Abstract
The genus Tulasnella often forms mycorrhizas with orchids and has worldwide distribution. Species of this genus are associated with a wide range of orchids, including endangered hosts. Initially, species identification relied mostly on morphological features and few cultures were preserved for later phylogenetic comparisons. In this study, a total of 50 Tulasnella isolates were collected from their natural sites in Minas Gerais, Brazil, cultured, and subjected to a phylogenetic analysis based on alignments of sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA. Our results, based on phylogeny, integrated with nucleotide divergence and morphology, revealed the diversity of isolated Tulasnella species, which included four new species, namely, Tulasnella brigadeiroensis, Tulasnella hadrolaeliae, Tulasnella orchidis and Tulasnella zygopetali. The conservation of these species is important due to their association with endangered orchid hosts and endemic features in the Brazilian Atlantic Forest.
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Introduction
Orchidaceae (or orchids) is the largest family of flowering plants, with approximately 27,000 species described1. The Neotropics is the region of greatest orchid diversity2 and approximately 205 genera and 2,650 species occur in Brazil, of which about 1,800 are endemic3. Many orchid species are endangered, mainly due to anthropogenic pressure and dependency between orchids and other organisms, i.e. pollinators or mycorrhizal fungi4,5.
Several endangered orchid species are listed in the Livro Vermelho da Flora do Brasil6. Among them, Hadrolaelia jongheana is an epiphytic orchid found in the Zona da Mata and Quadrilátero Ferrífero, two areas severely affected by anthropogenic activity. Zygopetalum maxillare is an epiphytic species which, although not officially endangered, grows almost exclusively in tree ferns7, which limits its distribution. Cattleya cinnabarina and Cattleya caulescens are rupicolous (i.e. grow on bare rocks) and endemic to the Southeastern Brazil8. These species belong to Brazilian Atlantic Forest, a highly diverse but endangered hotspot of biodiversity9. Like all orchids, they need mycorrhizal fungi for germination due to the limited reserves in seeds10. The symbiotic fungus supplies the embryo with carbon and other nutrients, which enable the germination and establishment of the orchid11. Orchids associate mainly with Basidiomycota often called rhizoctonia, a polyphyletic that includes taxa belonging to the families Sebacinaceae, Serendipitaceae, Ceratobasidiaceae and Tulasnellaceae12,13.
The specificity of orchid–mycorrhizal fungi varies among species12,14 and the distribution of mycorrhizal fungi can affect the patterns of distribution of orchids15. Species with low specificity for their fungal partner may be more successful in conservation strategies, such as assisted migration8. Despite this, specialist orchids might be widely distributed if their fungal partners are broadly distributed14,16. Indeed, the ecology of Tulasnella species orchid roots apart remains poorly known and even though they are often considered saprotrophic11 they may also colonize the roots of non-orchid plants17. The availability of compatible symbionts may directly impact the conservation of species4.
The genus Tulasnella is often observed as orchid mycorrhizal fungi in temperate and tropical regions12,18,19, and several isolates have been reported to increase seed germination and seedling growth20,21,22,23,24,25. Identification of mycorrhizal fungi in South American orchids, mostly conducted in Brazil, has often revealed Tulasnella symbionts: Tulasnella species were isolated from Epidendrum secundum26,27, Epidendrum dendrobioides and Sophronits milleri28, Oeceoclades maculata, Epidendrum rigidum and Polystachya concreta29, E. rigidum and P. concreta30. Yet little is known about Tulasnella in the hotspot of biodiversity of the Brazilian Atlantic Forest.
Tulasnella species have complex morphological characteristics, but rarely form fruitbodies in situ or sexual structures in vitro29,30,31,32,33. As morphological characteristics are not sufficient to describe Tulasnella species34, molecular approaches have been used too32,33,35,36,37,38. Species identification is mostly based on phylogenetic concordance of multiple unrelated genes/regions, but for this complex genus, the internal transcribed spacer (ITS) of the nuclear ribosomal DNA was shown to be highly suitable for species delimitation in Tulasnella31,38.
In a survey of cultivable mycorrhizal fungi associated with the roots of the rare-to-endangered Brazilian orchids H. jongheana, C. cinnabarina, C. caulescens and Z. maxillare, we obtained 50 isolates of Tulasnella. Herein, based on morphological and molecular analyses, we have evaluated the diversity of Tulasnella associated with these four orchids and describe potentially new Tulasnella species.
Results
Tulasnella isolates from Brazilian Atlantic Forest
Fifty isolates of the genus Tulasnella were obtained in this study (Table 1), namely, twenty isolates from C. cinnabarina roots, fourteen from C. caulescens roots, nine from H. jongheana (eight from Parque Estadual da Serra do Brigadeiro (PESB) and one from Parque Estadual da Serra Negra (PESN)) and seven isolates from Z. maxillare. As they were isolated from pelotons dissected from roots, they all are likely orchid mycorrhizal fungi. All isolates from C. cinnabarina and C. caulescens were identified as Tulasnella calospora, whereas isolates obtained from H. jongheana and Z. maxillare are described below as four new Tulasnella species.
Phylogeny
The ITS alignment consisted of 93 strains (including the outgroup sequence), of which 43 are from NCBI or UNITE and 50 from this study (Tables 1 and 2) and had a total length of 583 characters (including alignment gaps). Among these, 371 characters were parsimony-informative, 419 were variable and 147 were conserved.
Our phylogenetic analyses confirmed that mycorrhizal fungi isolated from the studied orchid species were Tulasnella (Fig. 1). Among these, four species are new in this genus and are described below, namely, Tulasnella hadrolaeliae, Tulasnella brigadeiroensis, Tulasnella orchidis and Tulasnella zygopetali. The newly proposed species are based on phylogenetic analyses, pairwise sequence divergence and morphological features (see below). The clades containing the Brazilian Tulasnella isolates are highlighted in the phylogenetic tree (Fig. 1).
Bayesian phylogenetic tree for Tulasnella based on ITS alignment. Maximum likelihood bootstrap support (ML > 60) and Bayesian posterior probabilities (PP) values are indicated next to the nodes (ML/PP). Species from Brazil are in the colored block and the new species described in this paper are indicated in bold face. Botryobasidium botryosum (AFTOL604) was used as the outgroup.
Phylogenetically, all isolates of Tulasnella from C. caulescens and C. cinnabarina are grouped in a clade including T. calospora isolates, close to another group composed of T. tubericola and T. bifrons (Fig. 1). The new species Tulasnella hadrolaeliae formed a well-supported clade (Maximum likelihood (ML)/Posterior probabilities (PP) = 100/1), which is a sister group of T. albida and T. pruinosa. Tulasnella brigadeiroensis isolates were grouped in a monophyletic clade. Tulasnella orchidis, isolated from Z. maxillare, clustered in a sister clade to T. brigadeiroensis and Tulasnella sp. COAD 2885. Finally, isolates of Tulasnella zygopetali obtained from Z. maxillare formed a strongly supported clade (ML/PP = 100/1), distinct from other Tulasnella species. Although the phylogenetic analyzes indicate that Tulasnella sp. COAD 2885 may represent a new species, it will not be formally described here since only one isolate was obtained during our study.
Divergence within and between clades
The Kimura-2-parameter distances between Tulasnella species ranged from 1.9 to 65.2% (Table 3). The divergence within Tulasnella species described here was lower than 0.6%. The nucleotide divergence between Tulasnella sp. COAD 2885 and T. brigadeiroensis was 7.5%, far above the 3% threshold suggested by Linde et al.31 in Tulasnella, and supposedly belong to two different species. For some species it was not possible to calculate the divergence within the clade, because only one isolate was used in analysis.
Taxonomy
Tulasnella brigadeiroensis E.F.S. Freitas, Meir. Silva & M.C.M. Kasuya, sp. nov. (Fig. 2)
Tulasnella brigadeiroensis (COAD2884). (a) Eight-day-old PDA culture. (b) Hyphae with branching at right angles. (c) Hyphae stained with SYBR Green I showing binucleate cells (N = nuclei; S = septa). Bars = 50 µm.
Mycobank: MB832785
Etymology:— Referring to Parque Estadual Serra do Brigadeiro, where the type species was isolated.
Diagnosis: Tulasnella brigadeiroensis is phylogenetically closely related to T. orchidis. In a comparison of the 583 ITS nucleotides, T. brigadeiroensis differs from T. orchidis by 47 bp (8.1%).
Type:—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, isolated from roots of the orchid Hadrolaelia jongheana, February 2018, E.F.S. Freitas (holotype VIC47299, ex-type culture COAD2884).
Description: Colonies on PDA attaining 31 mm diam after 8 d at 25 °C, white to cream, with undulate and submersed edge, aerial mycelium present. Reverse of the colony white to cream. Hyphae are regularly septate with branching at right angles, 1.5–2.5 µm diam (\(\bar{{\rm{X}}}\) ± SD = 2 ± 0.3 μm), hyaline, with binucleate cells. Molinioid cells not observed. Sexual morph not observed.
Substrate or host: Roots of Hadrolaelia jongheana.
Additional material examined.—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, from roots of Hadrolaelia jongheana, October 2019, E.F.S. Freitas (COAD3007, COAD3008). This species was isolated three times from two roots. There was no difference between the morphology of the isolates.
Tulasnella calospora Juel, Bih. K. svenska Vet-Akad. Handl. 23: 23 (1897). (Fig. 3)
Tulasnella calospora (COAD2869). (a) Eight-day-old PDA culture. (b) Hyphae stained with SYBR Green I showing binucleate cells (M= monilioid cell; N = nuclei; S = septa). (c) Hyphae with branching at right angles. (d) Monilioid cell chains in CMA. Bars = 50 µm.
Description: Colonies on PDA attaining 45–67 mm diam after 8 d, at 25 °C, white to cream, with undulate and submersed edge, some cultures showing aerial mycelium. Hyphae from cultures are regularly septate, with branching at right angles, 3–4 µm diam (\(\bar{{\rm{X}}}\) ± SD = 3.5 ± 0.3 μm), hyaline, with binucleate cells. Molinioid hyaline, barrel to elongated barrel-shaped, in branched chains with more than five cells. Sexual morph not observed.
Substrate or host: Roots of Cattleya caulescens and Cattleya cinnabarina.
Additional material examined—BRAZIL. Minas Gerais, Mariana, Mina da Alegria, Vale S.A., isolated from roots of Cattleya caulescens, COAD 2850–COAD2863; and from roots of Cattleya cinnabarina, COAD2864–2883, 2010, Bocayuva, M.F. There was no difference between the morphology of the isolates.
Tulasnella hadrolaeliae E.F.S. Freitas, Meir. Silva & M.C.M. Kasuya, sp. nov. (Fig. 4)
Tulasnella hadrolaeliae (COAD2889). (a) Thirty-day-old PDA culture. (b) Hyphae with branching at right angles. (c) Hyphae stained with SYBR Green I showing binucleate cells (N = nuclei; S = septa). Bars: B = 50 µm; C = 10 µm.
Mycobank: MB832786
Etymology: — Name derived from the plant host genus Hadrolaelia.
Diagnosis: Tulasnella hadrolaeliae is phylogenetically closely related to T. albida and T. pruinosa. In a comparison of the ITS nucleotides, T. hadrolaeliae differed from T. albida by 64 bp (11%) and from T. pruinosa by 73 bp (12.5%).
Type:—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, isolated from roots of orchid Hadrolaelia jongheana, February 2018, E.F.S. Freitas (holotype VIC47304, ex-type culture COAD2889).
Description: Colonies on PDA showed very slow-growing (56–59 mm diam after 30 d at 25 °C), white to cream, showing concentric rings, with undulate and submersed edge, aerial mycelium present. Reverse of the colony white to cream. Hyphae are regularly septate with branching at right angles, 2–3.5 µm diam (\(\bar{{\rm{X}}}\) ± SD = 2.5 ± 0.3 μm), hyaline, with binucleate cells and thin-walled. Molinioid cells not observed. Sexual morph not observed.
Substrate or host: Roots of Hadrolaelia jongheana.
Additional material examined.—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, from roots of Hadrolaelia jongheana, February 2018, E.F.S. Freitas (COAD2887, COAD2888, COAD2890, COAD2891). This species was isolated five times from three roots. There was no difference between the morphology of the isolates.
Tulasnella orchidis E.S. Cruz, E.F.S. Freitas, Meir. Silva & M.C.M. Kasuya, sp. nov. (Fig. 5)
Tulasnella orchidis (COAD2893). (a) Fourteen-day-old PDA culture. (b) Hyphae stained with SYBR Green I showing binucleate cells (N = nuclei; S = septa). (c) Hyphae with branching at right angles. (d) Monilioid cell chains in CMA. Bars = 50 µm.
Mycobank: MB832787
Etymology:— Name derived from the nature of host, an orchid, from which it was isolated.
Diagnosis: Tulasnella orchidis differs from T. brigadeiroensis by the culture characteristics on PDA, colonies forming concentric rings with undulate edge, whereas T. brigadeiroensis show uniform colonies with regular edge. In a comparison of the 583 ITS nucleotides, T. orchidis differed from T. brigadeiroensis by 47 bp (8%).
Type:—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, isolated from roots of Zygopetalum maxillare, February 2019, E.S. Cruz (holotype VIC47308, ex-type culture COAD2893).
Description: Colonies on PDA attaining 62–71 mm diam after 14 d, at 25 °C, white to cream, with undulate and submersed edge, showing concentric rings, no formation of aerial mycelium. Reverse of the colony white to cream. Hyphae are regularly septate with branching at right angles, 2.5–4.5 µm diam (\(\bar{{\rm{X}}}\) ± SD = 3.5 ± 0.5 μm), hyaline, with binucleate cells and thin-walled. Molinioid cells hyaline, barrel to elliptical-shaped, 5–11.5 µm diam (\(\bar{{\rm{X}}}\) ± SD = 8 ± 2 μm) and in branched chains. Sexual morph not observed.
Substrate or host: Roots of Zygopetalum maxillare.
Additional material examined.—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, from roots of Zygopetalum maxillare, February 2019, E.S. Cruz (COAD2894, COAD289). This species was isolated three times from the same root. There was no difference between the morphology of the isolates.
Tulasnella zygopetali E.S. Cruz, E.F.S. Freitas, Meir. Silva & M.C.M. Kasuya, sp. nov. (Fig. 6)
Tulasnella zygopetali (COAD2896). (a) Eight-day-old PDA culture. (b) Hyphae stained with SYBR Green I showing binucleate cells (N = nuclei; S = septa). (c) Hyphae with branching at right angles. (d) Monilioid cell chains in CMA. Bars = 50 µm.
Mycobank: MB832789
Etymology: — Name derived from the plant host genus Zygopetalum, from which it was first collected.
Diagnosis: Tulasnella zygopetali is phylogenetically different from other Tulasnella species. Morphologically, T. zygopetali differs from other Tulasnella species described here as it has wider hyphae (3–6 µm diam) and monilioid cells (6.5–12.5 µm diam). In a comparison of the 583 ITS nucleotides, T. zygopetali differed from T. brigadeiroensis by 134 bp (23%), from T. hadrolaeliae by 148 bp (25.4%) and from T. orchidis by 135 bp (23%).
Type:—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, isolated from roots of Zygopetalum maxillare, February 2019, E.S. Cruz (holotype VIC47311, ex-type culture COAD2896).
Description: Colonies on PDA attaining 86 mm diam after 8 d, at 25 °C, white to cream, with regular and submersed edge, dense aerial mycelium. Reverse of the colony white to cream. Hyphae are regularly septate with branching at right angles, 3–6 µm diam (\(\bar{{\rm{X}}}\) ± SD = 4 ± 0.9 μm), hyaline, with binucleate cells and thin-walled. Molinioid cells hyaline, elongated barrel-shaped, 6.5–12.5 µm diam (\(\bar{{\rm{X}}}\) ± SD = 10 ± 1.5 μm), in branched chains with more than five cells. Sexual morph not observed.
Substrate or host: Roots of Zygopetalum maxillare.
Additional material examined—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, from roots of Zygopetalum maxillare, February 2019, E.S. Cruz (COAD2897, COAD2898, COAD2899). This species was isolated four times from the same root. There was no difference between the morphology of the isolates.
Discussion
We investigated Tulasnella species associated with the roots of four Brazilian orchids from different vegetations of the Atlantic Forest, where this fungal genus is little known. A previous study of the same area, based only on the molecular approach, observed high fungal community diversity in roots of H. jongheana, C. caulescens and C. cinnabarina orchids, but no Tulasnella was identified8. The authors suggested that Tulasnella sequences were not detected due to the primers used. Indeed, universal fungal primers such as ITS1F/ITS4 often do not detect Tulasnella species due to a high rate of molecular evolution of nuclear rDNA genes in this genus35,39.
The genus Tulasnella (Tulasnellaceae) was described in 1888 by Schröter, with Tulasnella lilacina J. Schröt. as the type species, and nowadays there are 73 accepted species in Index Fungorum40. Due to the lack of molecular data from the type specimen, many Tulasnella species are described only by morphological-based approaches38. Morphological characters, such as size and shape of hyphae, basidia, sterigmata and basidiospore, when used alone, may lead to incorrect species identification34, e.g. because they are affected by cultural conditions. For species delimitation, we have combined both molecular and morphological data as recommended by Cruz et al.34,36, using ITS as suggested by Linde et al.38.
Among the species of the genus Tulasnella, T. calospora is considered as a nearly universal orchid symbiont41. It has been isolated from orchids in Asia42,43, Australia44,45, Europe46 and South America47,48. However, the definition of T. calospora species is still unclear, since phylogenies have shown taxonomic problems concerning this species35. In Brazil, T. calospora was obtained from the roots of the orchids Oeceoclades maculata29, Epidendrum secundum, Acianthera limae and Polystachya concreta48 in the Zona da Mata and Quadrilátero Ferrífero regions of the state of Minas Gerais. Herein, T. calospora was isolated from C. caulescens and C. cinnabarina roots also sampled in the Quadrilátero Ferrífero region. These results suggest that T. calospora is a nonspecific orchid symbiont broadly distributed in the studied region.
The present study also yielded information for four species, which likely are only a small fraction of the unknown Tulasnella species diversity. Tulasnella hadrolaeliae and T. brigadeiroensis are mycorrhizal fungi isolated from pelotons in the roots of H. jongheana, an endangered epiphytic orchid. Tulasnella brigadeiroensis was collected at two different times: first (February 2018) just one isolate was obtained, and second (October 2019) two additional isolates of the new species T. brigadeiroensis were collected. Tulasnella zygopetali and T. orchidis were isolated from pelotons from the same individual of Zygopetallum maxillare. Zygopetalum maxillare is an epiphytic orchid with high specificity in a host tree relationship7. In PESB, Z. maxillare grows exclusively on the stems of tree ferns.
The new Tulasnella species studied here were described using a polyphasic approach. Phylogenetically, T. hadrolaeliae formed a sister clade with T. albida and T. pruinosa. However, the definition of the phylogenetic species of T. albida cannot be confirmed due to the absence of molecular data from the type specimen49. Additionally, morphological characters cannot distinguish T. albida and T. pruinosa34. Therefore, as for T. calospora, molecular data from the type specimen are required to confirm the delimitation of the species T. albida and T. pruinosa49.
Tulasnella brigadeiroensis and T. orchidis formed well-supported sisters clades. Tulasnella brigadeiroensis and Tulasnella sp. COAD 2885 showed high percentage sequence divergence between clades (7.5%). This value is higher than the 3% sequence divergence cut-off value proposed for species delimitation50 or 3–5% divergence used for Tulasnella species38. Regarding the other new species described here, the interspecific nucleotide divergence ranged from 5.4 to 41.6%. These values are comparable to or even higher than those found in previous studies on Tulasnella33,34,38.
Knowledge of the diversity of orchid mycorrhizal fungi is important for successful conservation strategies4, together with their maintenance in culture collection. Our study contributes to the description of diversity of Tulasnella associated with orchids of the Brazilian Atlantic Forest, which is relevant for conservation of these orchids and for knowledge of fungal richness in this hotspot of biodiversity. Further studies are required to verify the potential of new species to support seed germination, seedling development and, consequently, orchid conservation programs.
Conclusions
Phylogenetic analyses, integrated with nucleotide divergence and morphological characteristics, showed the diversity of Tulasnella species associated with orchids of the Brazilian Atlantic Forest, including the description of four novel Tulasnella species. This is the first study using a polyphasic approach to the description of Tulasnella in Brazil, and it suggests that further studies will uncover more diversity. The cultivation of these species may help the strategies of conservation of endangered Brazilian orchids.
Methods
Sample collection and isolates
Root samples of the epiphytic orchid H. jongheana were collected from the PESB (Araponga – MG, Brazil) and PESN (Itamarandiba – MG, Brazil) (Fig. 7). Zygopetalum maxillare samples were also obtained from PESB, while C. cinnabarina and C. caulescens were sampled from iron mining areas in the Quadrilátero Ferrífero region (Mariana – MG, Brazil) (Fig. 7). Apparently healthy roots were analyzed at the Laboratório de Associações Micorrízicas (DMB/UFV). The root samples were gently washed under running tap water, cut into pieces of transversal root fragments, 2–3 mm thick, surface-sterilized in 70% ethanol for 1 min, 2% sodium hypochlorite for 3 min, followed by two successive rinses of sterile distilled water. These fragments were then examined under a stereomicroscope, after slicing into several thin transversal slices. Cells containing pelotons were placed on potato dextrose agar (PDA) medium without antibiotics and then incubated at 25 °C in the dark. Axenic cultures were preserved on rice grains in an ultrafreezer at −72 °C or silica gel and were deposited in the Coleção Oswaldo Almeida Drummond collection (COAD) at the Universidade Federal de Viçosa. Representative specimens were deposited at the Fungarium of the Universidade Federal de Viçosa (VIC).
Investigated orchids: (a), flower of Hadrolaelia jongheana; (b), Zygopetalum maxillare; (c), flower of Cattleya cinnabarina; (d), flower of Cattleya caulescens.
Morphology
The fungus and colony characteristics were described from cultures grown on PDA at 25 °C in the dark for 1–4 weeks depending on their growth rate. Measurements of colony diameters were taken using digital calipers. Color terminology followed Rayner51. The nuclear condition was observed from young hyphae after staining with SYBR Green I according to Meinhardt et al.52. The isolates were transferred to Corn Meal Agar (CMA) medium and incubated at 25 °C in the dark, for 4–6 weeks, to induce monilioid cell formation29. Observations, measurements and photographic images of microscopic fungal structures were recorded using an Olympus BX53 light microscope, with an Olympus Q-Color5TM digital high-definition color camera and differential interference contrast (DIC) illumination. Adobe Photoshop CS5 was used for the final editing of the acquired images and photographic preparations.
DNA extraction, PCR amplification and sequencing
The genomic DNA was extracted from fungal mycelia grown on PDA at 25 °C for 4 weeks, using the Nucleospin® Soil (MACHEREY-NAGEL GmbH & Co. KG), in accordance with the manufacturer’s instructions. The nuclear ribosomal internal transcribed spacer (ITS) region was amplified using primer pairs ITS1 and ITS453. Each polymerase chain reaction (PCR) was performed in 50 µL containing 10–20 ng of DNA template, 1× Taq buffer, 2 mM MgCl2, 0.2 μM of each primer, 0.4 mM of each dNTP, and 1.0 U Taq DNA polymerase (Cellco Biotec do Brasil Ltda, São Paulo, Brazil). PCR was carried out using a MyCyclerTM Thermal Cycler (Bio-Rad Laboratories B.V., Veenendal, The Netherlands) with an initial denaturation at 95 °C, for 2 min, followed by 39 PCR cycles (denaturation at 95 °C for 1 min; annealing at 50 °C for 1 min; extension at 72 °C for 1 min) before a final extension at 72 °C for 10 min.
The PCR products were visualized on 1% agarose gels stained with ethidium bromide to assess product size and quality, purified and then sequenced from the two strands using the primers ITS1 and ITS453. Consensus sequences were generated using the MEGA v.7.0.26 software tool54. All sequences were checked manually, and nucleotides with ambiguous positions were clarified using both primer direction sequences. The sequences were deposited in GenBank (see accession numbers in Table 1).
Phylogenetic analyses
Consensus sequences were compared against NCBI’s GenBank nucleotide databases by using the BLASTn algorithm. The most similar sequences were downloaded in FASTA format and aligned with our sequences by using the MAFFT v. 7 online portals55. The resulting sequence alignments were manually checked and adjusted in MEGA v.7.0.26 software tool54.
Bayesian inference (BI) analyses employing a Markov Chain Monte Carlo method were performed on all sequences. Nucleotide substitution models were determined using the MrModeltest 2.3 program56 and, once the likelihood scores had been calculated, the models were selected according to the Akaike information criterion (AIC). The results of MrModeltest recommended a GTR + G model for ITS, and a dirichlet (1,1,1,1) state frequency distribution and a gamma distributed rate variation were set. The phylogenetic analysis was performed using the CIPRES web portal57 and the MrBayes program v.3.1.158. Two sets of four MCMC chains were run simultaneously, starting from random trees for 1,000,000 generations and sampling every 1,000th generation. The first 25% of the trees were discarded as the burn-in phase for each analysis. Posterior probabilities59 were determined from the remaining trees and are presented on the left of each node. Maximum likelihood (ML) analysis was implemented using the RAxML-HPC v.8 on XSEDE (8.2.12) available on the CIPRES web portal. Parameters for maximum likelihood were set to rapid bootstrapping and the analysis was carried out using 1000 replicates. Alignments and trees were deposited in TreeBASE (http://treebase.org/treebase-web/) (25158). The trees were visualized in FigTree V1.4.460 and the layout of the tree for publication was done using Adobe Illustrator v. CC.
Divergence between clades and haplotype network
In order to assess the sequence divergence between and within the clades obtained in the phylogeny tree, the Kimura-2-parameter distances were calculated as implemented in MEGA v.7.0.2661. The analysis involved 85 nucleotide sequences. All positions containing gaps and missing data were eliminated. There was a total of 272 positions in the final dataset.
Data availability
All materials examined were deposited in the public culture collection of the Coleção Oswaldo Almeida Drummond (COAD), of the Universidade Federal de Viçosa. Alignments and tree files generated during the current study are available at TreeBASE (accession https://www.treebase.org/treebase-web/home.html; study 25158). All sequence files are available from the GenBank database. The complete list of accession numbers is included in Table 1. They will be made available to the public after the publication of the paper.
References
Govaerts, R. WCSP: World Checklist of Selected Plant Families. In: Species 2000 & ITIS catalogue of life, 2016 annual checklist. Leiden, the Netherlands (2016).
Pridgeon, A. The illustrated encyclopaedia of orchids. Timber, Portland (1995).
Giulietti, A. M., Harley, R. M., De Queiroz, L. P., Wanderley, M. G. L. & Berg, C. V. D. Biodiversity and conservation of plants in Brazil. Conservation Biology 19, 632–639, https://doi.org/10.1111/j.1523-1739.2005.00704.x (2005).
Swarts, N. D. & Dixon, K. W. Terrestrial orchid conservation in the age of extinction. Annals of Botany 104, 543–556, https://doi.org/10.1093/aob/mcp025 (2009).
Selosse, M.-A. The latest news from biological interactions in orchids: in love, head to toe. New Phytologist 202, 337–340, https://doi.org/10.1111/nph.12769 (2014).
Martinelli, G., Moraes, M. A. Livro Vermelho da Flora do Brasil, 1st edn. Rio de Janeiro (2013).
Pridgeon, A. M., Cribb, P. J., Chase, M. W. & Rasmussen, F. N. Genera Orchidacearum: Epidendroideae (Part Two), 5th vol. Oxford, New York (2009).
Oliveira, S. F. et al. Endophytic and mycorrhizal fungi associated with roots of endangered native orchids from the Atlantic Forest, Brazil. Mycorrhiza 24, 55–64, https://doi.org/10.1007/s00572-013-0512-0 (2014).
Myers, N., Mittermeier, R. A., Mittermeier, C. G., Fonseca, G. A. B. & Kent, J. Biodiversity hotspots for conservation priorities. Nature 403, 853–858 (2000).
Dearnaley, J. W. D., Perotto, S. & Selosse, M. A. Structure and development of orchid mycorrhizas. In: Martin, F. (ed) Molecular Mycorrhizal Symbiosis. Wiley-Blackwell, Hoboken, New Jersey, 63–86 (2016).
Smith, S. E., Read, D. J. Mycorrhizal Symbiosis, 3rd edn. Adelaide, Australia. (2008).
Dearnaley, J. D. W., Martos, F. & Selosse, M. A. Orchid mycorrhizas: molecular ecology, physiology, evolution and conservation aspects. In: Hock B (ed) The Mycota IX: Fungal Associations, 2nd edn. Springer, Berlin Heidelberg (2012).
Weiß, M., Waller, F., Zuccano, A. & Selosse, M. A. Sebacinales – one thousand and one interactions with land plants. New Phytologist 211, 20–40, https://doi.org/10.1111/nph.13977 (2016).
Jacquemyn, H., Honnay, O., Cammue, B. P. A., Brys, R. & Lievens, B. Low specificity and nested subset structure characterize mycorrhizal associations in five closely related species of the genus Orchis. Molecular Ecology 19, 4086–4095, https://doi.org/10.1111/j.1365-294X.2010.04785.x (2010).
McCormick, M. K., Whigham, D. F. & Canchani‐Viruet, A. Mycorrhizal fungi affect orchid distribution and population dynamics. New Phytologist 219, 1207–1215, https://doi.org/10.1111/nph.15223 (2018).
Otero, J. T., Flanagan, N. S., Herre, E. A., Ackerman, J. D. & Bayman, P. Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae). American Journal of Botany 94, 1944–1950, https://doi.org/10.3732/ajb.94.12.1944 (2007).
Girlanda, M. et al. Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations. American Journal of Botany 98, 1148–1163, https://doi.org/10.3732/ajb.1000486 (2011).
Martos, F. et al. The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids. Molecular Ecology 21, 5098–5109, https://doi.org/10.1111/j.1365-294X.2012.05692.x (2012).
Xing, X. et al. The impact of life form on the architecture of orchid mycorrhizal networks in tropical forest. Oikos 128, 1254–1264, https://doi.org/10.1111/oik.06363 (2019).
McCormick, M. K., Whigham, D. F. & O’Neill, J. Mycorrhizal diversity in photosynthetic terrestrial orchids. New Phytologist 163, 425–438, https://doi.org/10.1111/j.1469-8137.2004.01114.x (2004).
Pereira, M. C., Pereira, O. L., Costa, M. D., Rocha, R. B. & Kasuya, M. C. M. Diversidade de fungos micorrízicos Epulorhiza spp. isolados de Epidendrum secundum (Orchidaceae). Revista Brasileira de Ciência do Solo 33, 1187–1197, https://doi.org/10.1590/S0100-06832009000500012 (2009).
Zi, X. M., Sheng, C. L., Goodale, U. M., Shao, S. C. & Gao, J. Y. In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid, Dendrobium aphyllum (Orchidaceae). Mycorrhiza 24, 487–499, https://doi.org/10.1007/s00572-014-0565-8 (2014).
Khamchatra, N., Dixon, K. W., Tantiwiwat, S. & Piapukiew, J. Symbiotic seed germination of an endangered epiphytic slipper orchid, Paphiopedilum villosum (Lindl.) Stein. from Thailand. South African Journal Botany 104, 76–81, https://doi.org/10.1016/j.sajb.2015.11.012 (2016).
Meng, Y.-Y. et al. Symbiotic fungi undergo a taxonomic and functional bottleneck during orchid seed germination: a case study on Dendrobium moniliforme. Symbiosis 79, 205–212, https://doi.org/10.1007/s13199-019-00647-x (2019).
Meng, Y. Y., Zhang, W. L., Selosse, M. A. & Gao, J. Y. Are fungi from adult orchid roots the best symbionts at germination? A case study. Mycorrhiza 29, 541–547, https://doi.org/10.1007/s00572-019-00907-0 (2019).
Pereira, M. C. et al. Morphological and molecular characterization of Tulasnella spp. fungi isolated from the roots of Epidendrum secundum, a widespread Brazilian orchid. Symbiosis 62, 111–121, https://doi.org/10.1007/s13199-014-0276-0 (2014).
Pereira, M. C., Pereira, O. L., Costa, M. D., Rocha, R. B. & Kasuya, M. C. M. Diversidade de fungos micorrízicos Epulorhiza spp. isolados de Epidendrum secundum (Orchidaceae). Revista Brasileira de Ciência do Solo 33, 1187–1197 (2009).
Nogueira, R. E., Pereira, O. L., Kasuya, M. C. M., Lanna, M. C. S. & Mendonça, M. P. Fungos micorrízicos associados a orquídeas em campos rupestres na região do Quadrilátero Ferrífero, MG, Brasil. Acta Botanica Brasilica 19(3), 417–424, https://doi.org/10.1590/S0102-33062005000300001 (2005).
Pereira, O. L., Kasuya, M. C. M., Borges, A. C. & Araújo, E. F. Morphological and molecular characterization of mycorrhizal fungi isolated from neotropical orchids in Brazil. Canadian Journal Botany 83, 54–65, https://doi.org/10.1139/b04-151 (2005).
Pereira, O. L., Rollemberg, C. L., Borges, A. C., Matsuoka, K. & Kasuya, M. C. M. Epulorhiza epiphytica sp. nov. isolated from mycorrhizal roots of epiphytic orchids in Brazil. Mycoscience 44, 153–155, https://doi.org/10.1007/s10267-002-0087-7 (2003).
Linde, C. C., Phillips, R. D., Crisp, M. D. & Peakall, R. Congruent species delineation of Tulasnella using multiple loci and methods. New Phytologist 201, 6–12, https://doi.org/10.1111/nph.12492 (2014).
Solís, K., Barriuso, J. J., Garcés-claver, A. & González, V. Tulasnella tubericola (Tulasnellaceae, Cantharellales, Basidiomycota): a new Rhizoctonia-like fungus associated with mycorrhizal evergreen oak plants artificially inoculated with black truffle (Tuber melanosporum) in Spain. Phytotaxa 317, 175–187, https://doi.org/10.11646/phytotaxa.317.3.2 (2017).
Rachanarin, C. et al. A new endophytic fungus, Tulasnella phuhinrongklaensis (Cantharellales, Basidiomycota) isolated from roots of the terrestrial orchid, Phalaenopsis pulcherrima. Phytotaxa 374, 99–109, https://doi.org/10.11646/phytotaxa.374.2.1 (2018).
Cruz, D., Suárez, J. P., Kottke, I. & Piepenbring, M. Cryptic species revealed by molecular phylogenetic analysis of sequences obtained from basidiomata of Tulasnella. Mycologia 106, 708–722, https://doi.org/10.3852/12-386 (2014).
Suárez, J. P. et al. Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest. Mycological Research 110, 1257–1270, https://doi.org/10.1016/j.mycres.2006.08.004 (2006).
Cruz, D., Suárez, J. P., Kottke, I., Piepenbring, M. & Oberwinkler, F. Defining species in Tulasnella by correlating morphology and nrDNA ITS-5.8S sequence data of basidiomata from a tropical Andean forest. Mycological Progress 10, 229–238, https://doi.org/10.1007/s11557-010-0692-3 (2011).
Fujimori, S., Abe, J. P., Okane, I. & Yamaoka, Y. Three new species in the genus Tulasnella isolated from orchid mycorrhiza of Spiranthes sinensis var. amoena (Orchidaceae). Mycoscience 60, 71–81, https://doi.org/10.1016/j.myc.2018.09.003 (2019).
Linde, C. C. et al. New species of Tulasnella associated with terrestrial orchids in Australia. IMA Fungus 8, 27–47, https://doi.org/10.5598/imafungus.2017.08.01.03 (2017).
Moncalvo, J. M. et al. The cantharelloid clade: dealing with incongruent gene trees and phylogenetic reconstruction methods. Mycologia 98, 937–948, https://doi.org/10.1080/15572536.2006.11832623 (2006).
Kirk, P. & Bridge, P. Index Fungorum, http://www.indexfungorum.org/ (2019).
Hadley, G. Non-Specificity of symbiotic infection in orchid mycorrhiza. New Phytologist 69, 1015–1023 (1970).
Tan, X. M. et al. Isolation and identification of endophytic fungi in roots of nine Holcoglossum plants (Orchidaceae) collected from Yunnan, Guangxi, and Hainan Provinces of China. Current Microbiology 64, 140–147, https://doi.org/10.1007/s00284-011-0045-8 (2012).
Ding, R. et al. Identity and specificity of rhizoctonia-like fungi from different populations of Liparis japonica (Orchidaceae) in northeast China. Plos One 9, e105573, https://doi.org/10.1371/journal.pone.0105573 (2014).
Warcupii, J. H. The mycorrhizal relathionships of Australian orchids. New Phytologist 87, 371–381, https://doi.org/10.1111/j.1469-8137.1981.tb03208.x (1981).
De Long, J. R., Swarts, N. D., Dixon, K. W. & Egerton-Warburton, L. M. Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media. Annals of Botany 111, 409–418, https://doi.org/10.1093/aob/mcs294 (2013).
Warcupii, J. H. & Talbot, P. H. B. Perfect states of rhizoctonias associated with orchids. New Phytologist 66, 631–64, https://doi.org/10.1111/j.1469-8137.1967.tb05434.x (1967).
Steinfort, U., Verdugo, G., Besoain, X. & Cisternas, M. A. Mycorrhizal association and symbiotic germination of the terrestrial orchid Bipinnula fimbriata (Poepp.) Johnst (Orchidaceae). Flora 205, 811–817, https://doi.org/10.1016/j.flora.2010.01.005 (2010).
Nogueira, R. E., Berg, C., van den, Pereira, O. L. & Kasuya, M. C. M. Isolation and molecular characterization of Rhizoctonia-like fungi associated with orchid roots in the Quadrilátero Ferrífero and Zona da Mata regions of the state of Minas Gerais, Brazil. Acta Botanica Brasilica 28, 298–300, https://doi.org/10.1590/s0102-33062014000200017 (2014).
Cruz, D., Suárez, J. P. & Piepenbring, M. Morphological revision of Tulasnellaceae, with two new species of Tulasnella and new records of Tulasnella spp. for Ecuador. Nova Hedwigia 102, 279–338, https://doi.org/10.1127/nova_hedwigia/2015/0304 (2016).
Nilsson, R. H., Kristiansson, E., Ryberg, M., Hallenberg, N. & Larsson, K. H. Intraspecific ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evolutionary Bioinformatics 4, 193–201 (2008).
Rayner, R.W. A Mycological Color Chart. Commonwealth Mycological Institute, Kew, (1970).
Meinhardt, L. W., Bellato, C. D. M. & Tsai, S. M. SYBR® Green I used to evaluate the nuclei number of fungal mycelia. Biotechniques 31, 42–46 (2001).
White, T. J., Bruns, T., Lee, S. & Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J. (eds.) PCR Protocols: A Guide of Methods and Applications. Academic Press, San Diego, 315–322 (1990).
Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets brief communication. Molecular Evolutionary. Genetics 33, 1870–1874, https://doi.org/10.1093/molbev/msw054 (2016).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30, 772–778, https://doi.org/10.1093/molbev/mst010 (2013).
Nylander, J. A. A. MrModeltest version 2. Evolutionary Biology Centre, Uppsala, Sweden, https://github.com/nylander/MrModeltest2 (2004).
Miller, M. A., Pfeiffer, W. & Schwartz, T. Creating the CIPRES science gateway for inference of large phylogenetic trees. Gateway Computing Environments Workshop, https://doi.org/10.1109/GCE.2010.5676129 (2010).
Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: Beyesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574, https://doi.org/10.1093/bioinformatics/btg180 (2003).
Rannala, B. & Yang, Z. Proballity distribution of molecular evolutionary trees: A new method of phylogenetic inference. Journal of Molecular Evolution 43, 304–311 (1996).
Rambaut, A. FigTree version 1.4.4. In: Molecular evolution, phylogenetics and epidemiology, http://tree.bio.ed.ac.uk/software/figtree/ (2009).
Tamura, K. et al. MEGA5: Molecular evolutionary genetics analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony methods. Molecular Biology and Evolution 28, 2731–2739, https://doi.org/10.1093/molbev/msr121 (2011).
Acknowledgements
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-PROTAX); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior –Brasil (CAPES) – Finance Code 001; and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors also acknowledge Márcio Assis for assisting in the collections, and the administration and scientific staff of the Instituto Estadual de Floresta of the State of Minas Gerais, Serra do Brigadeiro State Park and Serra Negra State Park, for their provision of facilities and for permission to conduct exploratory surveys of mycodiversity in their protected areas.
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E.F.S.F. and M.S. designed the study. Material preparation and data collection were performed by E.F.S.F., E.S.C., M.F.B. and T.G.R.V. Analyses were performed by E.F.S.F., M.S. and E.M. The first draft of the manuscript was written by E.F.S.F. and was revised by M.S. All authors commented on previous versions of the manuscript. The work was substantially revised by M.-A.S. and supervised by M.C.M.K. All authors read and approved the final manuscript.
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Freitas, E.F.S., da Silva, M., Cruz, E.d.S. et al. Diversity of mycorrhizal Tulasnella associated with epiphytic and rupicolous orchids from the Brazilian Atlantic Forest, including four new species. Sci Rep 10, 7069 (2020). https://doi.org/10.1038/s41598-020-63885-w
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DOI: https://doi.org/10.1038/s41598-020-63885-w
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