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Filling a gap: endophytic fungal diversity in the leaves of Miconia mirabilis in the Atlantic Forest (Brazil) and the description of a new Muyocopron species

Abstract

Brazilian biomes, such as the Atlantic Forest, are hotspots for microbial diversity. Fungi at some stages of their life cycle can inhabit the interior of plants without apparent damage and are called endophytes. This study aimed to verify the culturable diversity of endophytic fungi present in the leaves of Miconia mirabilis in a conservation unit of the Atlantic Forest, in Northeast Brazil. A total of 273 endophytes were isolated, and 88 % of the estimated richness was recovered from 40 taxa with a Shannon diversity index of H′ = 2.79. The most abundant order was Xylariales (37.73 %), with Xylaria cf. cubensis being the most abundant species, followed by Glomerellales (14.65 %), Eurotiales (3.66 %), and Diaporthales (3.33 %). Additionally, a novel endophytic fungal species of Muyocopron was identified. Our results indicate the potential of endophytic diversity in the plants of tropical forests, reinforcing the need for further studies to better understand their ecological relationships.

Keywords:
Endophytic fungi; Phylogeny; Rainforest; Taxonomy; Xylariales

Introduction

Among Brazilian biomes, the Atlantic Forest is one of the 34 biodiversity hotspots in the world (Leitman et al. 2015Leitman P, Amorim AM, Sansevero JBB, Forzza RC. 2015. Floristic patterns of epiphytes in the Brazilian Atlantic Forest, a biodiversity hotspot. Botanical Journal of the Linnean Society 179: 587-601.). Approximately, 90 % of its original area is degraded and just 28 % of its original vegetation is protected in conservation units, generating implications at different ecological levels (Rezende et al. 2018Rezende CL, Scarano FR, Assad ED et al. 2018. From hotspot to hopespot: An opportunity for the Brazilian Atlantic Forest. Perspectives in Ecology and Conservation 16: 208-214.; Lembi et al. 2020Lembi RC, Cronemberger C, Picharillo C et al. 2020. Urban expansion in the Atlantic Forest: Applying the nature futures framework to develop a conceptual model and future scenarios. Biota Neotropica 20: e20190904.). Presently, the lack of conservation is a major problem, leading to a considerable loss of biodiversity and its services (Foden & Young 2016Foden WB, Young BE. 2016. IUCN SSC guidelines for assessing species' vulnerability to climate change. Cambridge, England and Gland, Switzerland, IUCN.). Therefore, it is necessary to preserve in situ this diversity in protected areas such as conservation units (Heywood 2019Heywood VH. 2019. Conserving plants within and beyond protected areas-still problematic and future uncertain. Plant Diversity 41: 36-49.).

Conservation units (UCs) are established to conserve and protect biodiversity (Mattar et al. 2018Mattar EPL, Barros TTV, Cunha BB, Souza JFD, Silva AMDC. 2018. Federal Conservation Units in Brazil: The situation of biomes and regions. Floresta e Ambiente 25: e20150051.); as a result, species that are threatened or vulnerable can be found in these areas. The UCs cover 18 % of Brazilian territory, with 334 federal UCs managed by the Chico Mendes Institute for Biodiversity Conservation. Of these, 131 are located in the Atlantic Forest (ICMBio 2022ICMBio - Instituto Chico Mendes de Conservação da Biodiversidade. 2022. Ministério do Meio Ambiente. https://www.gov.br/icmbio/pt-br. 9 Feb. 2022.
https://www.gov.br/icmbio/pt-br...
). In Brazil, UCs are divided into two categories (Strict Protected Areas and Protected Areas with sustainable use); however, they are mainly located in areas with large population concentrations (Lembi et al. 2020Lembi RC, Cronemberger C, Picharillo C et al. 2020. Urban expansion in the Atlantic Forest: Applying the nature futures framework to develop a conceptual model and future scenarios. Biota Neotropica 20: e20190904.) and, consequently, under pressure from anthropogenic activities (Bernard et al. 2014Bernard E, Penna, LAO, Araújo E. 2014. Downgrading, downsizing, degazettement, and reclassification of protected areas in Brazil. Conservation Biology 28: 939-950.). Among the UCs of the Atlantic Forest, the Reserva Biológica de Pedra Talhada (REBIO de Pedra Talhada) was established on 13 December 1989 and is located in a transitional area between the Seasonal Tropical Forest and the Caatinga, with humid forests surrounded by dry forests (Nusbaumer et al. 2015Nusbaumer L, Barbosa MRV, Thomas WW, Alves MV, Loizeau PA, Spichiger R. 2015. Flora e vegetação da Reserva Biológica de Pedra Talhada. In: Studer A, Nusbaumer L, Spichiger R (eds.). Biodiversidade da Reserva Biológica de Pedra Talhada (Alagoas, Pernambuco - Brasil). Boissiera. vol. 68, p. 59-121.; ICMBio 2022ICMBio - Instituto Chico Mendes de Conservação da Biodiversidade. 2022. Ministério do Meio Ambiente. https://www.gov.br/icmbio/pt-br. 9 Feb. 2022.
https://www.gov.br/icmbio/pt-br...
). Several species of animals, plants, and fungi have been reported in this reserve, including approximately 823 plant species (Studer et al. 2015Studer A, Nusbaumer L, Spichiger R (eds.). 2015. Biodiversidade da Reserva Biológica de Pedra Talhada (Alagoas, Pernambuco - Brasil). Boissiera. vol. 68.). Miconia mirabilis (Melastomataceae, Myrtales) is a plant found in this UC. It grows rapidly, restores degraded areas, and provides food resources for the fauna (Pessoa et al. 2012Pessoa MDS, Vleeschouwer KMD, Talora DC, Rocha L, Amorim AMA. 2012. Reproductive phenology of Miconia mirabilis (Melastomataceae) within three distinct physiognomies of Atlantic Forest, Bahia, Brazil. Biota Neotropica 12: 49-56.). Although there are few studies on the ecological aspects of this species, to the best of our knowledge, no study has reported the association of endophytic fungi with M. mirabilis. Complex microbial communities colonise plants at different stages of interaction and perform various functions, both beneficial and harmful to the plant, depending on environmental factors (biotic and abiotic) and the genetic compositions of the host and microorganism (Rey & Schornack 2013Rey T, Schornack S. 2013. Interactions of beneficial and detrimental root-colonizing filamentous microbes with plant hosts. Genome Biology 14: 121.; Brader et al. 2014Brader G, Compant S, Mitter B, Trognitz F, Sessitsch A. 2014. Metabolic potential of endophytic bacteria. Current Opinion in Biotechnology 27: 30-37. ). While studies have elucidated this interaction, it is known that when dealing with plant microbiome, fungi are an important part of plant development, growth, and adaptation. During all or part of their life cycle, they can exist within all plant tissues, without apparent damage, and are functionally defined as endophytic fungi (Khidir et al. 2010Khidir HH, Eudy DM, Porras-Alfaro A, Herrera J, Natvig DO, Sinsabaugh RL. 2010. A general suite of fungal endophytes dominate the roots of two dominant grasses in a semiarid grassland. Journal of Arid Environments 74: 35-42.; Hardoim et al. 2015Hardoim PR, Overbeek LSV, Berg G et al. 2015. The hidden world within plants: Ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiology and Molecular Biology Reviews 79: 293-320.).

Although there is a growing interest in the understanding of endophytic biology and diversity, these fungi still correspond to a neglected wealth; therefore, further studies are needed to explore its diversity and distribution (Arnold & Lutzoni 2007Arnold AE, Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology 88: 541-549.; Baucom et al. 2012Baucom DL, Romero M, Belfon R, Creamer R. 2012. Two new species of Undifilum, fungal endophytes of Astragalus (locoweeds) in the United States. Botany 90: 866-875.; Vaz et al. 2014Vaz ABM, Fontenla S, Rocha FS et al. 2014. Fungal endophyte β-diversity associated with Myrtaceae species in an Andean Patagonian forest (Argentina) and an Atlantic forest (Brazil). Fungal Ecology 8: 28-36.; Baldrian 2017Baldrian P. 2017. Forest microbiome: Diversity, complexity and dynamics. FEMS Microbiology reviews 4: 109-130.). The increase in conservation and accessibility to different biomes contribute to the increase in knowledge about the biology and life cycle of endophytic fungi. In particular, Brazilian biomes harbour substantial biodiversity and stand out due to specific characteristics (Noriler et al. 2018Noriler AS, Savi DC, Aluizio R, Palacio-Cortes AM, Possiede YM, Glienke C. 2018. Bioprospecting and structure of fungal endophyte communities found in the Brazilian biomes, Pantanal, and Cerrado. Frontiers in Microbiology 9: 1526.; Temperton et al. 2019Temperton VM, Buchamann N, Buisson E et al. 2019. Step back from the forest and step up to the Bonn Challenge: How a broad ecological perspective can promote successful landscape restoration. Restoration Ecology 27: 705-719.; Rosa 2021Rosa LH. 2021. Neotropical Endophytic Fungi. Springer International Publishing.).

This study aimed to verify the culturable diversity of endophytic fungi in the leaves of M. mirabilis growing in a UC of the Atlantic Forest in Brazil to contribute to the national and global fungal estimations and to understand the endophytic fungal diversity in a plant species of the Atlantic Forest in Brazil. We also introduced a new species of Muyocopron (Muyocopronaceae, Muyocopronales), demonstrating the capacity of M. mirabilis to harbour unknown fungal species.

Materials and methods

Study site

The leaves of M. mirabilis (Aubl.) L.O. Williams were collected during the rainy season (July 2018) from REBIO Pedra Talhada (36°22' to 36°28'W, 9°11' to 9°16'S) in the Atlantic Forest. The REBIO Pedra Talhada has an area of approximately 4469 ha and is located in the municipalities of Lagoa do Ouro and Correntes (Pernambuco) and Quebrangulo and Chã Preta (Alagoas) (Fig. 1). It is in the Borborema Plateau, a particular mountain formation in Northeast Brazil. The UC, a humid forest surrounded by dry forest, is in a transition area between the Seasonal Tropical Forest and Caatinga (Nusbaumer et al. 2015Nusbaumer L, Barbosa MRV, Thomas WW, Alves MV, Loizeau PA, Spichiger R. 2015. Flora e vegetação da Reserva Biológica de Pedra Talhada. In: Studer A, Nusbaumer L, Spichiger R (eds.). Biodiversidade da Reserva Biológica de Pedra Talhada (Alagoas, Pernambuco - Brasil). Boissiera. vol. 68, p. 59-121.). This UC has a defined rainy season (May, June, and July) with an average annual accumulation of 1628 mm (more than 250 mm per month), and the dry season extends from October to February, with less than 50 mm rainfall per month (Studer et al. 2015Studer A, Nusbaumer L, Spichiger R (eds.). 2015. Biodiversidade da Reserva Biológica de Pedra Talhada (Alagoas, Pernambuco - Brasil). Boissiera. vol. 68.; ICMBio 2022ICMBio - Instituto Chico Mendes de Conservação da Biodiversidade. 2022. Ministério do Meio Ambiente. https://www.gov.br/icmbio/pt-br. 9 Feb. 2022.
https://www.gov.br/icmbio/pt-br...
).

Figure 1
The geographical location of the Biological Reserve of Pedra Talhada, Atlantic Forest, Brazil (A), View of the main site of the studied area (B-C), and details of Miconia mirabilis (D).

Plant collection was authorised by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) under the Ministério do Meio Ambiente (MMA); the permission number 63010 and authentication code 55412112 were issued on 26 April 2018.

Sampling and processing of plant materials

Healthy leaves from four individuals (labelled M1, M2, M3, and M4) of M. mirabilis were randomly collected. Plant materials (three to four leaves per individual) were individually packed in paper bags and processed within 48 h. To isolate endophytic fungi, the leaves were disinfected using the methods described by Bezerra et al. (2015)Bezerra JDP, Nascimento CCF, Barbosa RN et al. 2015. Endophytic fungi from medicinal plant Bauhinia forficata: Diversity and biotechnological potential. Brazilian Journal of Microbiology 46: 49-57.. Briefly, asepsis of the leaves was performed by initial washing with water and detergent to remove impurities, followed by rinsing with distilled water. Subsequently, the leaves were washed in 70 % alcohol (1 min), 2-2.5 % sodium hypochlorite (3 min), and 70 % alcohol (1 min). Finally, the leaves were washed thrice (1 min each) in distilled and sterilized water.

The leaves were fragmented using a sterile scalpel blade, and 180 fragments of approximately 0.5 cm² were selected (45 fragments from each individual). The fragments were transferred to Petri dishes containing potato dextrose agar (PDA) and chloramphenicol (50 mgL-1). The Petri dishes were incubated at 28 ± 2 °C for 10 days in a natural light-dark cycle. To verify the effectiveness of disinfection, 1 mL of the last washing water was transferred to the surface of the PDA, and the Petri dishes were incubated under the same conditions. Fungal growth was observed daily, and all fungal colonies were isolated and purified. After isolation, endophytes were preserved in water containing 10 % glycerol for later morphological and molecular analyses.

Identification of fungal endophytes

Endophytic fungi were identified based on their morphology by observing the macro- and micro-morphological characteristics of somatic and reproductive structures (Barnett & Hunter 1987Barnett HL, Hunter BB. 1987. Illustrated Genera of Imperfect Fungi. 4th. edn. New York, MacMillan Publishing Company.; Hanlin 1990Hanlin RT. 1990. Illustrated Genera of Ascomycetes. St Paul, APS Press.; Samson & Frisvad 2004Samson RA, Frisvad JC. 2004. Penicillium Subgenus Penicillium: New Taxonomics 210 Schemes, Mycotoxins and Other Extrolites. Studies in Micology 49: 260.; Hsieh et al. 2010Hsieh HM, Lin CR, Fang MJ et al. 2010. Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily. Molecular Phylogenetics and Evolution 54: 957-969.; Gomes et al. 2013Gomes RR, Glienke C, Videira SI, Lombard L, Groenewald JZ, Crous PW. 2013. Diaporthe: A genus of endophytic, saprobic and plant pathogenic fungi. Persoonia-Molecular Phylogeny and Evolution of Fungi 31: 1-41.; Phillips et al. 2013Phillips AJL, Alves A, Abdollahzadeh J et al. 2013. The Botryosphaeriaceae: Genera and species known from culture. Studies in Mycology 76: 51-167.; Hernández-Restrepo et al. 2019Hernández-Restrepo M, Bezerra JDP, Tan YP et al. 2019. Re-evaluation of Mycoleptodiscus species and morphologically similar fungi. Persoonia-Molecular Phylogeny and Evolution of Fungi 42: 205-227.; Jayawardena et al. 2020Jayawardena RS, Hyde KD, Chen YJ et al. 2020. One stop shop IV: Taxonomic update with molecular phylogeny for important phytopathogenic genera: 76-100. Fungal Diversity 103: 87-218.). Later, the endophytic isolates were subcultured for DNA extraction and sequence analyses.

We deposited representative cultures of the endophytic isolates in the culture collection Micoteca URM Profa. Maria Auxiliadora Cavalcanti at the Universidade Federal de Pernambuco (UFPE), Recife, Brazil, and the permanent microscopic slides (holotype) of the new species in the herbarium at the Universidade Federal de Goiás (UFG), Goiânia, Brazil. In addition, we deposited all isolates in the working collection of the Laboratório de Micologia Ambiental at the UFPE and the ex-type strain of the new species in the working collection FCCUFG housed at the Laboratório de Micologia of the Instituto de Patologia Tropical e Saúde Pública of the UFG.

Morphological analysis of the new species

Endophytic isolates were cultured on malt extract agar (MEA) and PDA and incubated for two weeks at 28 °C in the dark. Colony colours were analysed using the Rayner colour chart (Rayner 1970Rayner RW. 1970. A mycological colour chart. Kew, Commonwealth Mycological Institute and British Mycological Society.). Lactic acid (60-80 %) was used as the mounting fluid to observe the micromorphological features. Macromorphological characteristics of the colonies were observed and described in two weeks. Micromorphological observations (such as conidiogenous cells and conidia) were performed after sporulation of the isolates on PDA for approximately 45 days, with the inclusion of sterile plant tissue in the agar plates (Hernández-Restrepo et al. 2019Hernández-Restrepo M, Bezerra JDP, Tan YP et al. 2019. Re-evaluation of Mycoleptodiscus species and morphologically similar fungi. Persoonia-Molecular Phylogeny and Evolution of Fungi 42: 205-227.).

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from seven-day cultures of endophytic fungi grown on PDA using the Wizard® SV Genomic DNA Purification System (Promega) Extraction Kit, following the manufacturer's instructions. Sequences of internal transcribed spacer (ITS) and intervening 5.8S rRNA, large subunit (LSU) rDNA, RNA polymerase II second largest subunit (rpb2), β-tubulin (tub2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using the primers ITS1/ITS4 or ITS5/ITS4 (White et al. 1990White TJ, Bruns T, Lee S et al. 1990. Amplification and direct sequencing of fungal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds.). PCR protocols: A guide to methods and applications. San Diego, Academic Press. p. 315-322.), LR0R/LR5 (Vilgalys & Hester 1990Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4239-4246.; Vilgalys & Sun 1994Vilgalys R, Sun BL. 1994. Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA sequences. Proceedings of the National Academy of Sciences 91: 4599-4603.), fRPB2-5F2/fRPB2-7cR (Liu et al. 1999Liu YJ, Whelen S, Hall B. 1999. Phylogenetic relationship among ascomycetes: Evidence from an RNA Polymerase II Subunit. Molecular Biology and Evolution 16: 1799-1808.; Sung et al. 2007Sung GH, Sung JM, Hywel-Jones NL, Spatafora JW. 2007. A multi-gene phylogeny of Clavicipitaceae (Ascomycota, Fungi): Identification of localized incongruence using a combinational bootstrap approach. Molecular Phylogenetics and Evolution 44: 1204-1223.), Bt2a/Bt2b (Glass & Donaldson 1995Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes. Applied and Environmental Microbiology 61: 1323-1330.) and GDF1/GDR1 (Weir et al. 2012Weir BS, Johnston PR, Damm U. 2012. The Colletotrichum gloeosporioides species complex. Studies in Mycology 73: 115-180.), respectively.

Amplification, sequencing, and sequence editing were performed according to the methodology described by Bezerra et al. (2017)Bezerra JDP, Oliveira RJV, Paiva LM et al. 2017. Bezerromycetales and Wiesneriomycetales ord nov (class Dothideomycetes), with two novel genera to accommodate endophytic fungi from Brazilian cactus. Mycological Progress 16: 297-309. and Oliveira et al. (2020)Oliveira TG, Bezerra JD, da Silva IR et al. 2020. Diversity of endophytic fungi in the leaflets and branches of Poincianella pyramidalis, an endemic species of Brazilian tropical dry forest. Acta Botanica Brasilica 34: 755-764.. The sequences obtained in this study were deposited in the NCBI GenBank database (Table S1 Table S1. GenBank accession numbers of sequences obtained in this study. ).

Phylogenetic analyses

Phylogenetic analyses were performed using our sequences and reference sequences retrieved from the GenBank database (Hernández-Restrepo et al. 2019Hernández-Restrepo M, Bezerra JDP, Tan YP et al. 2019. Re-evaluation of Mycoleptodiscus species and morphologically similar fungi. Persoonia-Molecular Phylogeny and Evolution of Fungi 42: 205-227.; Hyde et al. 2020Hyde KD, Dong Y, Phookamsak R et al. 2020. Fungal diversity notes 1151-1276: Taxonomic and phylogenetic contributions on genera and species of fungal taxa. Fungal Diversity 100: 5-277.). The sequences were aligned using the MAFFT v.7 online interface (Katoh & Standley 2013Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30: 772-780.) and manually edited with the MEGA v.7 software (Kumar et al. 2016Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.).

For fungal sequence analyses, we built alignments for each fungal genus and performed a maximum likelihood (ML) analysis (data not shown) following the instructions of Bezerra et al. (2017)Bezerra JDP, Oliveira RJV, Paiva LM et al. 2017. Bezerromycetales and Wiesneriomycetales ord nov (class Dothideomycetes), with two novel genera to accommodate endophytic fungi from Brazilian cactus. Mycological Progress 16: 297-309.. For the phylogenetic inference of the new species, Bayesian inference (BI) and ML analyses were conducted using MrBayes v. 3.2.7a on XSEDE and RAxML-HPC BlackBox v. 8.2.12, respectively, in the CIPRES Science Portal (Miller et al. 2010Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES science gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments workshop (GCE). p. 1-8.; Ronquist et al. 2012Ronquist F, Teslenko M, Van Der Mark P et al. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539-542.; Stamatakis 2014Stamatakis A. 2014. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.). BI analysis was conducted with 1 × 106 generations and a burning value of 25 %, with chains sampled every 1000 generations, and ML analysis was conducted with 1000 bootstrap replicates. The best nucleotide model for BI analysis was estimated using MrModelTest v.2.3 software (Nylander 2004Nylander JAA. 2004. Modeltest v2. Program distributed by the author. Uppsala, Evolutionary Biology Centre, Uppsala University.). The resulting phylogenetic trees were visualised using FigTree Software (Rambaut 2012Rambaut A. 2012. FigTree version 1.4.0. http://treebioedacuk/software/figtree/. 10 Jan. 2021.
http://treebioedacuk/software/figtree/...
). Values equal to or greater than 0.95 BI posterior probability (PP) and 70 % ML bootstrap support (ML-BS) were shown near the nodes. The alignment of Muyocopron was deposited in TreeBASE (Study ID S30510).

Data analyses

The endophytic colonisation rate (CR) of the fragments was calculated as the ratio of the number of fungal growth fragments (Nf) to the total number of fragments (Nt), that is CR = (Nf/Nt × 100) (Araújo et al. 2002Araújo WL, Lima ADS, Azevedo JL, Marcon J, Sobral JK, Lacava PT. 2002. Manual: isolamento de microrganismos endofíticos. Piracicaba, CALQ.). The total number of endophytes isolated was considered as absolute frequency, and the ratio of the number of isolates of each species to the total number of isolates gave the relative frequency (Photita et al. 2001Photita W, Lumyong S, Lumyong P, Hyde KD. 2001. Endophytic fungi of wild banana (Musa acuminata) at Doi Suthep Pui National Park, Thailand. Mycological Research 105: 1508-1513.).

Only endophytic isolates identified at the genus level were included in the ecological analyses. Richness (S) and Shannon-Wiener (H′) indices were estimated for each sample (individuals M1, M2, M3, and M4). Richness was defined as the number of species in each sample. Shannon-Wiener index was calculated based on the equation H′ = −∑ Ni ln(Ni) (Shannon & Weaver 1949Shannon C, Weaver W. 1949. The mathematical theory of communication. Urbana, Chicago, University of Illinois Press.). All the analyses were performed using the software R v. 4.1.0 (R Core Team 2021R Core Team. 2021. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. 10 Jan. 2021.
https://www.R-project.org/...
) under ‘vegan’, ‘agricolae’, and ‘lsmeans’ R packages (Lenth 2016Lenth RV. 2016. Least-squares means: The R Package lsmeans. Journal of Statistical Software 69: 33.; Oksanen et al. 2020Oksanen JFG, Blanchet M, Friendly R et al. 2020. Vegan: Community Ecology Package (R package Version 2.5-5). https://CRAN.R-project. org/package¼ vegan. 9 Feb. 2022.
https://CRAN.R-project. org/package¼ veg...
; Mendiburu 2021Mendiburu F. 2021. Agricolae: Statistical procedures for agricultural research. R package version 1.3-5. https://CRAN.R-project.org/package=agricolae. 9 Feb. 2022.
https://CRAN.R-project.org/package=agric...
). To determine the species richness, abundance, and diversity indices, we used the functions ‘biodiversity’, ‘specpool’, and ‘colSums’, respectively. Additionally, for Jaccard cluster analysis, the function ‘vegdist’ under the R package ‘vegan’ was used. Venn diagrams were created using the online software InteractiVenn (Heberle et al. 2015Heberle H, Meirelles GV, da Silva FR et al. 2015. InteractiVenn: A web-based tool for the analysis of sets through Venn diagrams. BMC Bioinformatics 16: 169.). To verify the sufficiency of the sampling effort based on the observed richness, curves of the accumulation and extrapolation of endophytic species were determined using iNEXT online (Chao et al. 2016Chao A, Ma KH, Hsieh TC. 2016. Inext (iNterpolation and EXTrapolation) Online: Software for interpolation and Extrapolation of species diversity. Code 30043: 1-14.).

Results

Fungal endophytes

In total, 168 leaf fragments of M. mirabilis were colonised (CR = 93.33 %), and 285 endophytic fungi were isolated. Twelve of the isolates did not grow after subculturing, and 273 endophytes were identified based on their morphology and DNA sequence analyses (Table S1 Table S1. GenBank accession numbers of sequences obtained in this study. ). We were not successful in the morphological analysis and DNA amplification of 104 isolates, most of which were tentatively grouped as xylariaceous fungi (83 isolates), therefore, these isolates were not included in our ecological analyses. The isolates identified at in some specific level were considered and organized within the four samples (M1-M4) that represented each individual plant collected. Twenty-five isolates were only identified at the order level (Xylariales) and one at the family rank (Sporormiaceae) because they were phylogenetically distinct from the other isolates and DNA sequences included in our analyses (data not shown).

Endophytic isolates belonged to eight orders of Ascomycota named: Botryosphaeriales, Diaporthales, Eurotiales, Glomerellales, Muyocopronales, Pleosporales, Trichosphaeriales, and Xylariales. The order Xylariales showed the highest number of isolates (103), followed by Glomerellales (40), Eurotiales (10), and Diaporthales (9). Endophytes of other orders (such as Botryosphaeriales and Muyocopronales) were also obtained but with a low isolation frequency (Table 1). Two isolates of Muyocopron (Muyocopronaceae, Muyocopronales) were described as new species.

Table 1
Absolute (f) and relative (fr) frequency of endophytic fungi isolated from Miconia mirabilis leaves in an area of the Atlantic Forest, Brazil.

Diversity and richness of endophytes

Forty taxa comprised the total richness of endophytic fungi associated with the leaves of M. mirabilis. They were distributed in eight orders and ten families of Ascomycota. The species richness observed for each individual of M. mirabilis did not differ , with M1 (S = 15), M2 (S = 17), M3 (S = 17), and M4 (S = 18); however, the diversity of endophytic fungi based on the Shannon index (H′ = 2.79) showed substantial differences, with M3 (H′ = 2.55) being the most diverse among the individuals studied (Table 2).

Table 2
Richness and Diversity based on Shannon-Wiener indices of endophytic fungi from leaves of Miconia mirabilis in an area of the Atlantic Forest, Brazil.

Species accumulation curve and sampling sufficiency

Based on Simpson's diversity, two species accumulation and extrapolation curves were constructed: one for the four individuals of M. mirabilis and the other for the endophytic fungi isolated from M. mirabilis leaves. The sampling sufficiency curve in each case did not reach a plateau; however, by observing each sampled individual, the sampling effort was sufficient to recover 86 % of the expected richness in M1, 81 % in M4, 71 % in M2, and 72 % in M3 (Fig. 2A). In total, the sampling effort was sufficient to recover 88 % of the estimated richness of endophytic fungi in M. mirabilis leaves (Fig. 2A-B).

Figure 2
Taxa accumulation and extrapolation curve, based on Simpsons Diversity, for endophytic fungi recovered from leaves of Miconia mirabilis in an area of the Atlantic Forest, Brazil (A) and taxa accumulation and extrapolation curve, based on Simpsons Diversity, for the four individuals of Miconia mirabilis in an area of the Atlantic Forest, Brazil (B).

Total abundance of species

Based on the number of species, the total abundance of the species index was determined as the sum of all sampled areas. The results showed that Xylaria cf. cubensis was the most abundant species, followed by Colletotrichum karstii of the C. boninense species complex, Colletotrichum sp. 4 of the C. gloeosporioides species complex, and Xylariales sp. 1. In contrast, Diaporthe ueckerae showed the lowest abundance index; however, it is important to highlight that Diaporthe was one of the most specious genus. In addition, an abundance chart was created for each individual to better understand the distribution of abundance among the samples. There were small differences in the total abundance of species, with M1 being the most abundant, followed by M4, M2, and M3 (Fig. 3).

Figure 3
Abundance of endophytic fungi species, according to ascending order, per individual studied in Miconia mirabilis leaves of in an area of the Atlantic Forest, Brazil.

Composition of the sampled individuals

A comparison of the taxa distribution revealed that only C. karstii of the C. boninense species complex, Colletotrichum sp. 4 of the C. gloeosporioides species complex, Xylaria cf. cubensis, and Xylariales sp. 4 were shared among the sampled Miconia individuals. Notably, M3 and M4 shared five taxa (Xylariales sp. 5, Xylariales sp. 6, Xylariales sp. 3, Xylaria cf. arbuscula, and Diaporthe sp. 1), and M1 and M2 shared two species (Xylaria cf. curta and Nemania cf. abortiva). M1-M2-M3 and M1-M3-M4 shared only one species, Xylaria cf. multiplex and Xylariales sp. 1, respectively (Fig. 4A). Moreover, some species occurred exclusively in a single sampled individual: M1 (seven species), M2 (seven species), M3 (four species), and M4 (six species). The new species of Muyocopron occurred in only one individual (M2), and Penicillium alagoense, a species recently described from M. mirabilis as an endophyte (Crous et al. 2019Crous PW, Carnegie AJ, Wingfield MJ et al. 2019. Fungal Planet description sheets: 868-950. Persoonia: Molecular Phylogeny and Evolution of Fungi 42: 291.), was isolated from two individuals (M2 and M4). Because the studied individuals shared a small number of species, the Jaccard cluster analysis method was used to investigate the similarity of the sampled individuals of M. mirabilis. Considering the species distribution, two main clades (Fig. 4B) were observed. The individuals M1-M2 similarity of 0.720 and M3-M4 a similarity of 0.781.

Figure 4
Venn diagram showing the richness of taxa of endophytic fungi from leaves of four individuals of Miconia mirabilis (exclusive and shared) in an area of the Atlantic Forest, Brazil (A) and Jaccard similarity index of the four individuals collected from Miconia mirabilis leaves in an area of the Atlantic Forest, Brazil. P = 0.96 (B).

New species

The sequences of the endophytic fungi URM 8306 = FCCUFG 22 and URM 8724 = FCCUFG 23 were compared with corresponding sequences using the BLASTn tool searches, demonstrated their relationship with Muyocopron species (Muyocopronaceae, Muyocopronales). The ITS sequences had low similarity with those of M. ficinum MFLU 18-2515 (92.81 %), the LSU sequence had high similarity with that of M. geniculatum CBS 721.95 (98.12 %), and the rpb2 sequence had low identity to M. atromaculans MUCL 34983 (87.64 %). The matrix of Muyocopron sequences consisted of 18 species and two outgroup sequences (Table S2 Table S2. GenBank accession numbers of sequences of Muyocopronaceae species used in the phylogenetic analyses. ), and the combined alignment contained 1516 characters (ITS = 686 and LSU = 830), including gaps. The GTR + I + G model was used for all ML analyses. For the BI analysis GTR + G and GTR + I + G models were used for of ITS and LSU, respectively.

Phylogenetic analyses using an individual dataset for each gene (data not shown) and a combined ITS-LSU matrix showed that the sequences of the new species belong to an independent lineage related to M. atromaculans and M. ficinum (ML-BS = 100 and PP = 1). (Fig. 5).

Figure 5
Bayesian phylogenetic tree based on a combined of LSU and ITS rDNA dataset of species included in Muyocopron. The new species described in this study (Muyocopron lulasilvae) is highlighted in red. Ex-type strains are in bold. Posterior probabilities from BI above 0.95 and ML bootstrap support values above 70 % are shown near nodes. The tree was rooted to Mycoleptodiscus terrestris CBS 231.53 and Neomycoleptodiscus venezuelense CBS 100519.

Taxonomy

Classification: Muyocopronaceae, Muyocopronales, Dothideomycetes

Muyocopron lulasilvae L.O. Ferro, Souza-Motta & J.D.P. Bezerra, sp. nov.Fig. 6

Figure 6
Muyocopron lulasilvae UFG 34288 (holotype). A. Conidiomata sporodochium-like. B-M. Conidiogenous cells and conidia. Scale bars: 10 μm.

Type: Brazil, Alagoas state, Quebrangulo municipality, Reserva Biológica de Pedra Talhada, 09°15'26.8"S and 36°25'53.7"W, as an endophyte from the leaves of Miconia mirabilis (Melastomataceae), July 2018, L.O. Ferro (Holotype Herbarium UFG 34288), ex-type culture in Micoteca URM 8306 = FCCUFG 22. MycoBank number: MB 849193.

Description: Asexual morph: Hyphae septate, branched, smooth, hyaline, pale brown and dark brown, 3-5.5(-8) µm wide. Conidiomata sporodochium-like, superficial, varying from a few combined cells to large aggregations, variable shape and size, pale to dark brown. Conidiogenous cells globose to ampulliform, smooth, with a collarette, pale to dark brown, (13.5-)16-19 × (8-)13(-15) µm. Conidia aseptate, lunate, curved, hyaline, smooth, guttulate, 16(-20) × 8(-11) µm. Appressoria not observed. Sexual morph not observed.

Culture characters: The colonies on MEA reached 85 mm diameter after 2 weeks at 28 °C, aerial mycelium was scarce, brown, reverse without pigmentation. On PDA, colonies reached 67 mm diameter after 2 weeks at 28 °C, and had concentric circles, greyish, brown at the centre, and entire margin of purple colour; reverse orange to brown, without pigment. After approximately 45 days, on PDA, few reproductive structures were observed on the plant tissue surface.

Distribution and habitat: Endophytic fungus in the leaves of Miconia mirabilis found in the Atlantic Forest, Brazil.

Etymology: The name refers to Luiz Inácio Lula da Silva, the 39th President of Brazil, elected when the new species was described.

GenBank numbers: URM 8306 = FCCUFG 22, ITS = OR147103, LSU = OR157961, and RPB2 = OR413339. URM 8724 = FCCUFG 23, ITS = OR147104, LSU = OR157962, and RPB2 = OR413340.

Other materials examined: Brazil, Alagoas state, Quebrangulo municipality, Reserva Biológica de Pedra Talhada, 09°15'26.8"S and 36°25'53.7"W, as an endophyte from the leaves of Miconia mirabilis (Melastomataceae), July 2018, L.O. Ferro (URM 8724 = FCCUFG 23).

Notes: Muyocopron lulasilvae showed phylogenetic similarities with M. atromaculans and M. ficinum. Our new species differs from M. atromaculans, which was isolated as an endophyte in the USA, by having larger conidiogenous cells (13.5-19 × 8-15 µm in M. lulasilvae and 9.5-14.5 µm diameter in M. atromaculans) and conidia with no prominent appendages in M. lulasilvae and with terminal appendages in M. atromaculans (Hernández-Restrepo et al. 2019Hernández-Restrepo M, Bezerra JDP, Tan YP et al. 2019. Re-evaluation of Mycoleptodiscus species and morphologically similar fungi. Persoonia-Molecular Phylogeny and Evolution of Fungi 42: 205-227.). Muyocopron lulasilvae also differs from M. ficinum, reported as a decomposer of dead leaves in China, which is known only based on the sexual morph (Tennakoon et al. 2021Tennakoon DS, Kuo CH, Maharachchikumbura SS et al. 2021. Taxonomic and phylogenetic contributions to Celtis formosana, Ficus ampelas, F. septica, Macaranga tanarius and Morus australis leaf litter inhabiting microfungi. Fungal Diversity 108: 215.).

Discussion

Tropical forests are important hotspots of microbial diversity, which is estimated to account more than half of the terrestrial biodiversity (Lewis et al. 2015Lewis SL, Edwards DP, Galbraith D. 2015. Increasing human dominance of tropical forests. Science 349: 827-832.). In the early 20th century, researchers began to investigate whether these tropical areas are great reservoirs of endophytic fungal diversity (Arnold et al. 2000Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA. 2000. Are tropical fungal endophytes hyperdiverse? Ecology Letters 3: 267-274.; Arnold & Lutzoni 2007Arnold AE, Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology 88: 541-549.). However, the ecological role of endophytic fungi and plant microbiome in these regions remains poorly understood. Several plants and biomes with high endophytic diversity have been investigated in an attempt not only to explore and increase fungal estimates, but also to elucidate the ecological patterns behind this diversity (Oita et al. 2021Oita S, Ibáñez A, Lutzoni F et al. 2021. Climate and seasonality drive the richness and composition of tropical fungal endophytes at a landscape scale. Communications Biology 4: 313. ). To the best of our knowledge, ours is the first study to report the endophytic fungal diversity in the leaves of M. mirabilis growing in the Atlantic Forest, Brazil.

In our study, 285 endophytic fungi were isolated from 93.33 % of the colonised fragments. The high rate of endophytic colonisation may be associated with the properties of rainforests (Arnold et al. 2000Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA. 2000. Are tropical fungal endophytes hyperdiverse? Ecology Letters 3: 267-274.). We obtained a similar number of fungal isolates compared with that of other studies on endophytes from rainforests (Arnold & Lutzoni 2007Arnold AE, Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology 88: 541-549.; Lima et al. 2012Lima JS, Figueiredo JG, Gomes RG et al. 2012. Genetic diversity of Colletotrichum spp. an endophytic fungi in a medicinal plant, Brazilian pepper tree. International Scholarly Research Notices 2012: 215716.; Nascimento et al. 2015Nascimento TL, Oki Y, Lima DMM, Almeida-Cortez JS, Wilson-Fernandes G, Souza-Motta CM. 2015. Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity. Fungal Ecology 14: 79-86.; Bonfim et al. 2016Bonfim JA, Vasconcellos RLF, Baldesin LF, Sieber TN, Cardoso EJBN. 2016. Dark septate endophytic fungi of native plants along an altitudinal gradient in the Brazilian Atlantic forest. Fungal Ecology 20: 202-210.; Correia et al. 2017Correia AM, Lira SP, Assis MA, Rodrigues A. 2017. Fungal endophyte communities in Begonia species from the Brazilian Atlantic rainforest. Current Microbiology 75: 441-449.). However, the number of individuals and leaf fragments used to isolate the endophytes was lower than those of other studies. Generally, the richness and colonisation rate of endophytic fungi may not be ideal for full comparison, when we take into consideration the complexity of the endophytic microbiota, types of isolation methods, as well as the type of plant tissue and the effort of sampling (Cannon & Simmons 2002Cannon PF, Simmons CM. 2002. Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana. Mycologia 94: 210-220.; Vieira et al. 2011Vieira PDDS, Souza-Motta CM, Lima D et al. 2011. Endophytic fungi associated with transgenic and non-transgenic cotton. Mycology 2: 91-97.).

The endophytic fungi were identified as belonging to 10 families, eight orders, and three classes (Dothideomycetes, Eurotiomycetes, and Sordariomycetes) of Ascomycota. The number of classes was similar to that presented by Arnold and Lutzoni (2007)Arnold AE, Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology 88: 541-549., who showed that endophytes from high latitudes belonged to few species and several classes of Ascomycota, whereas endophytic communities from tropical areas had several species within few classes of this phylum. Xylariales was the most abundant order among the endophytic fungi identified in this study, similar results can be seen in other studies and this order seems to prefer humid environments (Arnold & Lutzoni 2007Arnold AE, Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology 88: 541-549.; Daranagama et al. 2018Daranagama DA, Hyde KD, Sir EB et al. 2018. Towards a natural classification and backbone tree for Graphostromataceae, Hypoxylaceae, Lopadostomataceae and Xylariaceae. Fungal Diversity 88: 165.; Zhou et al. 2019Zhou H, Abuduaini A, Xie H, Kang R, Suo F, Huang L. 2019. The complete mitochondrial genome of wood-rotting fungus Xylaria hypoxylon. Mitochondrial DNA B Resource 4: 3848-3849.). Also, Xylariales is one of the most studied orders of filamentous fungi owing to its worldwide distribution and potential to produce bioactive compounds (Ma et al. 2022Ma X, Chomnunti P, Doilom M, Daranagama DA, Kang J. 2022. Multigene Phylogeny Reveals Endophytic Xylariales Novelties from Dendrobium Species from Southwestern China and Northern Thailand. Journal of Fungi 8: 248.). Xylaria cf. cubensis was the most frequently isolated taxon, similar to that in other studies (Davis et al. 2003Davis C, Franklin JB, Shaw AJ. 2003. Biogeographic and phylogenetic patterns in diversity of liverwort-associated endophytes. American Journal of Botany 90: 1661-1667.; Costa et al. 2012Costa IP, Maia LC, Cavalcanti MA. 2012. Diversity of leaf endophytic fungi in mangrove plants of northeast Brazil. Brazilian Journal of Microbiology 43: 1165-1173.; Chen et al. 2013Chen J, Zhang LC, Xing YM et al. 2013. Diversity and taxonomy of endophytic xylariaceous fungi from medicinal plants of Dendrobium (Orchidaceae). PLoS One 8: e58268.; Correia et al. 2017Correia AM, Lira SP, Assis MA, Rodrigues A. 2017. Fungal endophyte communities in Begonia species from the Brazilian Atlantic rainforest. Current Microbiology 75: 441-449.). Several studies have attempted to explain the emergence of xylariaceous fungi as endophytes. For example, Petrini (1995)Petrini O. 1995. Xylariaceous endophytes: An exercise in biodiversity. Fitopatologia Brasileira 20: 531-539. observed that the role of these fungi could be linked to the decomposition of cellulose and lignin when plants begin to senesce, and since the past X. cf. cubensis is broadly known and studied as saprobe (Rogers 1984Rogers JD. 1984. Xylaria cubensis and its anamorph Xylocoremium flabelliforme, Xylaria allantoidea, and Xylaria poitei in continental United States. Mycologia 76: 912-923.). In our study, the high prevalence of these fungi could be associated with the plant and environment from which they were collected. Miconia mirabilis is a common plant species in the degraded areas of humid tropical forests and has been used in the reforestation process (Pessoa et al. 2012Pessoa MDS, Vleeschouwer KMD, Talora DC, Rocha L, Amorim AMA. 2012. Reproductive phenology of Miconia mirabilis (Melastomataceae) within three distinct physiognomies of Atlantic Forest, Bahia, Brazil. Biota Neotropica 12: 49-56.).

Some fungal genera reported as pathogenic fungi can also be isolated as endophytes (Suryanarayanan et al. 2005Suryanarayanan TS, Wittlinger SK, Faeth SH. 2005. Endophytic fungi associated with cacti in Arizona. Mycological Research 109: 635-639.; Hyde & Soytong 2008Hyde KD, Soytong K. 2008. The fungal endophyte dilemma. Fungal Diversity 33: 173.; Hardoim et al. 2015Hardoim PR, Overbeek LSV, Berg G et al. 2015. The hidden world within plants: Ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiology and Molecular Biology Reviews 79: 293-320.) and were isolated from M. mirabilis, belonging mainly to Glomerellales and Diaporthales. As a representative of a latent pathogens, Colletotrichum was the second most abundant genus in our study and has already been reported associated with several plant species (Siqueira et al. 2011Siqueira VM, Conti R, Araújo JM, Souza-Motta CM. 2011. Endophytic fungi from the medicinal plant Lippia sidoides Cham. and their antimicrobial activity. Symbiosis 53: 89-95.; Mussi-Dias et al. 2012Mussi-Dias V, Araújo ACO, Silveira SF, Rocabado JMA, Araújo KL. 2012. Fungos endofíticos associados a plantas medicinais. Revista Brasileira de Plantas Medicinais 14: 261-266.; Chow & Ting 2015Chow Y, Ting AS. 2015. Endophytic L-asparaginase-producing fungi from plants associated with anticancer properties. Journal of Advanced Research 6: 869-876.; Singh et al. 2017Singh DK, Sharma VK, Kumar J et al. 2017. Diversity of endophytic mycobiota of tropical tree Tectona grandis Linn. f.: Spatiotemporal and tissue type effects. Scientific Reports 7: 3745.).

Other abundantly isolated endophytes include species of Diaporthe, a cosmopolitan genus usually found as saprobes, phytopathogens, and endophytically in plants of different environments (Hilarino et al. 2011Hilarino MPA, Oki Y, Rodrigues L et al. 2011. Distribution of the endophytic fungi Community in leaves of Bauhinia brevipes (Fabaceae). Acta Botanica Brasilica 25: 815-821.; Suwannarach et al. 2012Suwannarach N, Bussaban B, Nuangmek W, McKenzie EH, Hyde KD, Lumyong S. 2012. Diversity of endophytic fungi associated with Cinnamomum bejolghota (Lauraceae) in Northern Thailand. Chiang Mai Journal of Science 39: 389-398. ; Dissanayake et al. 2017Dissanayake AJ, Phillips AJL, Hyde KD, Yan JY, Li XH. 2017. The current status of species in Diaporthe. Mycosphere 8: 1106-1156.; Pádua et al. 2018Pádua APSL, Freire KTLS, Oliveira TGL et al. 2018. Fungal endophyte diversity in the leaves of the medicinal plant Myracrodruon urundeuva in a Brazilian dry tropical forest and their capacity to produce L-asparaginase. Acta Botanica Brasilica 33: 39-49.). Although Diaporthe proved to be one of the most specious genera of the study, D. ueckerae showed the lowest abundance, with only one isolate. Silva et al. (2022)Silva LF, Pádua APSL, Ferro LO et al. 2022. Cacti as low-cost substrates to produce L-asparaginase by endophytic fungi. World Journal of Microbiology and Biotechnology 38: 247. testing the production of L-asparaginase enzyme from different genus of endophytic fungi, presented as the best producer the Diaporthe isolate URM 8321 obtained originally in this study.

Isolates with lower abundance had only one or two representatives of each taxon, considered as rare taxa. These rare taxa are commonly found in endophytic studies, and this reduced number may be associated with the predominance of other genera in the endophytic community (Siqueira et al. 2011Siqueira VM, Conti R, Araújo JM, Souza-Motta CM. 2011. Endophytic fungi from the medicinal plant Lippia sidoides Cham. and their antimicrobial activity. Symbiosis 53: 89-95.; Dos Santos et al. 2015Dos Santos IP, Bezerra JDP, Souza-Motta CM, Cavalcanti MS, Lima VLM. 2015. Endophytic mycobiota from leaves of Indigofera suffruticosa Miller (Fabaceae): The relationship between seasonal change in Atlantic Coastal Forest and tropical dry forest (Caatinga), Brazil. African Journal of Microbiology Research 9: 1227-1235.). Another common fact in endophytic community studies is the discovery of new taxa (Bezerra et al. 2017Bezerra JDP, Oliveira RJV, Paiva LM et al. 2017. Bezerromycetales and Wiesneriomycetales ord nov (class Dothideomycetes), with two novel genera to accommodate endophytic fungi from Brazilian cactus. Mycological Progress 16: 297-309.; 2019Bezerra JD, Padua APSL, Oliveira TG et al. 2019. Pseudoplagiostoma myracrodruonis (Pseudoplagiostomataceae, Diaporthales): A new endophytic species from Brazil. Mycological Progress 18: 1329-1339.; Cavalcanti et al. 2020Cavalcanti AD, Silva ACS, Ferro LO, Bezerra JD, Souza-Motta CM, Magalhães OMC. 2020. Fusarium massalimae sp. nov. (F. lateritium species complex) occurs endophytically in leaves of Handroanthus chrysotrichus. Mycological Progress 19: 1133-1142.). Penicillium alagoense, isolated in the present study was previously described as a new species (Crous et al. 2019Crous PW, Carnegie AJ, Wingfield MJ et al. 2019. Fungal Planet description sheets: 868-950. Persoonia: Molecular Phylogeny and Evolution of Fungi 42: 291.) and Muyocopron lulasilvae is described here as a new endophytic species. Furthermore, the remaining possible novelties might be investigated in the future.

In this study we only analysed four individuals, however the ecological indices were relevant and showed good distribution and diversity of the endophytic fungi of M. mirabilis in the studied area. The richness observed in this study was similar to that reported by Correia et al. (2017)Correia AM, Lira SP, Assis MA, Rodrigues A. 2017. Fungal endophyte communities in Begonia species from the Brazilian Atlantic rainforest. Current Microbiology 75: 441-449., who analysed three species of Begonia in the Atlantic Forest (São Paulo state, Brazil). They found 71 morphospecies, and after phylogenetic analyses, they obtained a richness of 46 taxa with a Shannon index below that of our study (H′ = 1.41). In another study in the Atlantic Forest with plants of the same order (Myrtales) as that of M. mirabilis, similar Shannon indices ranging from 2.25 to 2.39 were found (Vaz et al. 2014Vaz ABM, Fontenla S, Rocha FS et al. 2014. Fungal endophyte β-diversity associated with Myrtaceae species in an Andean Patagonian forest (Argentina) and an Atlantic forest (Brazil). Fungal Ecology 8: 28-36.). In contrast to endophytic diversity in the Atlantic Forest, studies in the Caatinga forest showed lower diversity indices (Bezerra et al. 2015Bezerra JDP, Nascimento CCF, Barbosa RN et al. 2015. Endophytic fungi from medicinal plant Bauhinia forficata: Diversity and biotechnological potential. Brazilian Journal of Microbiology 46: 49-57.; Oliveira et al. 2020Oliveira TG, Bezerra JD, da Silva IR et al. 2020. Diversity of endophytic fungi in the leaflets and branches of Poincianella pyramidalis, an endemic species of Brazilian tropical dry forest. Acta Botanica Brasilica 34: 755-764.; Araújo-Magalhães et al. 2021Araújo-Magalhães GR, Maciel MHS, Silva LF et al. 2021. Fungal endophytes from leaves of Mandevilla catimbauensis (Apocynaceae): Diversity and potential for L-asparaginase production. Brazilian Journal of Microbiology 52: 1431-1441.); however, Pádua et al. (2018)Pádua APSL, Freire KTLS, Oliveira TGL et al. 2018. Fungal endophyte diversity in the leaves of the medicinal plant Myracrodruon urundeuva in a Brazilian dry tropical forest and their capacity to produce L-asparaginase. Acta Botanica Brasilica 33: 39-49. recorded a higher diversity estimation (H′ = 3.41) than our study when analysing the ‘Brejo de altitude’, an isolated fragment of humid forest (Atlantic Forest) found in the Caatinga forest. Although 88 % richness was recovered, more samples are needed to estimate the actual diversity of endophytic fungi. However, this pattern has been frequently observed in other studies of endophytic fungal communities (Gazis & Chaverri 2010Gazis R, Chaverri P. 2010. Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis ) in Peru. Fungal Ecology 3: 240-254.; Correia et al. 2017Correia AM, Lira SP, Assis MA, Rodrigues A. 2017. Fungal endophyte communities in Begonia species from the Brazilian Atlantic rainforest. Current Microbiology 75: 441-449.; Pádua et al. 2018Pádua APSL, Freire KTLS, Oliveira TGL et al. 2018. Fungal endophyte diversity in the leaves of the medicinal plant Myracrodruon urundeuva in a Brazilian dry tropical forest and their capacity to produce L-asparaginase. Acta Botanica Brasilica 33: 39-49.).

Our data provide the first insight into the endophytic community of M. mirabilis and may complement previous works on factors that determine endophytic assemblage in tropical forests, such as the Atlantic Forest, Brazil. The studied microbiome, which is part of an entire ecosystem, will help in understanding the complexity of the ecological interactions. Our results reinforce the urge for conducting studies on mycobiome in environments with high biodiversity in order to highlight the need of conservation of protected areas.

Acknowledgements

We thank the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE, processes IBPG-0104-2.03/20, APQ-0003-2.03/18, and APQ-0350-2.12/19), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process 421241/2017-9), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001), the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), and the teams of the Micoteca URM and the Laboratório de Micologia Ambiental/UFPE for their support. C.M. Souza-Motta and J.D.P. Bezerra have a fellowship from CNPq.

References

  • Araújo WL, Lima ADS, Azevedo JL, Marcon J, Sobral JK, Lacava PT. 2002. Manual: isolamento de microrganismos endofíticos. Piracicaba, CALQ.
  • Araújo-Magalhães GR, Maciel MHS, Silva LF et al 2021. Fungal endophytes from leaves of Mandevilla catimbauensis (Apocynaceae): Diversity and potential for L-asparaginase production. Brazilian Journal of Microbiology 52: 1431-1441.
  • Arnold AE, Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology 88: 541-549.
  • Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA. 2000. Are tropical fungal endophytes hyperdiverse? Ecology Letters 3: 267-274.
  • Baldrian P. 2017. Forest microbiome: Diversity, complexity and dynamics. FEMS Microbiology reviews 4: 109-130.
  • Barnett HL, Hunter BB. 1987. Illustrated Genera of Imperfect Fungi. 4th. edn. New York, MacMillan Publishing Company.
  • Baucom DL, Romero M, Belfon R, Creamer R. 2012. Two new species of Undifilum, fungal endophytes of Astragalus (locoweeds) in the United States. Botany 90: 866-875.
  • Bernard E, Penna, LAO, Araújo E. 2014. Downgrading, downsizing, degazettement, and reclassification of protected areas in Brazil. Conservation Biology 28: 939-950.
  • Bezerra JD, Padua APSL, Oliveira TG et al 2019. Pseudoplagiostoma myracrodruonis (Pseudoplagiostomataceae, Diaporthales): A new endophytic species from Brazil. Mycological Progress 18: 1329-1339.
  • Bezerra JDP, Nascimento CCF, Barbosa RN et al 2015. Endophytic fungi from medicinal plant Bauhinia forficata: Diversity and biotechnological potential. Brazilian Journal of Microbiology 46: 49-57.
  • Bezerra JDP, Oliveira RJV, Paiva LM et al 2017. Bezerromycetales and Wiesneriomycetales ord nov (class Dothideomycetes), with two novel genera to accommodate endophytic fungi from Brazilian cactus. Mycological Progress 16: 297-309.
  • Bonfim JA, Vasconcellos RLF, Baldesin LF, Sieber TN, Cardoso EJBN. 2016. Dark septate endophytic fungi of native plants along an altitudinal gradient in the Brazilian Atlantic forest. Fungal Ecology 20: 202-210.
  • Brader G, Compant S, Mitter B, Trognitz F, Sessitsch A. 2014. Metabolic potential of endophytic bacteria. Current Opinion in Biotechnology 27: 30-37.
  • Cannon PF, Simmons CM. 2002. Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana. Mycologia 94: 210-220.
  • Cavalcanti AD, Silva ACS, Ferro LO, Bezerra JD, Souza-Motta CM, Magalhães OMC. 2020. Fusarium massalimae sp. nov. (F. lateritium species complex) occurs endophytically in leaves of Handroanthus chrysotrichus Mycological Progress 19: 1133-1142.
  • Chao A, Ma KH, Hsieh TC. 2016. Inext (iNterpolation and EXTrapolation) Online: Software for interpolation and Extrapolation of species diversity. Code 30043: 1-14.
  • Chen J, Zhang LC, Xing YM et al 2013. Diversity and taxonomy of endophytic xylariaceous fungi from medicinal plants of Dendrobium (Orchidaceae). PLoS One 8: e58268.
  • Chow Y, Ting AS. 2015. Endophytic L-asparaginase-producing fungi from plants associated with anticancer properties. Journal of Advanced Research 6: 869-876.
  • Correia AM, Lira SP, Assis MA, Rodrigues A. 2017. Fungal endophyte communities in Begonia species from the Brazilian Atlantic rainforest. Current Microbiology 75: 441-449.
  • Costa IP, Maia LC, Cavalcanti MA. 2012. Diversity of leaf endophytic fungi in mangrove plants of northeast Brazil. Brazilian Journal of Microbiology 43: 1165-1173.
  • Crous PW, Carnegie AJ, Wingfield MJ et al 2019. Fungal Planet description sheets: 868-950. Persoonia: Molecular Phylogeny and Evolution of Fungi 42: 291.
  • Daranagama DA, Hyde KD, Sir EB et al 2018. Towards a natural classification and backbone tree for Graphostromataceae, Hypoxylaceae, Lopadostomataceae and Xylariaceae. Fungal Diversity 88: 165.
  • Davis C, Franklin JB, Shaw AJ. 2003. Biogeographic and phylogenetic patterns in diversity of liverwort-associated endophytes. American Journal of Botany 90: 1661-1667.
  • Dissanayake AJ, Phillips AJL, Hyde KD, Yan JY, Li XH. 2017. The current status of species in Diaporthe Mycosphere 8: 1106-1156.
  • Dos Santos IP, Bezerra JDP, Souza-Motta CM, Cavalcanti MS, Lima VLM. 2015. Endophytic mycobiota from leaves of Indigofera suffruticosa Miller (Fabaceae): The relationship between seasonal change in Atlantic Coastal Forest and tropical dry forest (Caatinga), Brazil. African Journal of Microbiology Research 9: 1227-1235.
  • Foden WB, Young BE. 2016. IUCN SSC guidelines for assessing species' vulnerability to climate change. Cambridge, England and Gland, Switzerland, IUCN.
  • Gazis R, Chaverri P. 2010. Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis ) in Peru. Fungal Ecology 3: 240-254.
  • Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes. Applied and Environmental Microbiology 61: 1323-1330.
  • Gomes RR, Glienke C, Videira SI, Lombard L, Groenewald JZ, Crous PW. 2013. Diaporthe: A genus of endophytic, saprobic and plant pathogenic fungi. Persoonia-Molecular Phylogeny and Evolution of Fungi 31: 1-41.
  • Hanlin RT. 1990. Illustrated Genera of Ascomycetes. St Paul, APS Press.
  • Hardoim PR, Overbeek LSV, Berg G et al 2015. The hidden world within plants: Ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiology and Molecular Biology Reviews 79: 293-320.
  • Heberle H, Meirelles GV, da Silva FR et al 2015. InteractiVenn: A web-based tool for the analysis of sets through Venn diagrams. BMC Bioinformatics 16: 169.
  • Hernández-Restrepo M, Bezerra JDP, Tan YP et al 2019. Re-evaluation of Mycoleptodiscus species and morphologically similar fungi. Persoonia-Molecular Phylogeny and Evolution of Fungi 42: 205-227.
  • Heywood VH. 2019. Conserving plants within and beyond protected areas-still problematic and future uncertain. Plant Diversity 41: 36-49.
  • Hilarino MPA, Oki Y, Rodrigues L et al 2011. Distribution of the endophytic fungi Community in leaves of Bauhinia brevipes (Fabaceae). Acta Botanica Brasilica 25: 815-821.
  • Hsieh HM, Lin CR, Fang MJ et al 2010. Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily. Molecular Phylogenetics and Evolution 54: 957-969.
  • Hyde KD, Dong Y, Phookamsak R et al 2020. Fungal diversity notes 1151-1276: Taxonomic and phylogenetic contributions on genera and species of fungal taxa. Fungal Diversity 100: 5-277.
  • Hyde KD, Soytong K. 2008. The fungal endophyte dilemma. Fungal Diversity 33: 173.
  • ICMBio - Instituto Chico Mendes de Conservação da Biodiversidade. 2022. Ministério do Meio Ambiente. https://www.gov.br/icmbio/pt-br 9 Feb. 2022.
    » https://www.gov.br/icmbio/pt-br
  • Jayawardena RS, Hyde KD, Chen YJ et al 2020. One stop shop IV: Taxonomic update with molecular phylogeny for important phytopathogenic genera: 76-100. Fungal Diversity 103: 87-218.
  • Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30: 772-780.
  • Khidir HH, Eudy DM, Porras-Alfaro A, Herrera J, Natvig DO, Sinsabaugh RL. 2010. A general suite of fungal endophytes dominate the roots of two dominant grasses in a semiarid grassland. Journal of Arid Environments 74: 35-42.
  • Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.
  • Leitman P, Amorim AM, Sansevero JBB, Forzza RC. 2015. Floristic patterns of epiphytes in the Brazilian Atlantic Forest, a biodiversity hotspot. Botanical Journal of the Linnean Society 179: 587-601.
  • Lembi RC, Cronemberger C, Picharillo C et al 2020. Urban expansion in the Atlantic Forest: Applying the nature futures framework to develop a conceptual model and future scenarios. Biota Neotropica 20: e20190904.
  • Lenth RV. 2016. Least-squares means: The R Package lsmeans. Journal of Statistical Software 69: 33.
  • Lewis SL, Edwards DP, Galbraith D. 2015. Increasing human dominance of tropical forests. Science 349: 827-832.
  • Lima JS, Figueiredo JG, Gomes RG et al 2012. Genetic diversity of Colletotrichum spp. an endophytic fungi in a medicinal plant, Brazilian pepper tree. International Scholarly Research Notices 2012: 215716.
  • Liu YJ, Whelen S, Hall B. 1999. Phylogenetic relationship among ascomycetes: Evidence from an RNA Polymerase II Subunit. Molecular Biology and Evolution 16: 1799-1808.
  • Ma X, Chomnunti P, Doilom M, Daranagama DA, Kang J. 2022. Multigene Phylogeny Reveals Endophytic Xylariales Novelties from Dendrobium Species from Southwestern China and Northern Thailand. Journal of Fungi 8: 248.
  • Mattar EPL, Barros TTV, Cunha BB, Souza JFD, Silva AMDC. 2018. Federal Conservation Units in Brazil: The situation of biomes and regions. Floresta e Ambiente 25: e20150051.
  • Mendiburu F. 2021. Agricolae: Statistical procedures for agricultural research. R package version 1.3-5. https://CRAN.R-project.org/package=agricolae 9 Feb. 2022.
    » https://CRAN.R-project.org/package=agricolae
  • Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES science gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments workshop (GCE). p. 1-8.
  • Mussi-Dias V, Araújo ACO, Silveira SF, Rocabado JMA, Araújo KL. 2012. Fungos endofíticos associados a plantas medicinais. Revista Brasileira de Plantas Medicinais 14: 261-266.
  • Nascimento TL, Oki Y, Lima DMM, Almeida-Cortez JS, Wilson-Fernandes G, Souza-Motta CM. 2015. Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity. Fungal Ecology 14: 79-86.
  • Noriler AS, Savi DC, Aluizio R, Palacio-Cortes AM, Possiede YM, Glienke C. 2018. Bioprospecting and structure of fungal endophyte communities found in the Brazilian biomes, Pantanal, and Cerrado. Frontiers in Microbiology 9: 1526.
  • Nusbaumer L, Barbosa MRV, Thomas WW, Alves MV, Loizeau PA, Spichiger R. 2015. Flora e vegetação da Reserva Biológica de Pedra Talhada. In: Studer A, Nusbaumer L, Spichiger R (eds.). Biodiversidade da Reserva Biológica de Pedra Talhada (Alagoas, Pernambuco - Brasil). Boissiera. vol. 68, p. 59-121.
  • Nylander JAA. 2004. Modeltest v2. Program distributed by the author. Uppsala, Evolutionary Biology Centre, Uppsala University.
  • Oita S, Ibáñez A, Lutzoni F et al 2021. Climate and seasonality drive the richness and composition of tropical fungal endophytes at a landscape scale. Communications Biology 4: 313.
  • Oksanen JFG, Blanchet M, Friendly R et al 2020. Vegan: Community Ecology Package (R package Version 2.5-5). https://CRAN.R-project. org/package¼ vegan 9 Feb. 2022.
    » https://CRAN.R-project. org/package¼ vegan
  • Oliveira TG, Bezerra JD, da Silva IR et al 2020. Diversity of endophytic fungi in the leaflets and branches of Poincianella pyramidalis, an endemic species of Brazilian tropical dry forest. Acta Botanica Brasilica 34: 755-764.
  • Pádua APSL, Freire KTLS, Oliveira TGL et al 2018. Fungal endophyte diversity in the leaves of the medicinal plant Myracrodruon urundeuva in a Brazilian dry tropical forest and their capacity to produce L-asparaginase. Acta Botanica Brasilica 33: 39-49.
  • Pessoa MDS, Vleeschouwer KMD, Talora DC, Rocha L, Amorim AMA. 2012. Reproductive phenology of Miconia mirabilis (Melastomataceae) within three distinct physiognomies of Atlantic Forest, Bahia, Brazil. Biota Neotropica 12: 49-56.
  • Petrini O. 1995. Xylariaceous endophytes: An exercise in biodiversity. Fitopatologia Brasileira 20: 531-539.
  • Phillips AJL, Alves A, Abdollahzadeh J et al 2013. The Botryosphaeriaceae: Genera and species known from culture. Studies in Mycology 76: 51-167.
  • Photita W, Lumyong S, Lumyong P, Hyde KD. 2001. Endophytic fungi of wild banana (Musa acuminata) at Doi Suthep Pui National Park, Thailand. Mycological Research 105: 1508-1513.
  • R Core Team. 2021. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/ 10 Jan. 2021.
    » https://www.R-project.org/
  • Rambaut A. 2012. FigTree version 1.4.0. http://treebioedacuk/software/figtree/ 10 Jan. 2021.
    » http://treebioedacuk/software/figtree/
  • Rayner RW. 1970. A mycological colour chart. Kew, Commonwealth Mycological Institute and British Mycological Society.
  • Rey T, Schornack S. 2013. Interactions of beneficial and detrimental root-colonizing filamentous microbes with plant hosts. Genome Biology 14: 121.
  • Rezende CL, Scarano FR, Assad ED et al 2018. From hotspot to hopespot: An opportunity for the Brazilian Atlantic Forest. Perspectives in Ecology and Conservation 16: 208-214.
  • Rogers JD. 1984. Xylaria cubensis and its anamorph Xylocoremium flabelliforme, Xylaria allantoidea, and Xylaria poitei in continental United States. Mycologia 76: 912-923.
  • Ronquist F, Teslenko M, Van Der Mark P et al 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539-542.
  • Rosa LH. 2021. Neotropical Endophytic Fungi. Springer International Publishing.
  • Samson RA, Frisvad JC. 2004. Penicillium Subgenus Penicillium: New Taxonomics 210 Schemes, Mycotoxins and Other Extrolites. Studies in Micology 49: 260.
  • Shannon C, Weaver W. 1949. The mathematical theory of communication. Urbana, Chicago, University of Illinois Press.
  • Silva LF, Pádua APSL, Ferro LO et al 2022. Cacti as low-cost substrates to produce L-asparaginase by endophytic fungi. World Journal of Microbiology and Biotechnology 38: 247.
  • Singh DK, Sharma VK, Kumar J et al 2017. Diversity of endophytic mycobiota of tropical tree Tectona grandis Linn. f.: Spatiotemporal and tissue type effects. Scientific Reports 7: 3745.
  • Siqueira VM, Conti R, Araújo JM, Souza-Motta CM. 2011. Endophytic fungi from the medicinal plant Lippia sidoides Cham. and their antimicrobial activity. Symbiosis 53: 89-95.
  • Stamatakis A. 2014. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.
  • Studer A, Nusbaumer L, Spichiger R (eds.). 2015. Biodiversidade da Reserva Biológica de Pedra Talhada (Alagoas, Pernambuco - Brasil). Boissiera. vol. 68.
  • Sung GH, Sung JM, Hywel-Jones NL, Spatafora JW. 2007. A multi-gene phylogeny of Clavicipitaceae (Ascomycota, Fungi): Identification of localized incongruence using a combinational bootstrap approach. Molecular Phylogenetics and Evolution 44: 1204-1223.
  • Suryanarayanan TS, Wittlinger SK, Faeth SH. 2005. Endophytic fungi associated with cacti in Arizona. Mycological Research 109: 635-639.
  • Suwannarach N, Bussaban B, Nuangmek W, McKenzie EH, Hyde KD, Lumyong S. 2012. Diversity of endophytic fungi associated with Cinnamomum bejolghota (Lauraceae) in Northern Thailand. Chiang Mai Journal of Science 39: 389-398.
  • Temperton VM, Buchamann N, Buisson E et al 2019. Step back from the forest and step up to the Bonn Challenge: How a broad ecological perspective can promote successful landscape restoration. Restoration Ecology 27: 705-719.
  • Tennakoon DS, Kuo CH, Maharachchikumbura SS et al 2021. Taxonomic and phylogenetic contributions to Celtis formosana, Ficus ampelas, F. septica, Macaranga tanarius and Morus australis leaf litter inhabiting microfungi. Fungal Diversity 108: 215.
  • Vaz ABM, Fontenla S, Rocha FS et al 2014. Fungal endophyte β-diversity associated with Myrtaceae species in an Andean Patagonian forest (Argentina) and an Atlantic forest (Brazil). Fungal Ecology 8: 28-36.
  • Vieira PDDS, Souza-Motta CM, Lima D et al 2011. Endophytic fungi associated with transgenic and non-transgenic cotton. Mycology 2: 91-97.
  • Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4239-4246.
  • Vilgalys R, Sun BL. 1994. Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA sequences. Proceedings of the National Academy of Sciences 91: 4599-4603.
  • Weir BS, Johnston PR, Damm U. 2012. The Colletotrichum gloeosporioides species complex. Studies in Mycology 73: 115-180.
  • White TJ, Bruns T, Lee S et al 1990. Amplification and direct sequencing of fungal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds.). PCR protocols: A guide to methods and applications. San Diego, Academic Press. p. 315-322.
  • Zhou H, Abuduaini A, Xie H, Kang R, Suo F, Huang L. 2019. The complete mitochondrial genome of wood-rotting fungus Xylaria hypoxylon Mitochondrial DNA B Resource 4: 3848-3849.

Publication Dates

  • Publication in this collection
    11 Dec 2023
  • Date of issue
    2023

History

  • Received
    29 Apr 2023
  • Accepted
    11 Sept 2023
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