Two new Cladosporium species from a quartzite cave in Brazil

Abstract

Caves are underground and natural environments mainly found in rocky terrain. Caves have a very specific microclimate, which benefits the occurrence of specific fungi. In recent studies, researchers have observed that caves harbour a great diversity of fungi. However, studies on fungal diversity in Brazilian caves are still incipient. In September 2019, airborne spore and soil samples were collected from the Monte Cristo cave, in the Southern Espinhaço Range, Diamantina, Minas Gerais state, Brazil. Two Cladosporium single‐spore isolates, among other genera, were obtained from these samples. This study aimed to characterise these two fungal isolates based on their DNA sequence data and morphology. Phylogenetic analyses of the rDNA-ITS, ACT and TEF1-α loci revealed that the isolates belonged to the Cladosporium cladosporioides species complex. Both isolates did not cluster with any known species and were formally described and named herein as C. diamantinense and C. speluncae. This study presents taxonomic novelties and contributes to the knowledge about the fungal diversity in Brazilian caves.

Introduction

Caves are underground and natural environments that are mainly found in rocky terrain. The interior of caves can be divided into three zones: the entrance, twilight and aphotic. The latter is characterised by the absence of light, low temperatures, a relative humidity close to 100% and scarcity of organic matter [1]. Caves provide a unique environment that promotes the growth of specific fungi. According to current research, caverns are home to a diverse range of fungi [2–5].

Although knowledge about the fungal diversity associated with caves is still incipient, approximately 2000 species of fungi have been reported in caves and similar environments [4–8]. Cladosporium Link is one of the most common fungi reported in caves in several countries, such as Brazil [7–12], China [4, 5], Poland [3] and Spain [2].

Cladosporium is a member of the family Cladosporiaceae within the order Cladosporiales, class Dothideomycetes and phylum Ascomycota [13]. This fungus has holoblastic conidiogenesis and forms amerospores or phragmospores in unbranched or branched acropetal chains [14]. The conidiogenous loci and conidial hila in Cladosporium are coronate, meaning they are protuberant with a central convex dome surrounded by a raised periclinal rim. These characteristics distinguish this genus from similar genera [14–18]. Cladosporium is characterised by the formation of various conidia, including ramoconidia, secondary ramoconidia, intercalary conidia and small terminal conidia [19]. This genus is divided into three species complexes, namely, Cladosporium cladosporioides [20], C. herbarum [19] and C. sphaerospermum [21]. A polyphasic approach is required to delimit the species in each complex.

Cladosporium has a global distribution, with species found on a variety of substrates, including soil, leaves, wood, food and textiles [14, 17, 22, 23]. Cladosporium species can be saprophytic, phytopathogenic, pathogenic in humans and animals and endophytic or hyperparasitic [14, 17, 24]. Therefore, they may be used as biological control agents against other fungi, such as those that cause rust, or insect pests [25–27]. Some species can inhabit harsh environments and can be halophilic, halotolerant or moderately tolerant to alkaloids [14, 28, 29]. Cladosporium spores are the most common spores found in indoor environments [2, 30, 31]. Several Cladosporium species have been reported in closed environments and clinical samples [18, 32]. Other species of the genus are also found in caves, e.g. C. anthropophilum, C. cavernicola, C. cladosporioides, C. halotolerans, C. pernambucoense, C. puris, C. sphaerospermum, C. subuliforme and C. tenuissimum [5, 8]. However, studies on fungal diversity in Brazilian caves are still incipient [7–12].

Researchers from around the world recognise the importance of knowledge about fungal diversity in tropical countries, which may harbour several species unknown to science [33, 34]. In this study, we characterised two fungal isolates obtained from airborne spores and soil samples collected from Monte Cristo cave, Minas Gerais state, Brazil. Through DNA sequence analyses and morphology, we recognised them as new Cladosporium species.

Material and methods

Study area

In September 2019, samplings were carried out in the Monte Cristo cave (18°17′49.79″S 43°33′30.5″W), which belongs to the Southern Espinhaço Mountain Range (SEMR), located in the municipality of Diamantina, Minas Gerais state, Brazil (Fig. 1), approximately 294 km far from the state capital, Belo Horizonte. In general, the climate of the study area (SEMR) is marked by mild, humid summers and cool, dry winters. The average annual temperature is 18–19 °C. Meanwhile, the average annual precipitation ranges from 1250 to 1550 mm. The rainy season, which endures from November to March, has an average rainfall of over 223 mm [35].

Fig. 1.

Monte Cristo cave. A Geographical location of the cave at the Southern Espinhaço Mountain Range, Diamantina, Minas Gerais state, Brazil. B, C Overview of the rock formation where the cave is located and the presence of native vegetation on the site. D Access to the cave entrance. E Overview of the main entrance to the cave

The Monte Cristo cave comprises an important lithostratigraphic set, in terms of volume and orographic expression of the Southern Espinhaço Range. This supergroup is mainly composed of quartzites and, subordinately, metasiltites, metaconglomerates, phyllites and metavulcanites [36]. This quartzite cave extends 250 m in length and is characterised as a predominantly horizontal cavity with linear development and photic, twilight and aphotic zones [37, 38]. The Monte Cristo cave has two entrances, one has approximately 30 m in width, and the other has about 40 cm in diameter [38]. The cavity is composed of two rooms characterised in some sections by the presence of a gently sloping ceiling and floor and other segments with a horizontalised floor (Fig. 1).

Sampling

A sampling of air and soil was performed along the Monte Cristo cave at different sampling sites. Each sampling site was approximately 50 m distant from each other starting from the cave entrance.

The Koch sedimentation method was used for the sampling of airborne spores [39, 40]. Therefore, three Petri dishes (90 × 15 mm) containing sterilised potato dextrose agar culture medium (PDA, Sigma Aldrich) supplemented with chloramphenicol (0.1 g/L) were randomly spread at each sampling site. Plates were exposed for 15 min to allow the fungal spores present in the cave air to settle on the culture medium surface due to gravity.

The topsoil was removed for soil sampling (0.5–1 cm) and around 10 g of soil were collected at 1–5-cm depth in randomly selected spots inside the cave [4]. From each selected location, three soil samples were collected, two near the walls and one in the middle region. All samples were stored in sterile Falcon tubes, labelled, placed in coolers, and transported to the Laboratório de Micologia e Etiologia de Doenças Fúngicas de Plantas of the Universidade Federal de Viçosa, Minas Gerais state, Brazil. The sampling was authorised by the Ministério do Meio Ambiente (MMA)/Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) (SISBIO number 70978–1).

Isolation of cavernicolous fungi

In the laboratory, the Petri dishes, which were opened inside the cave to collect airborne fungi, were incubated at 25 °C, in the dark, for 7 days. After incubation, single colonies were transferred to other Petri dishes containing PDA and incubated at room temperature in the dark, for a period of 7 to 10 days.

Fungi were isolated from soil samples using the serial dilution in plates method according to Zhang et al. [4]. Briefly, 1 g of the soil sample was suspended in 9 mL of sterile water in a sterile 20-mL Falcon tube. The tube was vortexed, and the suspension was diluted at different concentrations (10⁻¹,10⁻², 10⁻³ and 10⁻⁴). Two hundred microliters of suspension of each concentration were spread on plates containing PDA medium by the spread plate method. Two antibiotics, chloramphenicol (0.1 g/L) and rifamycin (150 μg/mL), were used to avoid bacterial growth. For each dilution, three replicates were performed. The plates were incubated aerobically at 25 °C in the dark for up to fifteen days.

These cultures were subcultured by the single hyphal-tip method prepared on 2% water-agar medium (WA) [41] and then maintained on PDA medium. All isolates were stored on anhydrous silica gel at 5 °C, in tubes with PDA culture medium and 2-mL microtubes containing 10% glycerol solution [42]. The isolates were deposited to Coleção Octávio Almeida Drummond (COAD), and metabolically inactive cultures of the holotypes were deposited in the VIC herbarium. Both repositories are housed at the Universidade Federal de Viçosa, Minas Gerais state, Brazil. Descriptions of the new species were deposited in MycoBank.

DNA extraction, PCR, and sequencing

Fungal genomic DNA was extracted from the single hyphal-tip cultures, previously grown in plates with PDA medium + cellophane, at 25 °C, in the dark, for 7 days. The mycelium was macerated in microtubes using L-Beader-6 (a Cell Disruptor of Loccus Biotecnologia). DNA extraction was conducted with the Wizard Genomic DNA Purification Kit (Promega’s Corporation, WI, USA), according to Pinho et al. [43].

The nuclear ribosomal internal transcribed spacer (rDNA-ITS) region, and the partial fragments of the actin (ACT) and translation elongation factor 1-α (TEF1- α) loci were amplified by polymerase chain reaction (PCR) using the following oligonucleotide pairs: ITS5/LR6 [44], ACT-512F/ACT-783R [45] and EF-728F/EF-986R [45], respectively. The PCR of the rDNA-ITS region was performed with 12.5 µL of DreamTaq PCR Master Mix 2 × (Thermo Fisher Scientific, Vilnius, Lithuania), 1 µL of each forward and reverse primer, 5 µL of bovine serum albumin (BSA), 1 µL of dimethyl sulfoxide (DMSO), 2.5 µL of nuclease-free water and 2 µL of genomic DNA (25 ng/µL). Nuclease-free water was used for the negative control, instead of genomic DNA. Amplification was performed with an initial denaturation of 95 °C for 5 min, followed by 35 cycles of denaturation of 95 °C for 1 min, annealing at 49 °C for 2 min, extension at 72 °C for 2 min and a final extension at 72 °C for 10 min. On the other hand, for ACT and TEF1-α, the PCRs were prepared with 18 µL of DNA Polymerase Platinum Taq, 0.4 µL of each primer and 1.2 µL of genomic DNA (20 ng/µL). The negative controls for these PCRs were performed as mentioned above. Amplifications of ACT and TEF1-α were performed with an initial denaturation at 94 °C for 30 s, annealing at 58 °C and 55 °C, respectively, for 30 s, extension at 72 °C for 2 min, and a final extension at 72 °C for 2 min. PCR products were analysed by electrophoresis on 2% agarose gels stained with GelRed™ (Biotium Inc., Hayward, CA, USA) in TAE buffer (1 ×) and visualised under UV light, in a Photo-documenter L-Pix, to verify the purity and size of the fragments. The PCR products were purified and sequenced by Macrogen Inc., South Korea.

The nucleotide sequences were visualised with the FinchTV v.1.4.0 software (Geospiza Inc.). All sequences were manually assessed and deposited in the GenBank database. The new sequences were compared with sequences of GenBank using the megaBLAST. Sequences of ex-type cultures or reference isolates were obtained from GenBank and included in the phylogenetic analyses for the identification of the isolates following recent papers [8, 14, 18, 20, 22, 24, 46].

Sequence alignment and phylogenetic analyses

A preliminary phylogenetic analysis using rDNA-ITS sequences was performed to determine to which species complex the isolates belong (data not shown). It consisted of an alignment containing all described Cladosporium species and Cercospora beticola CBS 116456 as the external member (outgroup taxon). Subsequently, alignments were performed for each locus, using only sequences of the Cladosporium cladosporioides species complex and C. longissimum CBS 300.96 and C. sphaerospermum CBS 193.54 as the outgroup taxa. The sequences were aligned in MAFFT v.7 [47] and then manual adjustments were performed in the MEGA v. 7 software [48].

The best nucleotide substitution model for each locus was determined using MrModelTest 2.3 [49], according to the likelihood scores. Bayesian Inference (BI) analyses were performed using the Markov chain Monte Carlo (MCMC) simulation methods with all sequences for each locus individually and then with the concatenated sequences (rDNA-ITS, ACT and TEF1-α). Phylogenetic analyses were performed on the CIPRES Science Gateway V. 3.3 [50], using the MrBayes 3.2.7a [51]. Four MCMC chains were conducted simultaneously, starting from random trees up to 10,000,000 generations. The trees were sampled every 1000 generations, resulting in 10,001 trees. The first 2500 trees were discarded during the burn-in phase of each analysis. The values of posterior probability [52] were determined from the consensus tree generated from the remaining trees. At the end of the analyses, the average standard deviations of split frequencies were below 0.01. Maximum likelihood analyses were performed using IQTREE v. 2.1.2 [53] with ultrafast bootstrapping made using 1,000 replicates [54]. The resulting trees were visualised in FigTree v. 1.4.3 [55] and exported to graphics programs.

Morphological studies

The isolates were grown on potato dextrose agar (PDA), oatmeal agar (OA) and synthetic-nutrient-limited agar (SNA) incubated at 25 °C in the dark for 14 days. Then, the colonies were measured using a digital calliper. Surface and reverse colours were rated using the charts of Rayner [56].

Slide cultures were assembled according to Rosado et al. [46] and incubated at 25 °C, in the dark, for 7–14 days. After sporulation, hand-made microscopy slides were mounted in lactoglycerol for viewing under an Olympus CX31 light microscope. The images were obtained through an Olympus BX53 microscope, equipped with an Olympus Q-Color5™ digital camera. Thirty measurements of the relevant morphological features were performed using the Olympus cellSens Dimension 1.9 software, to confirm the identification of the species.

Results

Among the filamentous fungal isolates obtained from airborne spore and soil samples from the Monte Cristo cave, two (COAD 3108 and COAD 3116) were identified as Cladosporium through the morphology of conidia. Therefore, in this study, we focus on characterising these two isolates based on DNA sequence data and morphology.

Amplicons of approximately 500, 200, and 350 bp were generated for rDNA-ITS, ACT and TEF1-α, respectively. Sequencing was successfully performed for all samples, and the sequences were made available in the GenBank. The samples were compared based on the sequences of the rDNA-ITS region, using Bayesian analysis, to determine the species complex to which each one belongs. This analysis indicated that both samples belong to the Cladosporium cladosporioides species complex (data not shown).

The multilocus alignment using all species of the C. cladosporioides species complex included 139 taxa. The alignment of the sequences of the three loci resulted in a total of 1193 base pairs (bp), including gaps. This combined alignment consisted of 520 bp, 263 bp and 408 bp for rDNA-ITS, ACT or TEF1-α sequences, respectively. The SYM + I + G model of evolution was selected for rDNA-ITS, and GTR + I + G for ACT and TEF1-α genes. Except for the rDNA-ITS tree that formed a polytomy, the topologies of the individual trees were similar to the tree of the combined data. Thus, only the latter is presented. To present phylogenetic results, the maximum likelihood tree was used, and bootstrap values (bs) and posterior probabilities (pp) are shown for branches (Fig. 2). The phylogenetic inference indicated that both isolates did not cluster with any known species, which represents two new species.

Fig. 2.

Concatenated phylogenetic tree of the genus Cladosporium obtained by the maximum likelihood method inferred from rDNA-ITS, TEF1-α and ACT sequences. Posterior probabilities and bootstraps values are indicated next to nodes. The isolates obtained in this study are highlighted in bold. The tree was rooted with C. longissimum CBS 300.96 and C. sphaerospermum CBS 193.54. The tree was separated into two sections (A and B) for easier visualization. T = ex-type cultures or reference isolates

The fungi of this study had typical Cladosporium morphology, such as holoblastic conidiogenesis, and formed various types of conidia in acropetal chains, including ramoconidia, secondary ramoconidia, intercalary conidia and small terminal conidia, besides conidiogenous loci and conidial hila coronate. The main morphological characteristics of the Cladosporium species that are close to those investigated in this study are presented in Table 1. Descriptions of the new species are given below.

Table 1.

Comparison of the most relevant morphological features of some Cladosporium species

Species	Macronematous conidiophores (µm)	Conidiogenous cells (µm)	Ramoconidia (µm)	Secondary ramoconidia (µm)	Conidia (µm)	Reference
C. angulosum	Up to 150 × 3–4	8–46 × 2–3.5	24.5–46 × 2–3.5	8–17 × 2.5–3	3.5–6 × 2–3	31
C. aulonemiae	37–228.5 × 2–4.5	14–48 × 2–4	19–28 × 3–3.5	8–18 × 2.5–4	4.6–8 × 2.8–5	54
C. bambusicola	24–179 × 2–6	9–47 × 1.6–4	34–40 × 4–6	6–26 × 2–5.5	4–11 × 2.7–5	54
C. chusqueae	23–141 × 2.6–4	8.6–22 × 2–4	Absent	5.5–12 × 2–3.5	3.8–7 × 1.8–2.5	54
C. diamantinense COAD 3108	17–63 × 1–4.6	3.5–18.5 × 1–4	2–11 × 0.5–3	2.5–12.5 × 1–5	1.5–8 × 1–3	This study
C. perangustum	8 − 150 × 1.5 − 4	7 − 40 × 0.8 − 1.5	25 − 45 × 2.5 − 4.5	6 − 34 × 2 − 3.5	2 − 19 × 1.5 − 3.5	19
C. rugulovarians	Up to 475 × 3.5–5	Up to 60 × 1–1.5	20–55 × 3–4.5	9–30 × 3–5.5	3–10 × 3–6	23
C. speluncae COAD 3116	16.4–394 × 1.6–4.6	5–29.6 × 1.8–4	6–27 × 1.5–4	7–15.5 × 3–4.5	3.6–9 × 2–4.6	This study
C. xanthochromaticum	Up to 210 × 2–4	12–32 × 3–4	18–36 × 2–3.5	10–28 × 3–4	4–7 × 2–3.5	31

Taxonomy

Cladosporium diamantinense A.W.C. Rosado, Y.L.G. Dutra & O.L. Pereira sp. nov. (Fig. 3).

Fig. 3.

Cladosporium diamantinense (COAD 3108). A–F Colonies incubated at 25 °C in the dark for 14 days, from above and below. A, B On potato dextrose agar. C, D On oatmeal agar. E, F On synthetic-nutrient-limited agar. G–M Conidiophores and conidial chains in vitro. Scale bars = 20 µm

MycoBank: MB844436.

Etymology: Name reflects the Brazilian municipality (Diamantina), where the species was found.

Type: Brazil, Minas Gerais state, Diamantina, habitat: Monte Cristo cave, isolated from air, September 2019, Y.L.G. Dutra (holotype VIC 47485, preserved as metabolically inactive culture; ex-type living culture COAD 3108, preserved in silica gel and glycerol).

GenBank accession numbers: ON062328 (rDNA-ITS), ON141933 (ACT), ON982817 (TEF1-α).

Microscopic description: Mycelium superficial and immersed, composed of septate, branched, subhyaline to light brown, smooth and thick-walled, 1–4.4-μm wide hyphae, hyphae with anastomosis. Conidiophores, solitary, erect or somewhat flexuous, cylindrical, non-nodular, non-geniculate, septate, light brown to dark brown, septum conspicuous, thickened walls, 17–63-µm long and 1–4.6-µm wide. Conidiogenous cells, terminal, cylindrical or subcylindrical, thickened, darkened conidiogenous loci, 3.5–18.5 × 1–4 µm. Ramoconidia, aseptate, cylindrical, light brown, smooth, with protruding conidial scars, truncated base, 2–11 × 0.5–3 μm. Secondary ramoconidia, ellipsoidal to subcylindrical, aseptate, with a narrowed base, 2.5–12.5 × 1–5 μm. Conidia, numerous, formed in short and branched chains, aseptate, reticulated, subhyaline to pale olivaceous brown; intercalary conidia, ovoid or ellipsoid, 4.5–8 × 2–3 μm; Small terminal conidia, globose or obovoid, 1.5–5 × 1–3 μm.

Culture characteristics: Colonies reached an average of 23.7 mm, 36 mm and 17.9 mm on PDA, OA and SNA, respectively, after 14 days, at 25 °C, in the dark. Colonies on PDA iron grey to greenish black, rosy vinaceous at the centre, translucent towards margins, reverse iron grey to greenish black, sporulating. Colonies on OA olivaceous buff to grey olivaceous, rosy buff towards margins, reverse greenish black, sporulating. Colonies on SNA hazel near the edge becoming vinaceous buff towards the centre, vinaceous buff towards margins, reverse pale olivaceous grey, olivaceous grey to greenish black, sporulating.

Notes: Based on the phylogenetic analyses, C. diamantinense was found to be a basal species within the Cladosporium cladosporioides species complex. Cladosporium diamantinense formed smaller colonies compared to related phylogenetic species. The conidiophores, conidiogenous cells and ramoconidia of this new species are shorter than those of C. rugulovarians, C. aulonemiae, C. perangustum, C. chusqueae (ramoconidia absent), C. angulosum, and C. xanthochromaticum. All these species are distinguishable by phylogenetic analyses (Fig. 2). Cladosporium diamantinense was isolated from the air of a Brazilian cave, just as C. cavernicola and C. pernambucoense, which were recently introduced by Pereira et al. [8].

Cladosporium speluncae T.O. Condé, A.W.C. Rosado & O.L. Pereira sp. nov. (Fig. 4).

Fig. 4.

Cladosporium speluncae (COAD 3116). A-F Colonies incubated at 25 °C in the dark for 14 days, from above and below. A, B On potato dextrose agar. C, D On oatmeal agar. E, F On synthetic-nutrient-limited agar. G–M Conidiophores and conidial chains in vitro. Scale bars: G–I 20 µm and J–M 10 µm

MycoBank: MB844437.

Etymology: The epithet name refers to the cave, which is the habitat where this fungus was found.

Type: Brazil, Minas Gerais state, Diamantina, habitat: Monte Cristo cave, isolated from soil, September 2019, Y.L.G. Dutra (holotype VIC 47563, preserved as metabolically inactive culture; ex-type living culture COAD 3116, preserved in silica gel and glycerol).

GenBank accession numbers: ON062329 (rDNA-ITS), ON141934 (ACT), ON982818 (TEF1-α).

Microscopic description: Mycelium, superficial and immersed, composed of septate, branched, subhyaline to light green, smooth and thick-walled, 3 − 5-μm wide hyphae. Conidiophores, erect, cylindrical, non-nodular, geniculate, septate, usually branched, subhyaline, slightly roughened to verruculose towards the base, with thickened and refractive wall, 16.4–394-μm long and 1.6–4.6-μm wide. Conidiogenous cells, cylindrical or subcylindrical, thickened and somewhat darkened conidiogenous loci, 5–29.6 × 1.8–4 μm. Ramoconidia, aseptate, cylindrical, light brown, smooth, with protruding conidial scars, truncated base, 6–27 × 1.5–4 μm. Secondary ramoconidia, septate, ellipsoidal to subcylindrical, usually attenuated in the centre, 7–15.5 × 3–4.5 μm. Conidia, numerous, formed in short and branched chains, aseptate, subhyaline to pale olivaceous brown; intercalary conidia, limoniform to ellipsoidal, 4–9 × 2.4–4.6 μm; small terminal conidia, oval to ellipsoidal, 3.6–6 × 2–3 μm.

Culture characteristics: Colonies reached an average of 31 mm, 36 mm and 24 mm on PDA, OA and SNA, respectively, after 14 days, at 25 °C, in the dark. Colonies on PDA olivaceous grey to grey olivaceous, buff towards margins, reverse vinaceous buff, sporulating. Colonies on OA olivaceous to greenish olivaceous, rosy buff towards margins, reverse greenish black, sporulating. Colonies on SNA greenish olivaceous to grey olivaceous at the margins becoming greenish black towards to the centre, rosy buff towards margins, reverse grey olivaceous to greenish black, sporulating.

Notes: Cladosporium speluncae is phylogenetically close but distinct from C. bambusicola (Fig. 2), which differs in forming larger colonies, smaller conidiophores (up to 179 µm long vs. up to 394 μm in C. speluncae), longer conidiogenous cells (up to 47 µm long vs. up to 29.6 µm in C. speluncae), and larger ramoconidia (34–40 × 4–6 µm vs. 6–27 × 1.5–4 µm in C. speluncae). This new species was discovered in cave soil, and C. tenuissimum has also been found in the soil of a bat cave in Bali [20].

Discussion

In this study, we characterised two fungal isolates obtained from airborne spores and soil samples collected in the Monte Cristo cave, Brazil, based on DNA sequence data and morphology. Both isolates did not cluster with any known Cladosporium species and, therefore, were formally described and named herein as C. diamantinense and C. speluncae.

The rDNA-ITS region was selected as the fungal DNA barcode [57], and it was efficient to determine that the isolates of this study belong to the Cladosporium cladosporioides species complex. However, using this region alone, it is not possible to identify all species within this species complex [18, 20, 24, 27]. Therefore, it is necessary to include other loci in phylogenetic analyses [27, 32, 46, 58]. Corroborating previous studies [24, 46], actin was the locus with the best resolution to identify species within the Cladosporium cladosporioides species complex.

This fungal complex has demonstrated tremendous diversity, as evidenced by the discovery of at least eleven new species in 2023. Among these discoveries, seven distinct Cladosporium species were identified in association with fruit trees in China [59]. These newfound species are C. congjiangense, C. kaiyangense, C. nayongense, C. pruni-salicinae, C. punicae, C. ribis and C. wenganense. Furthermore, C. corticola was isolated from the bark of Melaleuca quinquenervia (Myrtaceae) in Australia [60], while three other species were introduced from samples collected in marine environments in the Republic of Korea, namely C. lagenariiforme, C. maltirimosum and C. marinum [61].

In Brazil, some studies have focused on the taxonomy of Cladosporium bringing to the identification of fourteen species, namely, C. maracuja [62], C. langeronii [28], C. rugulovarians [24], C. passiflorae, C. passifloricola [46], C. puris [63], C. cavernicola, C. pernambucoense [8], C. aulonemiae, C. bambusicola, C. benschii, C. brigadeirensis, C. chusqueae and C. pseudotenuissimum [64].

Cladosporium has been widely reported in Brazilian caves. In Meu Rei cave, located in the Caatinga dry forest biome, three Cladosporium isolates were identified using the ITS region from cave air and bats [7]. In the Furna do Morcego cave, two new Cladosporium species, C. cavernicola and C. pernambucoense, were described together with the reports of C. austrohemisphaericum, C. parahalotolerans, C. puris, C. sphaerospermum, C. subuliforme and C. tenuissimum [8]. In the same cave, Carvalho et al. [9] found C. halotolerans and C. subululiforme in ectoparasitic flies collected from bats that inhabited that cave. In the Abrigo do Letreiro cave, another cave from the Caatinga biome, were found the species C. oxysporum, C. subuliforme, C. tenuissimum and C. xanthocromaticum from airborne and soil samples [10]. In the Lapa Nova cave, located in Minas Gerais state, two Cladosporium isolates were identified as C. cladosporioides and C. herbarum from cave air and bat guano [11]. Also in Minas Gerais state, Taylor et al. [12] isolated C. cladosporioides from the soil of an iron cave. Our results, together with those of previous studies, suggest a high Cladosporium diversity associated with caves in Brazil.

This study focused on identifying two isolates from different substrates from the interior of the Monte Cristo cave. It led to the discovery of two new species within the Cladosporium cladosporioides species complex, namely, C. diamantinense and C. speluncae, which occur in the air and soil of Monte Cristo cave, respectively. This study contributes with taxonomic novelties to the diversity of the C. cladosporioides species complex and contributes new mycological insights from a Brazilian cave.

Author contribution

A. W. C. R., A. F. L., T. O. C. and O. L. P. collected the samples. S. d. C. N. and L. M. S. F. carried out the caving and sampling. Y. L. G. D. processed the samples, carried out the fungal isolations, extracted DNA and did amplifications with the help of A. F. L., A. W. C. R. and T. O. C. T. O. C., A. W. C. R., Y. L. G. D. and O. L. P carried out the phylogenetic analyses and data interpretation. M. C. M. K. and O. L. P. conceived and designed the research project. A. W. C. R. and Y. L. G. D. wrote the manuscript with input from all authors.

Funding

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES, Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG-VALE RDP-00017–18).

Data availability

DNA sequences generated in this study are available in GenBank (https://www.ncbi.nlm.nih.gov/genbank).

Declarations

Ethics approval

The collection was authorised by the Ministério do Meio Ambiente (MMA)/Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) (SISBIO number 70978–1).

Competing interests

The authors declare no competing interests.