Naming Potentially Endangered Parasites: Foliicolous Mycobiota of Dimorphandra wilsonii, a Highly Threatened Brazilian Tree Species

A survey of foliicolous fungi associated with Dimorphandra wilsonii and Dimorphandra mollis (Fabaceae) was conducted in the state of Minas Gerais, Brazil. Dimorphandra wilsonii is a tree species native to the Brazilian Cerrado that is listed as critically endangered. Fungi strictly depending on this plant species may be on the verge of co-extinction. Here, results of the pioneering description of this mycobiota are provided to contribute to the neglected field of microfungi conservation. The mycobiota of D. mollis, which is a common species with a broad geographical distribution that co-occurs with D. wilsonii, was examined simultaneously to exclude fungal species occurring on both species from further consideration for conservation because microfungi associated with D. wilsonii should not be regarded as under threat of co-extinction. Fourteen ascomycete fungal species were collected, identified, described and illustrated namely: Byssogene wilsoniae sp. nov., Geastrumia polystigmatis, Janetia dimorphandra-mollis sp. nov., Janetia wilsoniae sp. nov., Johansonia chapadiensis, Microcalliopsis dipterygis, Phillipsiella atra, Piricauda paraguayensis, Pseudocercospora dimorphandrae sp. nov., Pseudocercosporella dimorphandrae sp. nov., Ramichloridiopsis wilsoniae sp. and gen. nov., Stomiopeltis suttoniae, Trichomatomyces byrsonimae and Vesiculohyphomyces cerradensis. Three fungi were exclusively found on D. wilsonii and were regarded as potentially threatened of extinction: B. wilsoniae, J. wilsoniae and R. wilsoniae.


Introduction
The Cerrado is a savannah-like Brazilian biome that is second in area only to the Amazon forest. It covers 21% of the country (2 million km 2 ) and is largely coincident with the central plateau [1]. However, it is rapidly being replaced by crops, pastures and exotic forest plantations [2,3]. One of the many endemic plant species occurring in this biome that are now endangered is Dimorphandra wilsonii Rizzini (Fabaceae), which is a tree known as "faveiro de Wilson". Only12 individuals of this tree species were known in nature, all in highly disturbed sites in privately owned areas and most in two neighboring municipalities in the Brazilian state of Minas Gerais (Paraopeba and Caetanópolis) [4] Dimorphandra wilsonii is listed in the IUCN Red List (http://www.iucnredlist.org/) as being critically endangered, which is the highest level of risk for the survival of a species prior to extinction in nature.
The general lack of public knowledge and awareness about fungi and their significance and the cryptic nature of most fungi, which are either invisible to the naked eye or produce ephemeral macroscopic fruit bodies, has probably led to the mistaken impression that species belonging to the kingdom Fungi are capable of escaping environmental changes because they are easily dispersed, ubiquitous and broadly spread [5]. Nevertheless, surveys of environmental DNA have indicated that microbial communities have a well-defined structure, with populations that have a high level of endemism [6]. However, the practical difficulties for gathering evidence that individual fungal species are actually threatened and a lack of effort by scientists in general (including mycologists) have resulted in the virtual absence of fungi from the lists of endangered species and from policies aimed at preventing global loss of biodiversity. Minter [7] referred to fungi as "the orphans of Rio" because these organisms were left out of the agenda of the Earth Summit (United Nations Conference on Environment and Development-UNCED) that occurred in Rio de Janeiro in June, 1992. Some years ago, Rocha et al. [8] conjectured that it might be possible to gather convincing scientific evidence of the status of "threatened of extinction" for certain members of the kingdom Fungi by investigating highly hostspecific plant pathogenic fungi associated with endangered plant species. The loss of one rare plant species may lead to coextinction events that threaten a range of specialized organisms that depend strictly on that species for their survival. Such events are well documented for parasite-host interactions, such as pigeon lice, primate parasites, pollinizer wasps and herbivorous insects [9,10,11], but not for fungi.
It was only recently that the need for conservation of fungi was embraced as a duty by mycologists. The first international conference on the issue took place in Whitby, UK, in October, 2009 ("Fungal conservation science, infrastructure and politics"). The International Society for Fungal Conservation was founded (http://www.fungal-conservation.org/) on August 6, 2010, during the 9th International Mycological Congress. More recently, the Third International Congress on Fungal Conservation took place in Gökova Bay, Turkey, in 2013 (http:// www.fungal-conservation.org/icfc3).
The first report addressing the issue of fungal conservation in Brazil was published by Rocha et al. [8] and involved the study of the foliage mycobiota of Coussapoa flocosa Akkermans & C.C. (Cecropiaceae). The mycobiota was regarded by the authors as likely to be in danger of coextinction due to its dependence on this rare endemic tree in the Brazilian Atlantic Forest. This study led to the discovery of six new fungal species, including a new fungal genus.
The present work aims to expand the study begun by Rocha et al. [8] to encompass an additional endangered Brazilian plant species (Dimorphandra wilsonii) and its mycobiota, which may also be potentially endangered by coextinction. Additionally, we also studied the mycobiota of Dimorphandra mollis Benth., which is a common species with a broad geographical distribution that is closely related to D. wilsonii and coexists with that plant in its remaining area of occurrence in nature. This study was performed to determine which fungi occurring on D. wilsonii also occurred on the non-endangered D. mollis. Such species should not deserve further consideration for conservation because the microfungi associated with D. wilsonii should not be regarded as being in danger of co-extinction.
Hence, the objectives of this study were: I) to survey and describe the foliicolous mycobiota associated with Dimorphandra wilsonii; II) to survey and describe the mycobiota of D. mollis; III) to verify the possibility of the co-occurrence of fungi on D. wilsonii and D. mollis and; IV) to produce a preliminary list of fungal species on D. wilsonii that are potentially in danger of extinction based on this evidence.

Materials and Methods
Survey trips were conducted between 2009 and 2011 in the municipalities of Paraopeba, Caetanópolis, Juatuba, Fortuna de Minas, Sete Lagoas and Pequi ( Table 1).
The owners of each property allowed samples to be collected for study, and no special permits were required for this study other than the registration of the corresponding author in SISBIO-ICMBio, Ministerio do Meio Ambiente (Reg. number 1839773). The fungi collected during this study have no official status of endangered or protected species at this stage at any level. Existing information on the localities of the occurrence of D. wilsonii individuals in nature were provided by F. Fernandes based on his regular surveys for remaining individuals of this species that started in 2003 [12]. Whenever individuals belonging to the closely related species D. mollis were found growing in the vicinities of an individual of D. wilsonii, branches and foliage of individuals belonging to that species were also collected. Dimorphandra wilsonii is readily separated from D. mollis by having longer pods with a sweetish scent and paler gray bark that is not easily detached unlike D. mollis. Its leaflets are also larger as compared with D. mollis (3-5 cm long) (Fig 1). Pictures were taken in the field with a SONY DSC-H9 digital camera, and samples of branches bearing foliage were collected with a long-poled pruner and dried in a plant press. After screening in the lab, dried relevant specimens were deposited at the herbarium of Universidade Federal de Viçosa (VIC). Samples were examined under a stereomicroscope (OLYM-PUS SZX7) while still fresh, and fungal structures were either scraped with a scalpel from tissue surfaces or free-hand sections of fungal structures or sections prepared using a freezing microtome (Microm HM 520) were mounted in lactophenol or other mounting media. Observations,measurements and drawing were performed with an OLYMPUS BX 51 light microscope fitted with a digital camera (OLYMPUS E330) and a drawing tube.
Isolations of fungi in pure cultures were attempted by the direct transfer of spores or other fungal structures onto plates containing VBA (vegetable broth agar) as described by Pereira et al. [13] with the help of a sterile fine-point needle. Pure cultures were preserved in PCA slants or silica-gel as described by Dhingra and Sinclair [14] and were deposited in the culture collection of the Universidade Federal de Viçosa (COAD). For scanning electron microscopy, samples were placed in a critical point dryer (Baltec model 030) with CO 2 as the transition fluid; after drying, the samples were coated with gold (20 nm thick) with a sputter coater (Balzers 1 model FDU 010) and examined with a Carl-Zeiss Model LEO VP 1430 electron microscope.
Culture descriptions were based on observations of the colonies formed in plates containing potato dextrose agar (PDA) or potato carrot agar (PCA). The plates were incubated at 25°C under a 12 h daily light regimen (light provided by two white and one near-UV lamps placed 35 cm above the plates) for 23 days. The color terminology followed Rayner [15].
For the molecular phylogeny studies of selected fungi, pure cultures were grown on PDA at 25°C for up to four weeks depending on their growth rate. Genomic DNA was extracted from the mycelium using the Wizard 1 Genomic DNA Purification Kit (Promega Corporation, WI, USA) following the manufacturer's instructions. For the sequencing of Phillipsiella atra, DNA was extracted by removing fungal structures from the plant tissue with a fine glass needle and placing them in a microtube (1.5 ml) provided by the extraction kit. Each fungal structure was carefully examined under the highest power of a stereomicroscope to check for possible contamination with other fungi or mycoparasites and to exclude any plant material from the sample. PCR reactions included the following ingredients for each 25 μl reaction: 12.5 μl of 2X DreamTaq™ PCR Master Mix (MBI Fermentas, Vilnius, Lithuania), 1 μl of 10 μM of each forward and reverse primer synthesized by Invitrogen (Carlsbad, USA), a maximum of 25 ng/μl of genomic DNA, and nuclease-free water to complete the total volume. The primers ITS4 (5'-TCCTCCGCTTATTGATATGC -3') and ITS5 (5'-GGAAG TAAAAGTCGTAACAAGG -3') were used to amplify the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA operon, including the 3' end of the 18S rRNA, the first internal transcribed spacer region, the 5.8S rRNA gene, the second internal transcribed spacer region and the 5' end of the 28S rRNA gene [16]. The large ribosomal subunit (LSU) was amplified with the primer pair LR0R (5'-ACCCGCTGAACTTAAGC-3') and LR5 (5'-TCCTGAGGGAAACTTCG -3') [17]. The amplifications were performed with a BIO RAD C1000 (Thermal Cycler) with an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 60°C for ITS and 53°C for LSU for 45 s, extension at 72°C for 2 min and a final extension of 7 min at 72°C. The amplified products were visualized on a 1% agarose gel to check the product size and purity. PCR products were purified with the PEQLAB E.Z.N.A. 1 Cycle-Pure Kit following the manufacturer's protocol. The sequencing was performed directly from the purified PCR-amplified fragments using the automated sequencer MegaBACE 500TM. The nucleotide sequences were edited with the BioEdit software [18]. All sequences were checked manually, and nucleotides with ambiguous positions were clarified using primers targeting both sequence directions. New sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov). The large ribosomal subunit sequences of additional species were retrieved from GenBank ( Table 2).
Consensus regions were compared against GenBank's database using their Mega BLAST program. The closest sequence hits were downloaded in FASTA format and aligned using the multiple sequence alignment program MUSCLE 1 [19] with default parameters. MUSCLE 1 was implemented using the MEGA v.5 software [20]. The alignments were checked, and manual adjustments were made where necessary.
Bayesian inference (BI) analyses employing a Markov Chain Monte Carlo method were performed. MrMODELTEST 2.3 [21] was used to select the nucleotide substitution models for the BI analysis. We used the general time-reversible model of evolution [22], including the estimation of invariable sites and assuming a discrete gamma distribution with six rate categories (GTR+I+G). The BI analysis was performed with MrBayes v. 3.1.1 [23,24,25,26]. Four MCMC (Markov chain Monte Carlo) chains were run simultaneously starting from random trees for 10,000,000 generations. Trees were sampled every 1000th generation for a total of 10,000 trees. The first 2500 trees were discarded as the burn-in phase of each analysis. Posterior probabilities [23] were determined from a majority-rule consensus tree generated with the remaining 7500 trees. Convergence of the log likelihoods was analyzed with TRACER v. 1.4.1 (beast.bio.ed.ac.uk/Tracer); no indication of a lack of convergence was detected. This analysis was repeated three times starting from different random trees to ensure trees from the same tree space were sampled during each analysis. Trees were visualized in FigTree (http://tree.bio. ed.ac.uk/software/figtree/) and exported to graphics programs. The tree was rooted to Dothidea sambuci (Pers.) Fr. (AFTOL-ID274) (Fig 2).

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names proposed in this work were submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: [PubMed Central, LOCKSS etc].

Results and Discussion
Phylogeny Amplification of the partial LSU was selected for the molecular phylogenetic identification of the new species included in this study. The manually adjusted alignment included 70 taxa and contained 768 characters, of which 195 were parsimony-informative, 230 were variable and 534 were conserved. Although the ITS sequences were not used in the phylogenetic analyses, they were deposited in GenBank for future studies and DNA barcode purposes (Table 2).
Only Janetia wilsoniae was successfully isolated in a pure culture. The investigation of sequences obtained from that taxon indicated that it grouped closely with Zasmidium cellare The lack of any other sequences of a member of Janetia in sequence databases and the lack of knowledge of its sexual morph connection limits the understanding of the phylogenetic relationships within this taxon (Fig 2). Notes: J. L. Bezerra examined our material and recognized it as a taxon described by him more than fifty years ago. The fungus on D. wilsonii belongs to the obscure genus Microcalliopsis Bat. & Cif. (Chaetothyriaceae) [37]. There are only four known species of Microcalliopsis and our specimens fitted well within the description of M. dipterygis, which was previously reported on the leaves of Dipterys alata Vog. in Brazil [37]. The occurrence of this fungus on D. wilsonii suggests that it can be a polyphagous fungus on the leaves of leguminous hosts. This is the first record of Microcalliopsis dipterygis colonizing D. wilsonii.
Phillipsiella atra Cooke, Grevillea (1878) Fig 9 Colonies on living leaves, hypophyllous, sparsely spread, not associated with disease symptoms on host. Internal mycelium not observed. External mycelium hypogenous, 2.5-4 μm diam, branched, net-forming, slightly undulate, composed of septate, pale brown, smooth hyphae. Ascomata apothecioid, hypophyllous, superficial, solitary, discoid, non-ostiolate, 445-550×75-105 μm high, pseudoparenchymatous basal stroma 12.5-20 thick, walls of brown textura angularis, powdery, ill-differentiated, dark grey centrally with raised pale gray margins, pseudoparaphyses filiform, up to 2 μm wide, septate, unbranched, hyaline, but branching above the asci, becoming inflated and pigmented at apices to form an epithecium. Asci bitunicate, parallel, cylindrical, 30-40 × 7.5-11μm, 8-spored, endotunica flattened apically at a distance from apical exotunica but extending as a narrow column towards the conspicuously domed ascal apex. Ascospores biseriate to inordinate, fusiform to ellipsoid, 7.5-10 × 2.5-4 μm, 1-septate, upper cell slightly broader than lower cell, eguttulate, hyaline, smooth. Notes: The genus Phillipsiella was proposed by Cooke [38] with Phillipsiella atra as the type species was collected on Quercus virginiana Mill (Fagaceae) in Georgia [39]. Key features for the genus were: discoid ascomata containing numerous asci, asci bitunicate and hyaline,   bicellularascospores. There are twelve species accepted within this genus, but none of these species has been reported in association with members of the Fabaceae [40]. Our specimens collected on D. wilsonii are the first to be reported growing on a member of the Fabaceae, and they fitted well within the description of P. atra. Müller and von Arx [41] placed the genus Phillipsiella within Schizothyriaceae. Later, von Arx & Müller [23] transferred Phillipsiella to the Saccardiaceae. Posteriorly, Barr [42] revived the family Phillipsiellaceae of von Höhnel [43], and Eriksson [44] also discussed and accepted the Phillipsiellaceae [45]. However, the recent publication on the classification of families of Dothideomycetes does not discuss the relationship of Saccardiaceae and Phillipsiellaceae [46]. Based on the analysis of large ribosomal subunit DNA gene sequences and the resulting phylogeny generated in the present study (Fig 2), it is finally clarified that Phillipsiella belongs to the Dothideomycetes (Capnodiales). Another fungus (Johansonia chapadiensis Crous, R.W. Barreto, Alfenas & R.F. Alfenas) was recently described from leaves of Dimorphandra mollis collected in Chapada dos Guimarães, Mato Grosso, Brazil. The phylogenetic study based on DNA sequence data of the nuclear ribosomal DNA (LSU) showed that Johansonia is also a member of Dothideomycetes, Capnodiales [47]. The fact that Phillipsiella and J. chapadiensis are grouped in the same highly supported clade (Fig 2) indicates that these two genera belong to the same family (possibly the Saccardiaceae). Unfortunately, there is no molecular information for the type species of the family Saccardiaceae which may be used for a clarification of the affinities of these taxa. Therefore, it is not possible to circumscribe the Saccardiaceae adequately, and at present it is also impossible to confirm that these genera belong to this family as proposed by von Arx & Müller [27].
In culture: slow-growing (0.6-1.6 cm diam, after 27 days), either of scanty floccose aerial mycelium and growing very poorly or of mostly immersed lobate, flat, dentritic mycelium, colony composed of monilioid and filamentous hyphae, striate and granulose, dark mouse gray, to iron gray, reverse dark olivaceous with slight yellow pigmentation of medium, no sporulation. Notes: The morphology of fungi in the genus Piricauda Bubák according to the generic concept of Hughes [48] and Ellis [49] is micronematous conidiophores developing on superficial hyphae, conidiogenous cells monotretic and conidia tretic arising singly from a pore on the conidiogenous cell. There are eight species in the genus Piricauda: P. cochinensis fits well within the boundaries of P. paraguayensis as described by Ellis [48]. This species was previously reported on Bignonia sp., Citharexylum sp. and Duranta sp. in Brazil, Cuba and Paraguay [49,50]. This is the first report of this fungus on D. wilsonii and D. mollis. Phylogenetically, P. paraguayensis grouped in a clade in the Capnodiales, Mycosphaerellaceae (Fig 2). This is the first report of this fungus in culture. More sequences of species of Piricauda are necessary to better elucidate the phylogenetic position of this obscure genus.
In culture: On PCA slow-growing (2.1-2.8 cm diam, after 27 days), slightly to pronouncedly lobate at edges, flat to low convex and pale olivaceous grey cerebriform centrally, surrounded with a ring of honey colored immersed mycelium followed by a narrow ring of olivaceous mycelium, followed by a periphery of pale olivaceous grey mycelium, diurnal zonation either pronounced or subtle, reverse leaden black followed by a periphery of leaden gray mycelium; not sporulating. Notes: Pseudocercospora Speg. is one of the largest genera of fungi and includes more than 1200 species [51]. Approximately 200 are parasitic on members of the Fabaceae. Cercosporoid fungi bearing unthickened and not darkened conidiogenous loci and hila and having pigmented conidiophores and conidia are often placed in Pseudocercospora [52,53,54]. The fungus on Dimorphandra has the typical morphological features of members of Pseudocercospora and was compared with species of Pseudocercospora reported on hosts phylogenetically close to Dimorphandra, such as the Dimorphandra-group (Banks and Lewis 2009) including Burkea Benth., Erythrophleum Afzel. Ex R. Br., Mora Schomb. ex Benth., Pachyelasma Harms, Stachyothyrsus Harms and Sympetalandra Stapf. Only one species of Pseudocercospora is known to occur on a member of this group (Pseudocercospora erythrophlei Z.Q. Yuan reported on the leaves of Erythrophleum chlorostachys Baill) [55]. This is the first report of Pseudocercospora on a member of Dimorphandra, and the fungus found in this study clearly differs from P. erythrophlei by having shorter conidiophores and conidia (10-27 μm and 17.5-87.5 μm, respectively) and not having geniculate conidiogenous cells with short "denticles" as found in P. erythrophlei, thereby justifying the proposition of the new species. The morphology of specimens of Pseudocercopora on D. wilsonni and on D. mollis is identical. A comparison of the DNA sequences obtained from both species confirmed that the isolates obtained from the two hosts belonged to the same species (Fig 2). The Pseudocercospora on Dimorphandra spp. grouped in the same clade of the type species of the genus Pseudocercospora (P. vitis) and it is phylogenetically close to Mycosphaerella bixae Crous & Bench. Mycosphaerella bixae is a parasite of a distantly related host (Bixa orellana L.) belonging to the family Bixaceae and has a Passalora asexual morph Crous & Bench [52,56]. Additionally, sequences of the ITS region of Pseudocercospora dimorphandrae have only 97% of similarity with the ITS sequence of P. bixae deposited in GenBank (Accession No. AF362056). Etymology: In reference to the genus of the host Dimorphandra Lesions on living leaves amphigenous, starting as minute dark dots becoming circular to irregular, necrotic, coalescing and leading to leaf blight, 1.5-5 mm diam, brown, covered with white foamy-like fungal colonies, coalescing to cover the whole surface of the leaflets and leading to leaflet blight. Internal mycelium inter and intracellular, 1.5-2.5 μm, branched, septate, pale brown. External mycelium absent. Stromata superficial, 17.5-30 × 37.5-55 μm, composed of subhyaline textura angularis. Conidiophores mostly restricted to conidiogenous cells, hypophyllous arising from stromata, in dense fascicles, cylindrical, straight to slightly sinuous, 10-20 × 2.5-5 μm, 0-1 septate, unbranched, hyaline, smooth,. Conidiogenuous cells integrated, terminal, holoblastic, cylindrical, hyaline. Conidiogenous loci, truncate, up to 2.5 μm diam, neither thickened nor darkened. Conidia dry, solitary, cylindrical, slightly curved to curved, 22.5-57.5 × 4-6 μm, truncate at the base, apex rounded, 2-7 septate, hilum unthickened and not darkened, hyaline to olivaceous, guttulate, smooth.
In culture: On PCA slow-growing (2-3 cm diam, after 27 days), slightly lobate edges, flat, immersed on media at periphery, aerial mycelium buff cottonous either followed with an outer ring with irregular portions of buff or honey cottonose mycelium or followed by a narrow ring of white cottonous mycelium over a layer of olivaceous gray colony, followed by a periphery of pale olivaceous gray mycelium, reverse either rosy buff or primrose alternating with leaden black; not sporulating. Notes: The cercosporoid fungus found on D. wilsonii and D. mollis clearly belongs to the genus Pseudocercosporella Deighton. It bears the key morphological characteristics of the genus: hyaline conidiophores, bearing unthickened and not darkened scars, hyaline conidia, bearing unthickened and not darkened hila and released by schizolytic secession [57]. Four Pseudocercosporella species are known to occur on members of the Fabaceae [40]: P. astragali  [60,61,62,63], whereas no asexual morph is known for M. gregaria ( Phaeophleospora gregaria) [62,63]. Only M. endophytica has a Pseudocercosporella asexual morph, but this species possesses a clearly distinct morphology. It has longer and narrower conidiophores (20-60 × 3-4 μm), smaller and narrower conidia (13-50 × 1.5-2.5 μm), and is a parasite of a member of a distantly related host family (Eucalyptus, Myrtaceae). Moreover, ITS region sequences from Pseudocercosporella dimorphandrae has 67, 41, 41 and 40 nucleotide differences as compared to M. ellipsoidea, M. endophytica, M. gregaria and M. stromatosa, respectively. Additionally, the significant level of host specificity known for this group of fungi provides further evidence that the fungus associated with Dimorphandra is a new species. The overlap in the DNA sequence analysis (Fig 2) and the equivalent morphology found for Pseudocercosporella specimens on D. wilsonni and D. mollis clearly shows that they belong to the same species.
Ramichloridiopsis M. Silva      Notes: The monotypic genus Vesiculohyphomyces is characterized by the presence of discrete vesicle-shaped conidiogenous cells arranged in apical verticillate rings on conidiophores and conidia that are solitary, pigmented and striate when mature. The fungus collected on D. wilsonii fits well within the description of V. cerradensis. This species was previously known only from trichomes of Caryocar brasiliense Cambess. (Caryocaraceae) from the Brazilian Cerrado [68]. The occurrence of this fungus on two unrelated hosts suggests that it is a generalist epiphyte on Cerrado plants. Vesiculohyphomyces cerradensis is recorded here for the first time colonizing D. wilsonii.

Conclusions
A high level of fungal diversity was found during the exploration of the foliicolous mycobiota of D. wilsonii (fourteen species). This diversity is significantly higher than the estimated average of six fungal species expected for each plant species roughly suggested by [69]. Additionally, this list is clearly only partial because a whole range of fungi occupying other niches in the plant remained unexplored in this work. Interestingly, D. wilsonii and D. mollis shared few fungi in our study despite their taxonomic relatedness (Peudocercospora dimorphandraea, Pseudocercosporella dimorphandraea, P. paraguayensis and T. byrsonimae). Six fungal taxa were found on D. mollis, including one that was described in a separate publication as the new genus Alveariospora Meir. Silva, R.F. Castañeda, O.L. Pereira & R.W. Barreto [70] and Johansonia chapadensis [47], which had its host tentatively identified in the original publication as D. mollis (confirmed here after being recollected on D. mollis). Several of the fungi were recognized as polyphagous/generalist organisms of little relevance in terms of conservation (T. byrsonimae, P. atra, M. dipterygis and S. suttoniae). Interestingly, several of these fungi appeared to be particularly rare in our sampling. Several were limited to very few leaves (V. cerradensis, P. paraguayensis, and G. polystigmatis), whereas others were either abundant on each specimen, frequently collected or both (T. byrsonimae, P. atra, M. dipterygis and S. suttoniae). The discovery of three new fungal taxa seemingly restricted to Dimorphandra wilsonii, Naming Potentially Endangered Parasites: Mycobiota of a Threatened Brazilian Tree including one that was described as belonging to a new genus (Janetia wilsoniae, Byssogene wilsoniae, and Ramichloridiopsis wilsoniae) mirrors the results of the pioneering work on the mycobiota of the endangered Brazilian plant species C. floccosa by Rocha et al. [8]. In that work, six novel fungal taxa were discovered on this host, including a new genus. This may be the best indication that the loss of a single plant species such as D. wilsonii can have disastrous consequences for a unique portion of the mycosphere. Additional studies are needed to confirm this conjecture and to demonstrate that the taxa found only on D. wilsonii are strictly host-specific and not capable of surviving on other substrates (i.e., as saprophytes or endophytes on other hosts); moreover, studies should be conducted to test the hypothesis of the risk of co-extinction. Fortunately, the effort coordinated by F. M. Fernandes to survey and preserve existing individuals occurring in the wild and to reintroduce D. wilsonii in areas where it originally existed have produced good results. The original list of 12 remaining individuals of D. wilsonii was significantly raised by the continuation of the searches for the remaining plants (now the count is of 219 mature individuals occurring in nature) and area of distribution is larger than previously though (now 16 municipalities in the state of Minas Gerais are known to harbor this endangered plant). Additionally the plant is being progressively re-introduced in areas where it occurred in the past and a National Action Plan for its protection has been recently published [12]. There is hope that the actions included in the plan will eventually result in the long-term preservation of this tree species and as a consequence the preservation of its specialized mycobiota.