan distribution. A global survey on the dominant soil fungal communities of different biomes has shown that Cadophora is one of the most ubiquitous soil fungal taxa with significantly higher number of genes related to stress-tolerance and resource uptake (Egidi et al. 2019). In some cold Arctic and Antarctic sites, Cadophora species have been frequently isolated from soils, marine sediments and organisms, fresh water lakes, especially the historic wood huts and some mummified or submerged drift wood (Blanchette et al. 2004, 2010, 2016; Jurgens et al. 2009; Gonçalves et al. 2012; Furbino et al. 2014; Zhang et al. 2017; Nagano et al. 2017; Duran et al. 2019). They are hypothesized to be key organisms capable of initiating nutrient cycles and energy flows from dead organic materials in high latitudes (Blanchette et al. 2016). Meanwhile, the saprotrophic species, mainly C. malorum, C. luteo-olivacea, and C. fastigiata which were frequently isolated from polar regions are also detected as pathogens or endophytes from different living plants worldwide (Di Marco et al. 2004; Gramaje et al. 2011; Navarrete et al. 2011; Travadon et al. 2015). Enzyme tests of some Cadophora members have shown that C. luteo-olivacea and C. malorum are capable of degrading a range of carbon sources and releasing soluble phosphorus so that their trophic modes could vary depending on their nutrient needs from different substrata (Day and Currah 2011; Walsh et al. 2018).
The Qinghai-Tibet Plateau, lying across the center of Asia and having an average elevation of 4000 m, possesses large numbers of glacial groups that constitute the center of Asian Highland Glaciers. Based on hydrothermal conditions and physical properties, glaciers in China can be divided into continental glaciers and marine glaciers. Continental glaciers, which are also known as cold glaciers, develop in the continental climate areas where precipitation amount is limited; marine glaciers, which are also known as temperate glaciers, generally form in marine climate areas with abundant precipitation (Shi et al. 1964, 2000). Controlled by the marine monsoonal climate, nearly 9000 marine glaciers, which cover a total area of 13,200 square kilometers and account for 22.2% of the total glacier area in China, form at southeast margin of the Qinghai-Tibet Plateau. Under the background of global warming, glaciers all over the world are retreating significantly. In the next 100 years, marine glaciers in the Qinghai-Tibet Plateau, with the features of fast accumulation and melting and being more sensitive to the change of climate, will retreat more quickly (Yao et al. 2004; Chen et al. 2005). It is necessary and urgent to investigate fungal diversity and resources in this unique area.
Our first investigation (2009–2011) on cold-adapted fungi in the permafrost and alpine glaciers of Qinghai-Tibet Plateau indicates that the diversity of cold-adapted fungi from marine glaciers is especially high and many of them may represent unknown species (Wang et al. 2015). Another survey was conducted in 2017, focusing on the diversity of cold-adapted fungi from marine glaciers. Based on preliminary analyses of the generated ITS sequences, 41 strains representing nine Cadopora species including seven new species are described and phylogenetic relationships intra and among Cadophora and related genera are discussed in this study.
Materials and methods
Soil, ice and water samples were collected from four marine glaciers and two nearby snow-capped mountains in 2017 (Table 1). Sampling sites were selected at different elevations of the following marine glaciers and snow-capped mountains: Hailuogou Glacier, Yanzigou Glacier and Dagu Glacier in Sichuan Province, Yulong Snow Mountain, Baima Snow Mountain and Mingyong Glacier in Yunnan Province (Figs. 1, 2). For all samplings, clean hand tools were surface sterilized with 70% ethanol before use. After the removal of the top 5–10 cm of surface sediment, c. 500 g soil or ice sample was collected from the underlying layer and placed in a fresh Zip-lock plastic bag and sterilized plastic bottles. Melt water samples were directly collected and placed in sterilized centrifuge tubes or Zip-lock plastic bags. All the samples were maintained at 4 °C until arrival at the laboratory.
Strains were isolated from soil and water samples as soon as they were taken to the lab. Soil samples were isolated with traditional pour plate method: A 10 g quantity of each soil sample was suspended in sterile-distilled water in a flask, the volume was then increased to 100 mL before the suspension was shaken to disperse soil particles and then serially diluted to 10–2, 10–3 and 10–4; 100 mL of each water sample was filtrated by nitrocellulose filter membrane with pore size of 0.45 μm, the membrane with trapped fungi was put in a sterile 50 mL centrifuge tube which contained 10 mL distilled water and the tube was vigorously agitated to suspend the trapped mycelium and spores. About 0.1 mL of each final diluent or concentrate was placed on the surface of two 90 mm diam Petri plates containing 1/4 strength Potato Dextrose Agar (1/4 PDA; 9 g of Potato Dextrose Agar [BD Difco] and 15 g of Agar per L of demineralized water) supplemented with chloramphenicol (0.1 mg/mL) and streptomycin (0.1 mg/mL). The plates were sealed and incubated at 15 °C and 25 °C (one plate per temperature) and were examined for fungal growth at 1 wk intervals for 4 wk. Colonies that appeared on the plates were transferred to two new plates and then incubated at 15 °C and 25 °C. All fungal strains were stored at 4 °C for further studies.
41 isolates representing all of the Cadophora species isolated were studied in more detail. To enhance sporulation, strains were inoculated on potato dextrose agar (PDA; BD Difco), malt extract agar (MEA, BD Difco) and oatmeal agar (OA; BD Difco). Pine needle medium, H2O2 treatment and slide culture technique (Xu et al. 2009; Su et al. 2012) were also used to induce sporulation. For phenotypic determination, the strains were transferred to PDA, MEA and OA plates with three replicates and incubated in the dark at 25 °C. Optimal growth temperature (OGT) and maximum growth temperature (MGT) were also tested by culturing each isolate in triplicate on PDA at temperatures ranging from 5 to 35 °C at 5 °C increments. Colony diameters were measured in two perpendicular directions after 2 wk at different temperatures, and the mean diameter was obtained from three replicate plates cultivated at the same temperatures. Colony colors were determined using taxonomic description color charts (Rayner 1970). Microscopic preparations were made by mounting aerial hyphae in water or using the slide cultures directly. Hyphae, conidiophores, and conidia were observed, photographed, and measured with 1000 × magnification by using a Nikon 80i microscope with differential interference contrast (DIC) optics. Specimens were deposited in the Mycological Herbarium of Hebei University (HBU), while living cultures including ex-types were deposited in the China General Microbiological Culture Collection Center (CGMCC).
DNA extraction, PCR amplification, sequencing and phylogenetic analyses
Genomic DNA was extracted from the fungal mycelia following the protocol described by Wang and Zhuang (2004). The partial large subunit nrDNA (LSU), the internal transcribed spacer region of the nuclear ribosomal RNA gene (ITS), the partial translation elongation factor 1-α gene (TEF-1α) and the β-tubulin (β-TUB) gene were amplified and sequenced with the primer pairs of LROR/LR5 (Vilgalys and Hester 1990), ITS1/ITS4 (White et al. 1990), EF1-688F/EF1-1251R (Alves et al. 2008) and BTCadF/R (Travadon et al. 2015), respectively. PCR was performed in 50 μL reactions containing DNA template 1.0 μL, each forward and reverse primers 1.0 μL, 2 × MasterMix 25 μL (ThermoFisher scientific Co. Ltd., Shanghai, China) and 22 μL H2O, PCR parameters were as follows: denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 30 s and a final elongation step at 72 °C for 10 min. Annealing temperature for each gene were 54 °C for LSU and ITS, 51 °C for TEF-1α and 56 °C for TUB. The PCR products were sequenced with the primers mentioned above by BGI Tech Solutions Co., Ltd. (Shenzhen, China).
Nucleotide sequences were initially checked and edited using Chromas software ver. 2.6.6 (http://www.technelysium.com.au/chromas.html) and EdiSeq (Lasergene, DNASTAR) and then were compared to accessions in the GenBank database via BLASTn searching to find the most likely taxonomic designation. To reveal the family placements of the species described in this study, a LSU tree was constructed. To investigate more exact phylogenetic relationships and taxonomic distinctions of novel species, a multi-locus analysis was performed based on ITS, LSU, TUB and TEF1-α genes. Sequence data of the four genes especially those of ex-type strains, were downloaded from GenBank and added to the sequences generated in this study. The datasets were aligned automatically using MAFFT v. 7.471 (Katoh and Standley 2013) and further manual alignment was carried out with MEGA v. 7 (Kumar et al. 2016) and alignments were deposited in TreeBASE (www.treebase.org, submission no. S29383).
Phylogenetic analyses were conducted using Bayesian Inference (BI), Maximum Likelihood (ML) and Maximum Parsimony (MP) methods. For BI analyses, the best fit model of evolution for each partition was estimated by MEGA v. 7. Posterior probabilities were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.7a (Ronquist and Huelsenbeck 2003) using the estimated models of evolution. For the LSU/multi-locus trees, six simultaneous Markov chains were run for 4,000,000/8,000,000 generations and trees were sampled every 100th generation (resulting in 40,000/80,000 total trees). The first 10,000/20,000 trees represented the burn-in phase of the analyses were discarded and the remaining 30,000/60,000 trees were used for posterior probabilities (PP) calculation in the majority rule consensus trees. The ML analyses were performed by raxmlGUI 2.0.0-beta (Edler et al. 2019) using the GTRGAMMA model with the rapid bootstrapping and search for best scoring ML tree algorithm, including 1000 bootstrap replicates. The MP analyses were conducted using PAUP v. 4.0b10 (Swofford 2002) and an unweighted parsimony (UP) analysis was performed. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Branches of zero length were collapsed and all equally most parsimonious trees were saved. Descriptive tree statistics such as tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI), were calculated for trees generated. Clade stability was assessed using bootstrap analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa.
1208 fungal strains isolated from 120 samples of four glaciers and two snow-capped mountains were preliminarily identified based on BLAST comparison of ITS sequences against the GenBank database. As one of the most commonly encountered fungal groups, 41 isolates belonging to Cadophora were studied in detail.
Sequences of referential species, especially those of ex-type strains, were retrieved from GenBank and added to the sequences generated in this study (Table 2). The alignments of partial sequences of LSU (For LSU phylogenetic analysis), ITS, LSU (For muti-locus phylogenetic analysis), TUB and TEF1-α have 855, 452, 834, 582 and 694 characters, respectively.
According to the LSU phylogenetic tree, representative Cadophora strains of this study (marked with bold font) and the known Cadophora species are interspersed with species of other genera in Ploettnerulaceae and form a well-supported clade (BP/BP/PP = 90/98/100, ML/MP bootstrap and BI posterior probability support values, respectively) that distinctly separate from other family members in the Helotiales (Fig. 3).
A multi-gene phylogenetic tree is also employed to investigate further phylogenetic relationships intra and among Cadophora and allied genera (Fig. 4). All the representative species cluster into two main clades with high ML/MP bootstrap or BI posterior probability support values (85/100/100, 97/100/100 respectively). In the first main clade (Clade 1), 38 isolates of this study form six distinct subclades: isolates of YZ1026 and YZ1034 cluster in a lineage including the ex-type sequences of C. novi-eboraci with strong branch support; although strain MY902 and the known species of Hymenula cerealis form a well-supported subclade, they are obviously distinguished morphologically. The placement of H. cerealis should also be confirmed by protein coding genes which are currently unavailable; the other four subclades group seperately from previously described species. Combined with morphological characteristics, we propose five Cadophora species new to science: Cadophora caespitosa, C. daguensis, C. indistincta, C. magna and C. qinghai-tibetana. Clade 1 also includes most of the phialidic Cadophora species (including the type species of the genus) and three species (Hymenula cerealis, Mollisia cinerella and Phialophora dancoi) currently placed in other genera. The second main clade (Clade 2) contains the remaining Cadophora species and most of the other Ploettnerulaceae members. Three isolates of this study are included in this clade: strain YL412 clusters with C. malorum in a well supported lineage; strain MY759 and MY814 form two distinct single strain clades and we propose them as two new species (Cadophora inflata and Cadophora yulongensis). Cadophora species in Clade 2 have multiform conidiogenesis modes and form lineages interspersed by other Ploettnerulaceae members.
Cadophora caespitosa Q.-M. Wang, B.-Q. Zhang & M.-M. Wang, sp. nov.
MycoBank No.: MB837889.
Etymology: Tufted (Lat.: caespitosa). Referring to multiple phialides arranged in terminal fascicles.
Diagnosis: Morphologically distinct from other Cadophora species in having penicillately branched heads of multiple phialides.
Type: China: Yunnan Province: Mingyong Glacier, N28°27′25″ E98°45′51″, 2960 m, from water, 9 May 2017, M.-M. Wang (HBU20001 – holotype; MY156 = CGMCC3. 20,179 – ex-type cultures).
Description: Mycelium hyaline to brown, septate, smooth-walled, branched, 1–3 μm wide. Conidiophores pale brown or hyaline, straight, septate, smooth, branched or unbranched, distinct, dark stipe with multiple phialides terminating in a complexly penicillately branched apex commonly observed. Conidiogenous cells phialidic, located laterally on fertile hyphae or arranged in complex heads, cylindrical to navicular, often constricted at the base, upper subulate, hyaline, smooth-walled, 6.5–32.3 × 2.6–3.8 μm, collarettes distinct, funnel-shaped, 1.9–3.9 μm long, opening 1.9–3.4 μm wide. Conidia hyaline, aseptate, smooth-walled, sporulation abundant, ovate to dacryoid or ellipsoidal, single, with both ends rounded, straight, 3.4–7.1 × 1.7–3.4 μm (mean = 5.0 ± 0.9 × 2.6 ± 0.4 μm, n = 30), L/W ratio = 2.0.
Culture characteristics — Colonies on MEA reaching 33 mm diam after 14 d at 25 °C in the dark, on OA and PDA reaching 55 mm and 34 mm diam, respectively. OGT 25 °C and MGT 35 °C (Fig. 5). Colonies on MEA with a smooth margin, flat, grey-white, buff to light yellow at the margin, reverse olivaceous black. Colonies on OA with an entire margin, flat, greenish-black with a white margin, reverse same colours. Colonies on PDA with an entire margin, flat, hazel to yellow–brown with a white margin, reverse same colours.
Notes: According to Day et al. (2012), the genera Cadophora and Phialocephala are generally distinguished by phialide complexity and conidial length, with the former producing solitary phialides and conidia longer than 4 μm, while the latter producing densely packed heads of phialides and conidia shorter than 4 μm. This newly described species is morphologically distinct from other Cadophora species, because it has penicillately branched heads of multiple phialides. This character is similar to species of Phialocephala. However, C. caespitosa and species of Phialocephala vary in conidial length. Phylogenetic analyses based on sequences of LSU and combined ITS + LSU + TUB + TEF1-α regions (Figs. 3, 4) show that C. caespitosa is grouped with species of Cadophora in the family of Ploettnerulaceae and forms a well-supported lineage.
Additional specimens examined: China: Sichuan Province: Dagu Glacier, N32°14′23″ E 102°47′7″, 3610 m, from water, 1 May 2017, M.-M. Wang (culture DG1120 = CGMCC3.20192); Hailuogou Glacier, N29°33′10″ E101°58′10″, 3180 m, from water, 28 Apr. 2017, M.-M. Wang (culture HL674 = CGMCC3.20431. Yunnan Province: Baima Snow Mountain, N28°23′29″ E98°59′22″, 4125 m, from soil, 10 May 2017, M.-M. Wang (BM691 = CGMCC3.20432; Mingyong Glacier, N28°27′25″ E98°45′51″, 2960 m, from water, 9 May 2017, M.-M. Wang (culture MY169 = CGMCC3.20180).
Cadophora daguensis Q.-M. Wang, B.-Q. Zhang & M.-M. Wang, sp. nov.
MycoBank No.: MB837890.
Etymology: Referring to the geographical location from where the isolates collected.
Diagnosis: Morphologically distinct from the phylogenetically related species of C. ramosa by having lower growth rate.
Type: China: Sichuan Province: Dagu Glacier, N32°14′21″ E102°47′5″, 3630 m, from soil, 1 May 2017, M.-M. Wang (HBU20040 – holotype; DG21 = CGMCC3.20846 – ex-type cultures).
Description: Mycelium black brown or hyaline, septate, smooth-walled, branched, 1–3 μm. Mycelial cell occasionally inflated in the middle, up to 5–8 μm wide, constricted at the septae. Conidiophores black brown or hyaline, septate, mesotonously branched or unbranched. Conidiogenous cells phialidic, hyaline, smooth-walled, tapering toward the tip and slightly constricted at the base, 13.4–23.5 × 2.2–3.8 μm, collarettes distinct and funnel-shaped, 2.8–4.8 μm long, opening 2.6–3.8 μm wide. Conidia hyaline, aseptate, smooth-walled, with subulate tip and round base, single, straight, 4.5–7.8 × 2.1–3.2 μm (mean = 5.5 ± 0.7 × 2.7 ± 0.3 μm, n = 30), L/W ratio = 2.1.
Culture characteristics — Colonies on MEA reaching 13 mm diam, after 14 d at 25 °C in the dark, on OA and PDA reaching 19 mm and 17 mm diam, respectively. OGT 20 °C and MGT 35 °C (Fig. 5). Colonies on MEA raised, glabrous, citrine to primrose, reverse same colours. Colonies on OA with a smooth margin, flat, olivaceous brown in the centre, light grey at the margin, reverse same colours. Colonies on PDA with a whitish margin, slight raised, pure yellow, reverse same colours.
Notes: Strains of DG5 and DG21, representing Cadophora daguensis, form a well-supported subclade. This newly described species is phylogenetically related to C. ramosa, but they are obviously distinguished in colony growth rates.
Additional specimen examined: China: Sichuan Province: Dagu Glacier, N32°14′21″ E102°47′5″, 3630 m, from soil, 1 May 2017, M.-M. Wang (culture DG5 = CGMCC3.20845).
Cadophora indistincta Q.-M. Wang, B.-Q. Zhang & M.-M. Wang, sp. nov.
MycoBank No.: MB837895.
Etymology: Referring to the indistinct collarettes of phialides.
Diagnosis: Cadophora indistincta characterized by the red coloured colony on PDA and indistinct collarettes.
Type: China: Sichuan Province: Dagu Glacier, N32°8′19″ E102°56′13″, 2380 m, from water, 1 May 2017, M.-M. Wang (HBU20012 – holotype; DG1014 = CGMCC3.20189 – ex-type cultures).
Description: Mycelium hyaline, septate, smooth-walled, branched, 1–4 μm. Conidiophores hyaline, septate, smooth, often solitary. Conidiogenous cells phialidic, located terminally or laterally, discrete, hyaline, smooth-walled, straight or curved, cylindrical to navicular, often inflated in the middle and constricted at the base, 5.3–31.4 × 1.6–3.7 μm, collarettes often indistinct. Conidia hyaline, aseptate, smooth-walled, cylindrical to oblong, 4.7–7.5 × 1.6–2.5 μm (mean = 5.5 ± 0.7 × 2.2 ± 0.2 μm, n = 30), L/W ratio = 2.5.
Culture characteristics — Colonies on MEA reaching 45 mm diam, after 14 d at 25 °C in the dark, on OA and PDA reaching 49 mm and 44 mm diam, respectively. OGT 25 °C and MGT 35 °C (Fig. 5). Colonies on MEA flat, primrose to pale citrine, white at the margin, reverse same colours. Colonies on OA with a yellow margin, surface black-brown, aerial mycelium sparse, reverse same colours. Colonies on PDA with a distinct and smooth margin, flat, grey to red, white at the edge, reverse dark-red.
Notes: Cadophora indistincta is phylogenetically related to C. qinghai-tibetana (Fig. 4), but they are especially different in colours of colony on PDA and the length of collarettes (Figs. 8, 13). Cadophora indistincta produces red coloured colony on PDA and this is also a distinct character different from other Cadophora species except C. ferruginea, but the colour of the colony produced by C. ferruginea is rust red and darker than that of C. indistincta.
Additional specimens examined: China: Sichuan Province: Dagu Glacier, N32°8′19″ E102°56′13″, 2380 m, from soil, 1 May 2017, M.-M. Wang (culture DG978 = CGMCC3.20233; DG1074 = CGMCC3.20196); N32°15′38″ E102°48′15″, 3510 m, from soil, 1 May 2017, M.-M. Wang (culture DG1017 = CGMCC3.20195); N32°14′23″ E102°47′7″, 3610 m, from water, 1 May 2017, M.-M. Wang (cculture DG1054 = CGMCC3.20234).
Cadophora inflata Q.-M. Wang, B.-Q. Zhang & M.-M. Wang, sp. nov.
MycoBank No.: MB837892.
Etymology: Referring to the characteristics of the inflated hyphae.
Diagnosis: Morphologically distinct from other Cadophora species by producing multiple chlamydospores and single holoblastic conidia attached to the hyphae with short conidiophores.
Type: China: Yunnan Province: Mingyong Glacier, N28°27′24″ E98°45′51″, 2976 m, from soil, 9 May 2017, M.-M. Wang (HBU20009 – holotype; MY759 = CGMCC3.20186 – ex-type cultures).
Description: Mycelium olivaceous or hyaline, septate, branched, smooth-walled, 2–4 μm wide. Hyphal cells often strongly inflated, up to 6–10 μm wide, form chains or microsclerotia-like bodies. Conidiophores very short or highly reduced. Conidiogenous cells holoblastic. Conidia hyaline, attached to mycelium, located laterally or terminally, smooth-walled, globular or spathulate, solitary, 2.9–7.1 × 3.0–4.4 μm (mean = 3.9 ± 0.8 × 3.7 ± 0.4 μm, n = 30), L/W ratio = 1.1.
Culture characteristics — Colonies on MEA reaching 28 mm diam, after 14 d at 25 °C in the dark, on OA and PDA reaching 47 mm and 37 mm diam, respectively. OGT 25 °C and MGT over 35 °C (Fig. 5). Colonies on MEA, with an entire margin, flat, white, lacking aerial mycelium, reverse same colours. Colonies on OA with a smooth margin, flat, black in the center, olivaceous to white from middle to edge, reverse same colours. Colonies on PDA with a smooth margin, felty, grey, pale yellow at the margin, reverse grey-brown with a pale buff to white margin.
Notes: Cadophora inflata is characterized by chains or microsclerotia-like inflated cells that are similar to Leptodophora gamsii and L. echinata which were first described as C. gamsii and C. echinata (Maciá-Vicente et al. 2020). The original authors interpreted these structures as holoblastic conidia but they may just as well described as inflated hyphal segments with dormancy functions. Our newly described species failed to produce conidia on MEA, OA, and PDA media. We also tried other methods such as treating the cultures with H2O2 or culturing the isolates on pine needle medium before a slide culture technique was used. Cadopohra inflata produces globose or ellipsoidal conidia attached directly to the hyphae with very short conidiophores that resemble those of Leptodophora orchidicola, which has been transferred from Cadophora to Leptodophora (Koukol & Maciá-Vicente, 2022). Thus, we presume that the inflated hyphal cells are really just chlamydospores.
Cadophora magna Q.-M. Wang, B.-Q. Zhang & M.-M. Wang, sp. nov.
MycoBank No.: MB837893.
Etymology: Big (Lat.: magna). Referring to the comparatively large conidia.
Diagnosis: Cadophora magna is distinct in producing the large conidia and strongly inflated hyphal cells.
Type: China: Yunnan Province: Mingyong Glacier, N28°27′24″ E98°45′51″, 2976 m, from soil, 9 May 2017, M.-M. Wang (HBU20011 – holotype; MY902 = CGMCC3.20188 – ex-type cultures).
Description: Mycelium hyaline to dark brown, septate, branched, smooth-walled, 1–3 μm, hyphal cells often strongly inflated, variable in shape. Conidiophores brown, smooth-walled, often reduced to conidiogenous cells. Conidiogenous cells phialidic, mostly single, arranged terminally or laterally on the hyphae, cylindrical to navicular, apex wedge, base truncate, smooth-walled, straight or slightly curved, 12.7–20.3 × 2.8–3.8 μm, collarettes funnel-shaped, 1.9–3.0 μm long, opening 2.8–2.9 μm wide. Conidia hyaline, aseptate, smooth-walled, ovoidal or dacryoid to ellipsoidal, upper wedge-shaped, base round, single, straight, 5.2–9.4 × 3.0–4.7 μm (mean = 7.3 ± 0.9 × 3.7 ± 0. 4 μm, n = 30), L/W ratio = 2.0.
Culture characteristics — Colonies on MEA reaching 30 mm diam after 14 d at 25 °C in the dark, on OA and PDA reaching 41 mm and 29 mm diam, respectively. OGT 20 °C and MGT 35 °C (Fig. 5). Colonies on MEA white, margin covered with white and velvety aerial mycelium, reverse white. Colonies on OA with a smooth margin, flat, whitish, pale olivaceous in the centre, reverse same colours. Colonies on PDA white, reverse same colours.
Notes: Cadophora magna is currently only known from a single isolate (MY902) from soil samples of Mingyong Glacier and is morphologically distinct from other Cadophora species in the huge single conidia. In the newly described species, both C. magna and C. inflata produce strongly inflated hyphae cells, but the hyphae cells of C. inflata are often thick-walled and form tuft-like bodies. C. magna is phylogenetically related to Hymenula cerealis, but they are obviously distinguished morphologically, as the latter often produces short chains of spores as well as spores enveloped in a mucus drop (Nisikado et al. 1934). Besides, the placement of H. cerealis should also be confirmed by more molecular data which are currently unavailable.
Cadophora malorum (Kidd & Beaumont) W. Gams, Stud. Mycol. 45: 188 (2000).
Description: Mycelium brown-black, septate, smooth-walled, branched, 2–3 μm. Conidiophores brown-black, septate, smooth. Conidiogenous cells phialidic, often forming clusters, terminally or laterally on the hyphae, smooth-walled, straight, ampulliform, often 9.5–16.0 × 2.9–3.5 μm, collarettes distinct, collarettes short tubular to funnel-shaped, 1.1–2.0 μm long, opening 1.6–1.9 μm wide. Conidia fuscous, aseptate, smooth-walled, ellipsoidal to elongate-ellipsoidal or subglobose, single, straight, 2.7–4.7 × 1.9–3.4 μm (mean = 3.7 ± 0.5 × 2.5 ± 0.4 μm, n = 30), L/W ratio = 1.5.
Culture characteristics — Colonies on MEA reaching 41 mm diam, after 14 d at 25 °C in the dark, on OA and PDA reaching 60 mm and 48 mm diam, respectively. OGT 25 °C and MGT 35 °C (Fig. 5). Colonies on MEA with a weakly undulate margin, brown-grey to yellow–brown, reverse same colours. Colonies on OA with a distinct and white margin, olivaceous to dull green, reverse same colours. Colonies on PDA with a distinct margin, felty, brown, reverse yellow–brown.
Notes: Cadophora malorum is a very common Cadophora species and has often been isolated as saprobes or pathogens worldwide (Blanchette et al. 2010; Gramaje et al. 2011; Sugar and Spotts 1992). Strain YL412 was isolated from soil samples collected from Yulong Snow Mountain and the morphological characteristics are similar with the description of the type (Gams, 2000).
Specimen examined: China: Yunnan Province: Yulong Snow Mountain, N27°11′17″ E100°22′43″, 3362 m, from soil, 7 May 2017, M.-M. Wang (culture YL412 = CGMCC3.20184).
Cadophora novi-eboraci Travadon et al., Fungal Biol. 119: 61 (2015).
Description: Mycelium hyaline to brown, septate, smooth-walled, branched, 1–3 μm. Conidiophores hyaline, aseptate, smooth, often solitary. Conidiogenous cells phialidic, terminally or laterally on the hyphae, discrete conidiogenous cells hyaline, smooth-walled, curved or straight, cylindrical to navicular, 6.2–19.9 × 2.4–3.0 μm, collarettes short, tubular, 1.0–1.9 μm long, opening 1.4–1.8 μm wide. Conidia hyaline, aseptate, smooth-walled, elongate-ellipsoidal to cylindrical, straight, 3.9–8.3 × 1.8–2.7 μm (mean = 5.8 ± 1.0 × 2.3 ± 0.3 μm, n = 30), L/W ratio = 2.6.
Culture characteristics — Colonies on MEA reaching 29 mm diam, after 14 d at 25 °C in the dark, on OA and PDA reaching 26 mm and 28 mm diam, respectively. OGT 25 °C and MGT 35 °C (Fig. 5). Colonies on MEA with an undulate margin, surface white, reverse same colours. Colonies on OA with a distinct margin, flat, citrine to pure yellow, white at edge, reverse same colours. Colonies on PDA with a distinct margin, raised, white to whitish, sometimes covered by floccose aerial mycelium, reverse same colours.
Notes: Cadophora novi-eboraci was originally described from decaying wood of Grapevine in North America mainly based on phylogenetic analyses of three nuclear loci (ITS, TUB and TEF1-α) (Travadon et al. 2015). It has also been isolated from Prunus wood and freshwater (Bien and Damm 2020; Lim et al. 2021). Strains observed in this study were isolated from soil samples of the Yanzigou Glacier in China.
Specimens examined: China: Sichuan Province: Yanzigou Glacier, N29°41′58″ E102°0′7″, 2620 m, from soil, 29 Apr. 2017, M.-M. Wang (culture YZ1026 = CGMCC3.20434; YZ1034 = CGMCC3.20190).
Cadophora qinghai-tibetana Q.-M. Wang, B.-Q. Zhang & M.-M. Wang, sp. nov.
MycoBank No.: MB837896.
Etymology: Referring to the geographical location from where the type strain was collected.
Diagnosis: Morphologically distinguished from the phylogenetically related species of C. indistincta in colony colours and the length of collarettes.
Type: China: Sichuan Province: Dagu Glacier, N32°8′19″ E102°56′13″, 2380 m, from soil, 1 May 2017, M.-M. Wang (HBU20019 – holotype; DG1156 = CGMCC3.20193 – ex-type cultures).
Description: Mycelium hyaline or brown-black, septate, smooth-walled, branched, 2–4 μm, often forming coils up to 34.9 μm diam. Conidiophores hyaline, smooth, frequently reduced to conidiogenous cells. Conidiogenous cells phialidic, laterally on the hyphae or hyphae coils, single or in groups of two or three, the mesotonously branched ones often reduced to mere openings with collarettes formed directly on conidiophores, cylindrical or navicular, inflated in the middle and attenuated at the base, hyaline or fuscous, smooth-walled, straight or curved, 6.8–19.9 × 2.0–3.9 μm, collarettes funnel-shaped or absent, 1.6–2.5 μm long, opening 1.6–2.7 μm wide. Sporulation abundant, conidia hyaline, aseptate, smooth-walled, cylindrical to elongate-ellipsoidal, 5.0–7.3 × 1.7–2.7 μm (mean = 6.0 ± 0.7 × 2.1 ± 0.2 μm, n = 30), L/W ratio = 2.8.
Culture characteristics — Colonies on MEA reaching 19 mm diam after 14 d at 25 °C in the dark, on OA reaching 31 mm and 18 mm diam, respectively. Colonies on MEA with a distinct margin, flat, colony surface buff, reverse same colours. Colonies on OA with a smooth margin, flat, surface olivaceous black, whitish at the margin, reverse same colours. Colonies on PDA with a distinct and regular margin, aerial mycelium sparse, grey in the centre, buff to whitish at the margin, reverse same colours.
Notes: More than half of the isolates in this study were identified as Cadophora qinghai-tibetana and these were isolated from soil and water samples of Yulong Glacier, Mingyong Glacier, Baima Snow Mountain in Yunnan Province and Dagu Glacier in Sichuan Province. Strains of YL73 (from Yulong Snow Mountain), DG1048, DG1073, DG1087, DG1105 and DG1156 (from Dagu Glacier), MY527, MY588, MY589 and MY873 (from Mingyong Glacier) have optimum growth temperature of 20 °C while the others have optimum growth temperature at 25 °C. Cadophora qinghai-tibetana has typical phialidic conidiogenesis and produces cylindrical to ellipsoidal conidia that are common in many Cadophora species, but morphologically distincts from the phylogenetically related species of C. indistincta in colony colours and the length of collarettes.
Additional specimens examined: China: Sichuan Province: Dagu Glacier, N32°13′14″ E102°45′29″, 4850 m, from soil, 1 May 2017, M.-M. Wang (culture DG975 = CGMCC3.20232); N32°8′19″ E102°56′13″, 2380 m, from soil, 1 May 2017, M.-M. Wang (culture DG1048 = CGMCC3.20191; DG1073 = CGMCC3.20235; DG1087 = CGMCC3.20236; DG1105 = CGMCC3.20197). Sichuan Province: Hailuogou Glacier, N29°34′8″ E101°59′36″, 3180 m, from soil, 28 Apr. 2017, M.-M. Wang (culture HL876 = CGMCC3.20437). Yunnan Province: Baima Snow Mountain, N29°23′1″ E99°0′20″, 4366 m, from soil, 10 May 2017, M.-M. Wang (culture BM327 = CGMCC3.20181; BM360 = CGMCC3.20183; BM523 = CGMCC3.20230; BM816 = CGMCC3.20436); N28°22′59″ E99°0′31″, 4343 m, from soil, 10 May 2017, M.-M. Wang (culture BM857 = CGMCC3.20433; Mingyong Glacier, N28°27′27″ E98°45′49″, 2976 m, from soil, 9 May 2017, M.-M. Wang (culture MY474 = CGMCC3.20185); N28°27′28″ E98°45′43″, 3067 m, from soil, 9 May 2017, M.-M. Wang (culture MY492 = CGMCC3.20847; MY527 = CGMCC3.20848; MY588 = CGMCC3.20849; MY589 = CGMCC3.20850; MY873 = CGMCC3.20231); Yulong Snow Mountain, N27°10′55″ E100°19′87″, 4531 m, from soil, 7 May 2017, M.-M. Wang (culture YL73 = CGMCC3.20228); N27°11′17″ E100°22′43″, 3362 m, from water, 7 May 2017, M.-M. Wang (culture YL305 = CGMCC3.20435; YL319 = CGMCC3.20229; YL357 = CGMCC3.20182; YL414 = CGMCC3.20194).
Cadophora yulongensis Q.-M. Wang, B.-Q. Zhang & M.-M. Wang, sp. nov.
MycoBank No.: MB837894.
Etymology: Referring to Yulong Snow Mountain, the geographic origin of the type strain.
Diagnosis: Morphologically distinguished from the phylogenetically related species of Leptodophora and Collembolispora in conidiogenesis and conidial shapes.
Type: China: Yunnan Province: Yulong Snow Mountain, N27°10′52″ E100°19′84″, 4531 m, from soil, 7 May 2017, M.-M. Wang (HBU20010 – holotype; YL814 = CGMCC3.20187 – ex-type cultures).
Description: Mycelium hyaline, septate, smooth-walled, branched, 1–3 μm wide. Conidiophores hyaline, smooth, often reduced to conidiogenous cells. Conidiogenous cells phialidic, located laterally or terminally, cylindrical or navicular, apex wedge, base truncate, hyaline, smooth-walled, straight or bent, 11.4–25.5 × 1.6–3.1 μm, collarettes evident, 2.1–4.5 μm long, opening 1.6–2.5 μm wide. Conidia hyaline, aseptate, smooth-walled, cylindrical, sporulation abundant, single, straight, 4.5–6.9 × 1.4–2.5 μm (mean = 5.5 ± 0.6 × 1.9 ± 0.3 μm, n = 30), L/W ratio = 2.9.
Culture characteristics — Colonies on MEA reaching 36 mm diam, after 14 d at 25 °C in the dark, on OA and PDA reaching 38 mm and 28 mm diam, respectively. Colonies on MEA pale pink to whitish, white at the margin, reverse same colours. Colonies on OA black-grey with light grey margin, reverse same colours. Colonies on PDA felty, grey to pale grey, reverse pale yellow.
Notes: Cadophora yulongensis failed to produce conidia when cultured on MEA, OA, and PDA media. Other efforts including pine needle medium culturing and H2O2 treatment (Xu et al. 2009) also failed to induce sporulation until we used a slide culture technique. In the multigene phylogenetic tree (Fig. 4), C. yulongensis is closely related to lineages formed by species of Leptodophora and Collembolispora. The genus Leptodophora is currently proposed to accommodate species firstly described as Cadophora. All Leptodophora species produce rarely seceding conidia and the conidial morphology differs markedly (Koukol & Maciá-Vicente, 2022). Species of Collembolispora often produce multicellular macroconidia with appendages and a synasexual morph of phialides (Marvanová et al. 2003). The newly described species is characterized by long cylindrical phialides and cylindrical conidia with comparatively high conidium length/width ratio (2.9).
Species of Cadophora have been reported from different locations worldwide, mainly as plant pathogens or root colonizers from northern temperate regions or decomposers from the cold Arctic and Antarctic environments (Blanchette et al. 2004, 2010, 2016, 2021; Duran et al. 2019; Gramaje et al. 2011; Maciá-Vicente et al. 2020; Travadon et al. 2015; Walsh et al. 2018). The Qinghai-Tibet Plateau, which is also called “the third pole”, is located in the southwest of China and is the highest and largest low-latitude region with permafrost in the world. The unique geographic location of high elevation and low latitude makes the Qinghai-Tibet Plateau a unique alpine ecosystem that is more sensitive to changes of climate and surface conditions (Cheng 1998). Warm, moist air from the Indian Ocean flows up the valleys and is then blocked by huge mountains, leading to abundant rainfall in the southeast range of the plateau. Large numbers of marine glaciers are formed in this area (Shi et al. 1964). During the investigation of cold-adapted fungi from marine glaciers in the Qinghai-Tibet Plateau in 2017, 1208 fungal strains were isolated and identified based on preliminary analyses of generated ITS sequences. Forty-one isolates belonging to Cadophora, one of the three most commonly encountered genera (Cadophora, Geomyces and Pseudogymnoascus; the latter two will be discussed in another paper) were studied in detail. Our results revealed seven Cadophora species, represented by 38 isolates, new to science and three isolates identified to two known species (C. malorum and C. novi-eboraci).
Because of the limited discriminating morphological characteristics existing among Cadophora and related genera, the genus has suffered taxonomic flux since the beginning of its establishment. DNA sequences have provided critical information for species delimitation. Some Cadophora species with multiform morphological characters deviate from the original generic concept, such as C. antarctica, C. fallopiae, C. fascicularis, and C. obovata, have been described mainly based on molecular data (Crous et al. 2017, 2020; Maciá-Vicente et al. 2020). Day et al. (2012) tried to find some consistencies between morphological characteristics and phylogenetic relationships in Cadophora and the related genera. They hypothesized that the ancestral state for these taxa was the production of sclerotium-like heads of multiple phialides and clades derived from phialide arrangements agreed with those generated from rDNA ITS sequence analyses. Although ITS is useful for most fungal species identification, it often fails to discriminate species or even results in misleading information in this group. For example, according to the ITS analyses, Cadophora malorum CBS 165.42 is nested within the Cadophora luteo-olivacea clade, but in the TEF tree, C. malorum CBS 165.42 is strongly supported as the sister group to C. luteo-olivacea (Travadon et al. 2015) and the RPB1 gene can also resolve species relationships between C. meredithiae and C. interclivum better than the ITS (Walsh et al. 2018); C. microspora only known from the sexual morph, was first identified based on ITS and morphological characteristics by Ekanayaka et al. (2019), but in recent studies, it was transferred to Rhexocercosporidium based on LSU and ITS analyses (Hyde et al. 2020). With more genes and species included, Maciá-Vicente et al. (2020) provided a more comprehensive overview about the ecology, morphology and phylogeny of Cadophora. Their results show that the genus is apparently paraphyletic and encompasses a broad spectrum of morphologies and life-styles. They tended to split the genus into three genera: one included those referred to as ‘Cadophora s. str. species’ that evolved from an ancestor with phialidic conidiogenesis; the second included species like C. interclivum, C. meredithiae, C. luteo-olivacea, C. malorum, and C. helianthi that produced conidia phialidically but are clustered in a separate clade; the third genus should take the name of Collembolispora including Cadophora species with holoblastic conidiogenesis. But this drastic restructuring still needs to be confirmed. Our multi-gene phylogenetic analyses confirmed paraphyly in Cadophora and all the species involved are clustered into two main clades (Fig. 4). Clade 1 comprised 21 Cadophora species (including five newly described in this study and the type species of the genus) and three species belonging to other genera (Hymenula cerealis, Mollisia cinerella, and Phialophora dancoi). This clade was similar to the ‘Cadophora s. str.’ clade defined by Maciá-Vicente et al. (2020), just with more species involved in our study. Although all species in Clade 1 have phialidic conidiogenesis, it is somewhat arbitrary to combine P. dancoi, M. cinerella, and H. cerealis into Cadophora at present, as we have just assembled the ITS data sets of these three species to maximize taxon coverage and more exact morphological examinations also need to be done for these fungi. Clade 2 includes most members of Ploettnerulaceae and the remaining Cadophora species. Cadophora constrictospora, C. gregata, C. helianthi, C. interclivum, C. luteo-olivacea, C. malorum, C. meredithiae, C. sabaouae, and C. vivarii which have phialidic conidiogenesis cluster with species including C. antarctica, C. fallopiae, C. inflata, C. obovata, and two species of Mastigosporium which produce conidia with putative enteroblastic or holoblastic conidogenesis. Specimens of C. lacrimiformis only known by the sexual morph is also in this lineage; Leptodophora gamsii, L. echinata, L. orchidicola, L. variabilis, and Collembolispora disimilis which are currently transferred from Cadophora form a subclade with C. yulongensis and two species of Collembolispora; Cadophora fascicularis clusters with species of Mycochaetophora in a distinct lineage. Thus, the currently circumscribed genus could be split into separate genera, but the introduction of more satisfying generic concepts depends on more phylogenetically related taxa in Ploettnerulaceae being involved.
Although Cadophora species are often encountered in cold environments, especially in the polar regions, most of them are psychrotolerant and have an optimum growth temperature (OGT) near or above 20 °C (Blanchette et al. 2021). The only psychrophilic species reported is C. antarctica which was isolated from a soil sample in King George Island (Antarctica) and had an OGT of 15 °C (Crous et al. 2017). Travadon et al. (2015) hypothesized that the geographic distribution patterns of Cadophora species in North America might reflect their adaptation to the contrasting environments: species recovered from cooler areas normally had an OGT at 20 °C and ones isolated from warmer regions tended to grow well at 25 °C. In our study, strains isolated from samples of Dagu Glacier (DG5, DG21, DG1048, DG1073, DG1087, DG1105 and DG1156), Mingyong Glacier (MY527, MY588, MY589, MY873) and Yulong Glacier (YL73) all had optimum growth rates at 20 °C, while others isolated from the same sampling sites had an OGT at 25 °C. Besides, strains being identified as the same species (C. qinghai-tibetana) have different OGTs (ranging from 20 °C to 25 °C). Environmental adaptations of fungal strains might be affected by many factors, such as temperature, humidity, radiation, and substrates. They have to evolve complex abilities to survive in adverse environments. Therefore, it is necessary to test more physiological, biochemical characteristics or perform genome analyses to illustrate adaptation mechanisms of this important fungal group.
Our study shows a very high diversity of Cadophora in the marine glaciers of Qinghai-Tibet Plateau and we described seven Cadophora species new to science. With more species involved, the genus has become apparently paraphyletic and requires phylogenetic reconstruction. Thus, more comprehensive sampling is necessary for the creation of new generic concepts which could accommodate species which deviate morphologically and phylogenetically in this important fungal group.
Availability of data and materials
All sequence data generated for this study (Table 2) can be accessed via GenBank: https://www.ncbi.nlm.nih.gov/genbank/. Alignments are available at TreeBase (http://www.treebase.org) and available online at https://doi.org/10.6084/m9.figshare.20230977.v1
China General Microbiological Culture Collection Center
Differential interference contrast
Mycological Herbarium of Hebei University
The internal transcribed spacer
The large ribosomal subunit (28S)
Markov Chain Monte Carlo sampling
Malt extract agar
National Center for Biotechnology Information
Potato dextrose agar
Rescaled consistency index
Translation elongation factor 1-α
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This study was supported by the National Science Foundation of China: [Grant Numbers 31600027 and 31961133020].
This study was supported by the National Science Foundation of China: [Grant Numbers 31600027 and 31961133020].
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Zhang, B., Li, X., Li, G. et al. Cadophora species from marine glaciers in the Qinghai-Tibet Plateau: an example of unsuspected hidden biodiversity. IMA Fungus 13, 15 (2022). https://doi.org/10.1186/s43008-022-00102-5
- Cold-adapted fungi
- Dark biodiversity
- Marine glacier
- Multi-gene phylogeny