Diversity of mycorrhizal Tulasnella associated with epiphytic and rupicolous orchids from the Brazilian Atlantic Forest, including four new species

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.


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).
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.   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 (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 (X ± SD = 10 ± 1.5 μm), in branched chains with more than five cells. Sexual morph not observed.

Description
Substrate or host: Roots of Zygopetalum maxillare.  . 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 identified 8 . 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 genus 35,39 .
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 Fungorum 40 . Due to the lack of molecular data from the type specimen, many Tulasnella species are described only by morphological-based approaches 38 . Morphological characters, such as size and shape of hyphae, basidia, sterigmata and basidiospore, when used alone, may lead to incorrect species identification 34 , 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 symbiont 41 . It has been isolated from orchids in Asia 42,43 , Australia 44,45 , Europe 46 and South America 47,48 . However, the definition of T. calospora species is still unclear, since phylogenies have shown taxonomic problems concerning this species 35 . In Brazil, T. calospora was obtained from the roots of the orchids Oeceoclades maculata 29 , Epidendrum secundum, Acianthera limae and Polystachya concreta 48 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   www.nature.com/scientificreports www.nature.com/scientificreports/ epiphytic orchid with high specificity in a host tree relationship 7 . 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 specimen 49 . Additionally, morphological characters cannot distinguish T. albida and T. pruinosa 34 . Therefore, as for T. calospora, molecular data from the type specimen are required to confirm the delimitation of the species T. albida and T. pruinosa 49 .
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 delimitation 50 or 3-5% divergence used for Tulasnella species 38 . 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 Tulasnella 33,34,38 .
Knowledge of the diversity of orchid mycorrhizal fungi is important for successful conservation strategies 4 , 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 www.nature.com/scientificreports www.nature.com/scientificreports/ 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).
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 Rayner 51 . 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 formation 29 . 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. 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 ITS4 53 . Consensus sequences were generated using the MEGA v.7.0.26 software tool 54 . 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 portals 55 . The resulting sequence alignments were manually checked and adjusted in MEGA v.7.0.26 software tool 54 .
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 program 56 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 portal 57 and the MrBayes program v.3.1.1 58 . 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 probabilities 59 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 V1.4.4 60 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.7.0.26 61 . 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.