Oligotrophic fungi from a carbonate cave, with three new species of Cephalotrichum

ABSTRACT Oligotrophs are microorganisms that can grow in environments where concentrations of nutrients are low or even absent. Caves are typical oligotrophic environments distinctly characterised by constant low temperature, high humidity, scarcity of organic matter and darkness, which encompass a high diversity of fungi. In our investigation of microorganisms from carbonate caves in China, 169 strains belonging to at least 84 taxa were isolated using oligotrophic carbon free silica gel medium (SGM). Cephalotrichum appeared to be one of the dominant genera. Further morphological comparisons and molecular phylogenetic analyses using DNA sequences of four loci (LSU, ITS, TUB2 and EF-1α) revealed that the 30 strains of Cephalotrichum represent three new species, which are described and named C. guizhouense, C. laeve and C. oligotriphicum. This study also significantly improved our understanding on fungi being able to grow on carbon free medium, with the known species increased from 18 to 99.


Introduction
Oligotrophs are microorganisms that can grow in environments where concentrations of nutrients are low or even absent (Wainwright 1993). Soil of extreme environments, ocean and polar environments have been universally regarded as low nutrient habitats, from which a variety of oligotrophic fungi have been isolated (Gundersen et al. 1976;Hiroyuki and Tsutomu 1983;Bergero et al. 1999;Godinho et al. 2015). Oligotrophic habitats are generally regarded with a nutrient flux from 1-15 mg of carbon per litre (Poindexter 1981), and medium containing a carbon concentration of 10 mg per litre was widely suggested to cultivate oligotrophic fungi (Martin and MacLeod 1984;Parkinson et al. 1989;Wainwright et al. 1997). Since agar contains available carbon, silica gel is more preferable for the isolation of oligocarbotrophic fungi for laboratory studies (Payton et al. 1976;Wainwright and Grayston 1988). Up to now, a wide range of fungi have been isolated from oligotrophic habitats, while only a few of them were confirmed possessing the ability for growth on carbon free Silica Gel Medium (SGM) (Mirocha and DeVay 1971;Wainwright and Grayston 1988;Parkinson et al. 1989Parkinson et al. , 1990Wainwright 1993;Wainwright and Al-Talhi 1999;Connell and Staudigel 2013;Liu et al. 2013;Godinho et al. 2015) ( Table 1).
Caves have been generally regarded as typical oligotrophic environments (Bastian et al. 2009), distinctly characterised by constantly low temperature, high humidity, scarcity of organic matter and darkness (Gabriel and Northup 2013). However, studies on oligotrophic fungi from caves are rare. The objective of this study was to verify oligotrophic fungi in a cave with ability of growing on carbon free SGM and describe several new oligotrophic Cephalotrichum species by means of morphological examinations and multi-locus phylogenetic analysis.

Materials and methods
Fungal isolation and growth on carbon free medium Fifteen air samples, 25 limestone samples, 6 water samples, and 14 soil samples were collected from a carbonate cave (28°13ʹ819ʹʹ N, 107°18ʹ041ʹʹ E; about 750 m deep; 19°C) located at the Shuanghe National Geographic Park, Guizhou Province, China. From the entrance of the cave, each sampling site was 1 Nectriaceae Hypocreales (Parkinson et al. 1990); This study F. solani Limestone 1 Nectriaceae Hypocreales (Parkinson et al. 1990); This study F. thapsinum Seeping, stream and pool water in karst cave  Continued ) approximately 200 m distant from the next. Collections of air samples followed the Koch sedimentation method described by Borda et al. (2004) and Kuzmina et al. (2012). Three Petri dishes that contained 2% potato-dextrose agar (PDA, Difco) were exposed to the atmosphere in the cave for 15 min at each sampling site, then sealed with parafilm and placed in zip-locked plastic bags. Limestones of the cave wall were collected following the method of Ruibal et al. (2005). Five pieces of limestone in different orientations were collected at each site. Seeping, stream and pool water was collected (10 ml) and kept in 15 ml sterile centrifuge tubes. Ten grams of soil samples were collected at shallow depth (1-5 cm) after removing surface layer (ca. 1 cm). Water and soil samples were placed onto PDA (potato dextrose agar) medium following the dilution plate method (a series of concentrations, i.e. 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 ) (Zhang et al. 2015). All isolates were cultured on 1/2000 strength PDA (Liu et al. 2013) and then inoculated on carbon free silica gel medium (SGM) to screen for oligotrophic strains at room temperature (approximately 20-25°C). SGM was prepared from the following three solutions: (a) mineral salts solution consisting of KH 2 PO 4 1.0 g, KCl 0.5 g, MgSO 4 . 7H 2 O 0.5 g and FeSO 4 . 7H 2 O 0.01 g in 1 L ultrapure water (UPW); (b) orthophosphoric acid, 20 mL in 100 mL of UPW; (c) silicic acid 10 g and KOH 7 g added to 100 mL of UPW. Gels were prepared by mixing 10 mL of solution (a) with 10 mL of solution (c) and 2 mL of solution (b) into an autoclaved plastic  (Wainwright 1993) Petri dish (9 cm) (Wainwright and Al-Talhi 1999). All glassware used in the preparation of silica gel medium were washed with chromic acid and then rinsed with ultrapure water (Parkinson et al. 1989). Type specimens of the novel species were deposited in Mycological Herbarium of Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), with the ex-type living cultures deposited in China General Microbiological Culture Collection Center (CGMCC) and LC culture collection (personal collection of Lei Cai housed in the Institute of Microbiology, Chinese Academy of Sciences).

Morphological observation of cephalotrichum isolates
Isolates of Cephalotrichum were incubated on SGM, oatmeal agar (OA, BD, France), malt extract agar (MEA, BD, France) and potato dextrose agar (PDA, BD, France) at different temperatures (0-40°C at intervals of 5°C, as well as 37°C) and examined at 7, 14 and 28 d to determine colony growth rates (Sandoval-Denis et al. 2016b). Measurements and photographs of colony and micromorphological descriptions were made according to methods described by Sandoval-Denis et al. (2016b), with colours assessed according to the colour chart of Rayner (1970). Observations were performed with a Leica M125 dissecting microscope and a Zeiss Axio Imager A2 compound microscope under differential interference contrast (DIC) illumination.

Molecular identification and phylogenetic analysis
Sequences from forward and reverse primers were assembled to obtain a consensus sequence with MEGA v. 6.0 (Tamura et al. 2013). All strains were megablast searched in NCBI and assigned to potential genera and species. For Cephalotrichum, the obtained sequences and related sequences downloaded from GenBank (Table 2) were aligned with MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/ index.html; Katoh and Standley 2013). Ambiguous regions were excluded from the analyses and gaps were treated as missing data. A 70% neighbour-   (Posada 2008) was used to determine the best nucleotide substitution model settings for each locus. The Bayesian analyses of the combined four-locus dataset and individual locus data were performed with MrBayes v. 3.2.1 (Ronquist et al. 2012). The Markov Chain Monte Carlo sampling (MCMC) analysis of four chains started in parallel from a random tree topology. The number of generations was set at 10 million and the run was stopped automatically when the average standard deviation of split frequencies fell below 0.01. Trees were saved each 1 000 generations. Burn-in was set at 25% after which the likelihood values were stationary and the remaining trees were used to calculate posterior probabilities. Maximum-likelihood analyses including 1 000 bootstrap replicates were conducted using RAxML v. 7.2.6 (Stamatakis and Alachiotis 2010). A general time reversible model (GTR) was applied with a gamma-distributed rate variation.

Results
A total of 510 strains were isolated from air, limestone, water and soil using PDA medium. Among them, only 169 strains were determined as oligocarbotrophic fungi through their cultivation on SGM. A preliminary identification based on ITS megablast searches in GenBank assigned these isolates to 84 taxa in 51 genera, 28 families and 20 orders. Species able to grow on carbon free medium, including those previously known and those determined in this study, are summarised in Table 1. The result demonstrated that oligotrophic fungi are of multiple evolutionarily origins. The most common genera obtained in this study include Cephalotrichum (3 species, 30 strains), Plectosphaerella (2 species, 16 strains), Clonostachys (1 species, 14 strains), Cladosporium (8 species, 13 strains) and Fusarium (6 species, 10 strains) ( Table 1). The most common species include Plectosphaerella cucumerina, Clonostachys rosea, Cephalotrichum oligotriphicum and C. guizhouense (Table 1). For the substrates of isolation, 85 oligotrophic strains from limestones belong to 41 species; 53 strains from air belong to 41 species; 25 strains from soil belong to 17 species; and 6 strains from water belong to 6 species (Table 1).
The multi-locus dataset of Cephalotrichum used for phylogenetic analysis included 55 in-group strains, including 30 oligotrophic strains obtained from the present study, and Wardomyces inflatus CBS 216.61 as an out-group. The dataset comprises 2708 characters including gaps (805 characters for LSU, 498 for ITS, 498 for TUB2 and 895 for EF-1α). TIM3 + I + G was selected as the bestfit model for the ITS dataset, while TrN+I for LSU, TrN+G for TUB2 and TIM2 + I + G for EF-1α were also selected. The phylogenetic trees obtained from Bayesian inference and RAxML were similar in topology. The ML consensus tree with Bayesian posterior probabilities (BPP) and RAxML bootstrap support (MLBS) values is shown in Figure 1. Thirty strains of Cephalotrichum from caves clustered in three distinct clades with high bootstrap supports.

Taxonomy
Cephalotrichum guizhouense J.R. Jiang, L. Cai & F. Liu, sp. nov. Figure 2 Fungal names: FN 570479. Etymology: named after its distribution: China, Guizhou province.  Figure 1. Phylogenetic tree inferred from a Maximum likelihood analysis based on a concatenated alignment of LSU, ITS, TUB2 and EF-1α sequences. The BPP and MLBS are given at the nodes (BPP/MLBS). Ex-type strains are marked by asterisks (*). The tree is rooted with Wardomyces inflatus (CBS 216.61).

Culture characteristics
OA 35-40 mm diam in 14 d at 25°C, margin regular, flat, velvety with scarce white aerial mycelia, olive-grey, synnemata abundant, more or less powdery; reverse buff, pale brown near the centre. Colonies on MEA 30-35 mm diam in 14 d at 25°C , flat, velvety with scarce aerial mycelia, front and reverse white. Colonies on carbon free SGM growing more slowly compared to that on PDA, OA and MEA, aerial mycelia scarce but forming hyphae networks, with brown and sparse synnemata in one month.

Notes
Isolates of C. guizhouense formed a well-supported clade distinct from its most closely related species C. dendrocephalum (Figure 1). Morphologically C. guizhouense is distinct from the latter in the absence of undulating setae and the number of annellides (2-3 vs. 4(-6)) (Udagawa et al. 1985).

Culture characteristics
Colonies on PDA 40-45 mm diam in 14 d at 25°C, flat, felty to floccose, pale olivaceous near the centre, with white regular margin; reverse pale brown near the centre, white near the margin. Colonies on OA 45-50 mm diam in 14 d at 25°C, margin regular, flat, velvety with scarce white aerial mycelia, leaden, synnemata abundant; reverse pale yellow, buff near the margin. Colonies on MEA 25-30 mm diam in 14 d at 25°C, flat, velvety with scarce aerial mycelia, front and reverse white. Colonies on carbon free SGM growing more slowly compared to that on PDA, OA and MEA, aerial mycelia scarce but forming hyphae networks, with pale brown and sparse synnemata in one month. Hyphae septate, hyaline to pale brown, smooth-and thin-walled, 2-3.5 μm wide. Conidiophores unbranched or branched, in groups of 3-5 annellides on basal cells, pale brown, smooth-and thin-walled, usually forming synnemata. Synnemata 600-1000 μm high, stipes brown, 10-28 μm wide, conidial heads olive-grey, ellipsoidal; setae absent. Annellides ampulliform, 5.5-8 × 3-4 μm, subhyaline, pale brown, smooth-and thin-walled. Conidia ellipsoidal to ovoid, 5.5-7 × 3-conidia (Jiang and Zhang 2008). The colony of C. laeve is felty to floccose with white regular margin on PDA, while that of C. oligotriphicum is velvety to felty with white crenate to fimbriate margin. The maximum growth temperature of C. laeve is 37°C, whereas that for C. oligotriphicum and C. verrucisporum are 30°C (Sandoval-Denis et al. 2016b).

Discussion
Caves are typical oligotrophic environments, with total organic carbon (TOC) < 0.5 mg/kg, two orders lower than an average terrestrial environment (Barton and Jurado 2007). Among the oligotrophic fungi tested with the carbon free medium SGM in this study, air and limestone harbour the highest fungal diversity (41 spp. respectively), followed by soil (17 spp.) and water (6 spp.). It is premature to speculate why these different substrates harboured different number of oligotrophic species, as the isolation protocols are probably more determinate for the obtained fungal communities.
Although numerous fungi have been reported from various oligotrophic habitats, most are likely facultative rather than obligate oligotrophic fungi (Parkinson et al. 1989(Parkinson et al. , 1990Wainwright 1993;Wainwright and Al-Talhi 1999;Connell and Staudigel 2013;Liu et al. 2013;Godinho et al. 2015;Jiang et al. 2017). Hitherto only 18 species have been proven for being able to grow on SGM (Table 1). The data presented here increased the known oligotrophic species to 99 (Table 1). Within the 84 species from the preliminary identification, up to 31 (exclude three new species in this study) are considered potential new species, and they will be examined in future studies.
Limestone is predominantly composed of calcium carbonate (Li et al. 2009), and the carbonate minerals play important roles in global carbon cycling. In the present study, 83% of 30 Cephalotrichum strains were obtained from limestone in caves. These fungi may utilise energy gained from inorganic oxidation (Wainwright and Grayston 1988), as well as play important roles in the limestone weathering and dissolution (Sterflinger 2000;Northup and Lavoie 2001;Burford et al. 2003;Gadd 2007). In addition to the three species of Cephalotrichum described in this study, C. stemonitis from Slovakia and Spain, and C. verrucisporum from Japan were also previously reported from caves environments (wall and ceiling, rodent feces, and rhizomorphs) (Nováková 2009;Kuzmina et al. 2012;Kiyuna et al. 2017).
Three novel species of Cephalotrichum, C. guizhouense, C. laeve, and C. oligotriphicum, detailed in this study produce little typical aerial mycelia under oligotrophic conditions, but their hyphae stick on the surface of the silica gel, forming fine hyphal networks (i.e. gossamers) (Figure 2(h); Figure 3(h); Figure 4(h)), which provided a large surface area aiding nutrient scavenging from the gel and atmosphere (Parkinson et al. 1989;Wainwright 1993). In contrast, they produced abundant synnemata and dry and light airborne conidia on the carbon-free silica gel, which might be a survival strategy to promote the spore dispersal in a nutrient-less environment (Wainwright 1993;Sandoval-Denis et al. 2016b