https://orcid.org/0000-0003-0187-3498
Figures
Abstract
Species of the genus Tulostoma are easily recognizable by the presence of a spore sac, with a mouth from which spores are released, attached to a stipe. Tulostoma is a species-diverse genus with a worldwide distribution, and some attempts were made to delimitate species and to evaluate reliable taxonomic-informative characteristics for species identification. However, there is a notable information gap regarding Neotropical species, especially for geographic distribution and DNA data, which hampers further understanding of the infrageneric diversity, evolution, and ecology of this genus. Based on morphological analysis, molecular phylogenetics and geographic distribution, we propose here two new species of Tulostoma with reticulated spores, from the two threatened Brazilian geographical areas, Atlantic Forest and “campos rupestres” (rupestrian grassland), as well as we provide notes on the taxonomic rank of Tulostoma exasperatum var. ridleyi.
Citation: Cabral TS, Silva BDBd, Vargas-Isla R, Oliveira JJSd, Ferreira JAdS, Castro L, et al. (2023) Diversity of Neotropical stalked-puffball: Two new species of Tulostoma with reticulated spores. PLoS ONE 18(12): e0294672. https://doi.org/10.1371/journal.pone.0294672
Editor: Erika Kothe, Friedrich Schiller University, GERMANY
Received: March 26, 2023; Accepted: October 31, 2023; Published: December 13, 2023
Copyright: © 2023 Cabral et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All files from phylogenetic analyses are available from the TreeBase database (ID 30727). DNA sequences were deposited at Genbank from NCBI (accession numbers OQ626704-OQ626707, OQ672312, OQ626710-OQ626712, OQ658839). Additional data were uploaded at Zenodo (10.5281/zenodo.8305573).
Funding: NKI received the grant from Fundação de Amparo à Pesquisa do Amazonas (Edital N. 008/2021–PROSPAM and Edital No. 007/2021 - BIODIVERSA) and Fazenda Bananal also funded this research (for NKI, RVI and JJSO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Commonly known as stalked-puffballs, species of the genus Tulostoma Pers. are easily recognizable by the presence of a spore sac, with a mouth from which spores are released, attached to a stipe, which itself may be visible or may grow belowground [1]. This genus was first proposed by Persoon [2], with Tulostoma brumale Pers. as the type species; the name Tulostoma was later sanctioned by Persoon [3]. Tulostoma is a species-diverse genus with a worldwide distribution, which includes 276 legitimate species according to MycoBank [4].
In an attempt to delimitate species, Wright [5] defined the mouth and spores (size and ornamentation) as reliable primary characteristics. Wright [1] expanded these studies and published a classification based on macro- and micromorphological characters, incrementing Pouzar’s proposition of four sections: Brumalia Pouzar, Poculata Pouzar, Fimbriata Pouzar, and Volvulata Pouzar [6]. Wright’s classification proposed new subgenera, series, and sections, based mainly on the type of mouth and exoperidium [1].
With the application of molecular techniques to fungal systematics, some new attempts to delimitate species have been implemented. Studying European specimens, Jeppson et al. [7] reported high species diversity, including five new species to science. They also found that some of the characters commonly used to differentiate species are homoplasious or plesiomorphic but suggested that spore ornamentation and endo- and exoperidium characteristics may be good features to delimit species. In addition to the morphological aspects, internal transcribed spacer (ITS) regions between small-subunit and large-subunit ribosomal RNA (rRNA) genes have widely been used in DNA barcoding to identify Tulostoma species [8–11].
However, there is a notable information gap regarding Neotropical species, a fact that Wright himself perceived [1]. Despite the efforts of taxonomists in studying Tulostoma diversity in this important geographic area [9, 12–18], there is a paucity of geographic distribution data and especially of DNA data, which hampers further understanding of the infrageneric diversity, evolution, and ecology of this genus. For example, species with smooth spores tend to occur in dry areas, while species with ornamented spores occur in humid areas [7, 19, 20], but this has been tested to date only for Argentinian and European species.
Compared to other localities and taxa, there are few studies on Tulostoma species diversity in Brazil. Currently, 20 species are registered in Brazil from five of the six Brazilian biomes: Amazonia rainforest, Atlantic Forest, Caatinga, Cerrado, and Pampa [13–15, 18, 21, 22]. Brazil is a global biodiversity hotspot, harboring a vast array of fungal, plant, and animal species found nowhere else on Earth. Therefore, it has a high potential for species discovery, as is probably the case for Tulostoma. The most recent new species for the country were Tulostoma irregulireticulatum Dourado-Barbosa, R.L. Oliveira, A.A. Lima, Baseia & R. Cruz [22], T. catimbauense A.A. Lima, Accioly, Baseia & M.P. Martín and T. deltaconcavum A.A. Lima, Accioly, Baseia & M.P. Martín [18].
From the Brazilian species, Tulostoma exasperatum Mont. is so far the most represented species. It was first described from Cuba and is shown to be easily distinguished by the spiny exoperidium, scaly stipe, and reticulated spores, and is one of the few species with lignicolous habitat [1]. Tulostoma ridleyi Massee, described in 1899 from Malaysia is another very similar species, which differs mainly in the basidioma size and the sizes and shapes of the spores and endoperidial verruca [1]. Therefore, Wright [1] changed the species status to the variety T. exasperatum var. ridleyi (Massee) J.E. Wrigth. After the description of the species, several specimens of T. exasperatum were recorded, and it is now known to be widely distributed in the African, Asian, and American tropics [23–29]. On the other hand, the few records of T. exasperatum var. ridleyi from Malaysia, Singapore, and Thailand [30] suggest that this taxon is endemic to Southeast Asia, but further analyses are needed to elucidate the taxonomic rank. Similarly, because several widely distributed mushroom species may in fact be different evolutionary units [31–34], studies on the diversity of T. exasperatum sensu Wright [1] are also needed.
Here, we propose two new species of Tulostoma with reticulated spores found in Brazil, closely related to T. exasperatum, but from different phytophysiognomies and different substrates (wood and soil), based on morphology and DNA phylogenetic data. In addition, we provide notes on the taxonomic rank of T. exasperatum var. ridleyi.
Materials and methods
Sampling and morphological analysis
Specimens were collected during field trips to areas of the Atlantic Forest (AF) in southeast Brazil (Paraty, state of Rio de Janeiro–RJ–three specimens collected) and rocky fields (rupestrian grassland, “campos rupestres”) of northeast (Mucugê, in Chapada Diamantina range, state of Bahia–BA–a single specimen collected) Brazil (Fig 1). The AF is considered a biodiversity hotspot, found only in Brazil, with high levels of endemicity, although it has suffered severe habitat fragmentation and other threats [35, 36]. The sample area in RJ was Fazenda Bananal, a private reserve for ecotourism and nature education, which includes part of the AF. It is a coastal area with a temperate-dry winter with hot summer (“Cwa” in Köppen’s climate classification), at sea level, with a mean annual rainfall of 3407 mm and mean temperature of 21°C. The “campos rupestres” are characterized by herbaceous and bushy vegetation associated with rocky outcrops and sandy soil on the tops of mountains (above 900 m), with recognizable levels of endemicity [37]; the climate is semihumid (“Aw” in Köppen’s climate classification], with mean annual rainfall of 1100 mm and mean temperature of 20°C [38]. We followed Vargas-Isla et al. [39] for collection, storage, and herborization of specimens. This study is registered at SisGen (Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado) under ID A6DBD2C; collections made at Fazenda Bananal were authorized in writing by the managers and specimens were transported under SISBIO 85230–1 permission. Collections made at Parque Nacional de Mucugê does not require permissions.
Map: Clayton Bit-tencourt Junior. The map image is from GEBCO (General Bathymetric Chart of the Oceans) Gridded Bathymetric Data and it is in the public domain.
Morphological descriptions were obtained based on both fresh and dried basidiomata using an M205 C stereo microscope coupled with an MC 190 camera, and a DM 2500 optical microscope (Leica, Wetzlar, Germany). Colors were defined based on Küppers [40] and mycelial cord/rhizomorph descriptions were based on Yafetto [41]. For micromorphological characters, freehand sections of different structures (peridium, stipe, rhizomorph, spores) were mounted in 5% KOH or distilled water, and stained with cotton blue, Congo Red or Melzer’s reagent, and observed under a light microscope. Thirty spores were measured, including the ornamentation, where n = number of randomly measured basidiospores, x = mean ± standard deviation of basidiospore diameter and height (including ornamentation), and Qm = mean height/width quotient. We followed the spore shape definitions of Bas et al. [42]. Scanning electron microscopy (SEM) was performed at Centro Multiusuário para Análise de Fenômenos Biomédicos (CMABio) of Universidade Estadual do Amazonas (UEA) and Laboratório de Microscopia Eletrônica (LAMUME—Institute of Physics) of Universidade Federal da Bahia, following previously described methods [43]. Additional figures of macro and microcharacters can be found at Cabral et al. [44].
Phylogenetic analyses and species delimitation approach
To increase the geographic coverage of the sampling, one specimen registered as T. exasperatum was loaned from UFRN-Fungos herbarium and included in phylogenetic analyses, as well as a specimen from Singapore deposited at MA herbarium (MA-Fungi 83796). DNA was extracted from dried material using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany). Three DNA regions were obtained the internal transcribed spacer (ITS), the large subunit of the nuc-RNA gene (nucLSU), and translation elongation factor subunit 1 alpha (TEF1a), using primers pairs ITS1-ITS4 [45], LR0R-LR5 [46], and EF983F-F2218R [47], respectively, for both amplification and sequencing. Sequences generated in this study were visualized and assembled using Geneious R6.1 (Biomatters Ltd., Newton, New Zealand), and submitted to a BLAST search. The newly generated sequences were aligned to homologous sequences available in GenBank, mostly from Jeppson et al. [7] (S1 Table), and edited manually using the MUSCLE alignment tool in AliView v 1.26 [48]. Seventy-nine taxa were included in phylogenetic analysis, with Calvatia caatinguensis (UFRN-Fungos 2945) and Lycoperdon subperlatum (KA12-0981) forming the outgroup, following previous publications [7, 29]. Each DNA region was aligned separately and the substitution model was chosen for each matrix using MrModelTest [49]. Each matrix was composed of all specimens, and for the specimens lacking a DNA region, it was treated as missing data. Then the three matrices were concatenated into a single matrix, which was subjected to Bayesian phylogenetic analysis (BA) with MrBayes [50]. This consisted of two parallel runs executed with four incrementally heated simultaneous MCMC simulations over 10 million generations, with trees sampled every 1000 generations, with the first 25% discarded at the tree summarizing stage. Maximum likelihood (ML) analysis was run in RAxML [51] using the same partitioned dataset, with estimated proportion of invariable sites (GTRGAMMA + I). Support values are given as posterior probabilities (PP) for BA analysis and bootstrap (BP) for ML on the nodes of the tree. The substitution model and phylogenetic analyses were implemented in CIPRES Science Gateway [52]. Trees were visualized and edited in FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). All data are available in TreeBASE under submission ID 30727.
The Bayesian Posterior Tree Poisson (bPTP) analysis was applied for species delimitation [53]. The bPTP analysis was run in a web server (http://species.h-its.org/ptp/) with 500,000 MCMC generations and the remaining default settings, using the same Bayesian tree obtained in previous analysis. We also generated distances matrix based on ITS sequences, calculated between one representative of each species defined by bPTP, using dist.dna function of the ape package [54] with “raw” model, and MEGA5 software to calculate ingroup mean distances [55]; the distances were plotted in a heat map using heatmapSpp function of spider package [56] for R environment.
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 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 contained in this work have been 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 repository: PubMed Central, LOCKSS.
Results
Phylogenetic analyses
Nine new sequences from the analyzed Tulostoma were generated in this study (5 ITS, 3 nucLSU, and 1 TEF1a). The concatenated matrix consisted of 77 sequences of ITS, 64 nucLSU, and 24 TEF1a, with 2813 characters, of which 794 were from ITS, 886 were from nucLSU, and 1133 were from TEF1a. For the three alignments (ITS, nucLSU, and TEF1a), the GTR+I+G model was selected using MrModelTest. The resulted phylogenetic tree of both methods differ mainly in the most internal nodes, where Bayesian analysis resulted in an unresolved tree topology. On the other hand, Maximum Likelihood analysis resulted in a topology with better resolution, but with low support values mostly on internal nodes. However, the species clades were congruent in both analyses (ML phylogenetic tree can be found in S1 Fig). In the phylogenetic tree (Fig 2 and S1 Fig), most clades of the European species were consistent with Jeppson et al. [7]. However, one clade grouped the Neotropical and three South Asian specimen, all of which had reticulated spores and verrucose or spiny exoperidium (similar to T. exasperatum), with high a support value (PP = 1, BP = 100). Within the new clade with reticulated spores, we delimited five species-level clades, two of which are described here as new species and one that has a change in the status rank, as discussed in the following sections.
Colors indicate the new species proposed here: T. mucugeense in green and T. paratyense in blue. Thicker branches indicate posterior probabilities values ≥ 95%.
The bPTP method estimated a mean number of 59 partitions assigned to putative species by simple heuristic search (bayesian) and 58 by maximum likelihood. In both searches, the clade with reticulated-spores returned 5 species with high support values: T. mucugeense (1.00), T. ridleyi (0.87), T. aff. exasperatum from Brazil (1.00)., T. exasperatum from Thailand (0.99) and T. paratyense (1.00) (see S2 Data; the tree topologies are available at Cabral et al. [44]). Additionally, the genetic p-distances values ranged between 0% to 16%, with the intraspecific mean distances of 5.8%; interestingly, the mean distances between reticulated-spores group and other Tulostoma species is of 10%. Both the genetic distances matrix and a heat map can be found at S1 Data and S2 Fig.
Taxonomy
Tulostoma mucugeense B.D.B. Silva & T.S. Cabral, sp. nov., Figs 3a, 3b, 4a–4c and 5a.
Tulostoma mucugeense: a. macromorphology (bar = 5 mm), b. habitat type. Tulostoma paratyense: c. macromorphology and gregarious habit (bar = 10 mm), d. mouth and exoperidium (bar = 10 mm), e. socket (bar = 2 mm). Photos (c, d): Rafael E. de Freitas.
Micromorphology: a, d. spores (bars: a = 5 μm, d = 10 μm); b, e. hyphae of exoperidium (bars = 10 μm); c, f. hyphae of mycelial base (bars: c = 10 μm, f = 20 μm). a, b, c. Tulostoma mucugeense; d, e, f. Tulostoma paratyense.
a. Tulostoma mucugeense; b. Tulostoma paratyense; c. Tulostoma aff. exasperatum UFRN-Fungos 1908.
Mycobank: MB847695 [urn:lsid:mycobank.org: 847695]
Diagnosis: The species is characterized by spores with halos under light microscopy, helicoid with uneven alveoli under SEM, measuring 8 × 10 μm; fibrillose mouth, exoperidium verrucose, and found growing on sandy soil.
Holotype: Brazil, Bahia, Mucugê, Parque Nacional de Mucugê, -12.983333, -41.333056, 05 Apr. 2018, leg. Silva, B.D.B. et al. (ALCB 141142!, ITS, nucLSU Genbank sequences: OQ626704, OQ626710).
Etymology: In reference to the type locality, Mucugê (Bahia, Brazil).
Macromorphology: Spore sac subglobose, up to 8 mm high × 5 mm wide. Exoperidium verrucose, brown (N70Y90M50) with areolate surface, granulose-membranous on the lower third and persisting in parts of the rest, falling with maturity first at the apex and later at the base, leaving scars on the exoperidium. Endoperidium smooth, light brown (A70M20C10). Mouth fibrillose, concolor with the endoperidium. Socket slightly detached from the stipe, formed mainly by the pyramidal spines. Stipe short and slender, 10 mm in high × 3 mm wide, surface scaly, with longitudinal scales densely distributed, light brown (Y80M50C30), basally ending in a small bulb. Gleba beige to light brown (Y80M40C30) when mature, pulverulent.
Micromorphology: Basidiospores globose, ornamented, 8 × 10 μm (x = 8.8 ± 0.78 × 8.8 ± 0.8, Qm = 1, n = 30) including ornamentation, brown in 5% KOH, spiny under light microscopy, helicoid with uneven alveoli under SEM. Capillitium 3–5 μm, thick-walled, septate, lumen visible, hyaline in 5% KOH. Exoperidium formed by hyphae of variable shape, yellowish, 4–8 μm. Endoperidium formed by long hyphae, hyaline to yellowish, thick (2–4 μm), with widened septa. Stipe context formed by thin-walled hyphae, hyaline in 5% KOH, septate, with clamp connections, 2–4 μm wide. Scales of stipe formed by parallel organized hyphae, septate, light brown, 5–7 μm wide, brittle, thick-walled. Mycelial base formed by parallel organized hyphae, thin-walled, septate, 3–4 μm wide, thick-walled hyaline to light brown in 5% KOH.
Habitat and distribution: Found growing on sandy soil, known from highlands of the state of Bahia (“campos rupestres”).
Notes: Tulostoma mucugeense is a species with reticulated spores and a fimbriated mouth. It differs from Tulostoma paratyense n. sp., T. exasperatum, and T. ridleyi by its spiny exoperidium, from T. opacum Long and T. rickii Lloyd by its membranous exoperidium, and from T. portoricense J.E. Wright by its hyphal exoperidium. In addition to presenting reticulated spores and a fimbriated mouth, T. transvaalii Lloyd also has exoperidium verrucose as in T. mucugeense. However, it differs due to the larger spores in the latter (4.6–5.7 μm). Tulostoma exasperatosporum J.E. Wrigth also has reticulated spores but has a tubular mouth and membranous exoperidium [1].
Tulostoma paratyense T.S. Cabral & B.D.B. Silva, sp. nov., Figs 3c–3e, 4d–4f and 5b.
Mycobank: MB847959 [urn:lsid:mycobank.org: 847959]
Diagnosis: The species is characterized by spores with a halo under light microscopy, helicoid with uneven alveoli under SEM, measuring 7.5–8.2 × 10.3–10.6 μm; fibrillose-fimbriate mouth; exoperidium spiny and lignicolous habitat.
Type: Brazil, Rio de Janeiro, Paraty: Fazenda Bananal, -23.215611, -44.763167, 13 Dec. 2022, leg. Ferreira, J., Oliveira, J.J.S & Freitas, R.E..—JO2162 (Holotype: RB 845594!, ITS, nucLSU and TEF1a Genbank sequences: OQ626707, OQ626712, OQ658839).
Etymology: In reference to the type locality, Paraty (Rio de Janeiro, Brazil).
Macromorphology: Spore sac subglobose, up to 14 mm high × 21 mm wide. Exoperidium spiny, brown (N80A60M70) with areolate surface (mud-cracked), and presence of pyramidal spines up to 1.6 mm, each spine with a rounded ring at the base, more abundant closer to the mouth, falling down with maturity first at the apex and later at the base, leaving scars on the exoperidium. Endoperidium smooth, light brown (A70M20C10). Mouth fibrillose-fimbriate, concolor with endoperidium, but darker when mature. Socket slightly detached from the stipe, formed mainly by the pyramidal spines. Stipe long, 31–53 mm high × 3.5–4.5 mm wide, surface scaly, with longitudinal scales densely distributed, brown (N80A60M70), ending up in a cream yellow (N10A50M20) mycelium base. Gleba beige to light brown (N40A70M50) when mature, pulverulent.
Micromorphology: Basidiospores subglobose, ornamented, 7.5–8.2 × 10.3–10.6 μm (x = 9 ± 0.8 × 9.5 ± 0.6, Qm = 1,06, n = 30) including ornamentation, brown in 5% KOH, spores with a halo under light microscopy, helicoid with uneven alveoli under SEM. Capillitium 3–8 μm, thick-walled, septate, lumen visible, hyaline in 5% KOH. Presence of ribbon-like hyphae on gleba. Exoperidium formed by hyphae of variable shape, golden yellow in 5% KOH, same found on spines. Endoperidium formed by long hyphae, hyaline to yellowish, thick (2.5–6.5), with widened septa. Stipe context formed by thin-walled hyphae, hyaline in 5% KOH, septate, with clamp connections, 2.5–6.5 μm wide. Scales of stipe formed by parallel hyphae, septate, light brown, 3.5–7.9 μm wide, sometimes ramified, thick-walled. Mycelial base formed by hyphae thin-walled, septate, 2–4 μm wide, hyaline in 5% KOH.
Habitat and distribution: Found growing on wood (“Pau-jacaré”, Piptadenia gonoacantha (Mart.) J.F.Macbr.), gregarious; known so far from AF in Rio de Janeiro.
Notes: Tulostoma paratyense is morphologically very close to T. exasperatum but differs mainly in spore size and shape, growth habit, and size of scales on the stipe. In T. exasperatum, spores are globose and are up to 8 μm in length including ornamentation, under light microscopy with long spines projecting from the angles of the reticulum, which is not deciduous, may appear finger-like; cespitose growth habit [1], while in T. paratyense spores are subglobose measuring 7.5–8.2 × 10.3–10.6 μm, under light microscopy appearing spiny with a halo. Tutolostoma paratyense has larger and densely distributed scales on the stipe and gregarious growth habit, while in T. exasperatum the scales are smaller and the growth habitat is cespitose. Tulostoma exasperatum from Thailand [29] also has smaller basidiospores when compared to T. paratyense, and the spore ornamentation is more similar to T. paratyense than to T. exasperatum from Cuba [1]. Tulostoma ridleyi is also close morphologically but differs in spore shape and size (ovoid and up to 5–8 μm, respectively), larger stipe (120 mm), and the larger and more sparsely distributed spines on the exoperidium [1]. Tulostoma opacum and T. rickii also have a fimbriate mouth and reticulated spores; however, neither species presents a spiny exoperidium or a scaly stipe, and spores are strongly reticulated in T. opacum and apparently spiny under light microscopy in T. rickii [1]. Tulostoma exasperatosporum is another species with reticulated spores, but it has a tubular mouth, a lateritic exoperidium, and smaller spores [1] compared to T. paratyense. Tulostoma squamosum (J.F. Gmel.) Pers. has a stipe with dark brown scales but the scales have a scattered distribution throughout the stipe, mouth is tubular, and the spores are smaller and echinulate [10].
Tulostoma ridleyi Massee, Bull. Misc. Inf., Kew (nos 153–154): 173 (1899)
= Tulostoma exasperatum var. ridleyi (Massee) J.E. Wright, Biblthca Mycol. 113: 99 (1987)
Description: Wright [1] and Calonge et al. [57].
Habitat and distribution: Lignicolous habit, distributed in Southeast Asia.
Material examined: Singapore, Chek Jawa Coastal Hill, 6 Feb. 2012, leg. J. Lai, Sing 2012–45 (MA-Fungi 83796).
Notes: According to Wright [1], T. ridleyi is morphologically close to T. exasperatum but has a larger basidioma and stipe, larger spines on the exoperidium, and larger spores. It also differs from T. paratyense in the slightly smaller spores, larger basidiome, larger spines on the exoperidium, and the cespitose growth habit. Tulostoma mucugeense is different from T. ridleyi mainly in the verrucose exoperidium. We also noted that the ornamentation of the spores is different under SEM; in T. ridleyi, the walls of alveoli are thicker and they are continuous throughout the spores, while in T. exasperatum these walls are thin and uninterrupted, making the ornamentation look like spines under light microscopy.
Discussion
Tulostoma is a genus with a worldwide distribution but with little research of its neotropical diversity, especially including DNA data. Here, we focused on species from the Brazilian AF and rocky fields (“campos rupestres”), with reticulated spores, to evaluate their identity based on geographical distribution, phylogenetic position, and morphological characters. We identified two new species: T. mucugeense, characterized mainly by the verrucose exoperidium, reticulated spores, and fibrillose mouth in specimens from rocky fields (“campos rupestres”); and T. paratyense, characterized by the spiny exoperidium, reticulated spores, and fibrillose-fimbriate mouth in specimens from the AF. Although it is preferable to include multiple collections when describing a new fungal species, in some cases this is not possible (e.g., rare taxa, ephemeral basidiomata, etc.). For these cases, it is recommended that the species is described based on multiple line of evidence [58]. Although we have a single collection of T. mucugeense, we do provide macro and micro morphological and molecular data that supports the decision of describing it as a new species. Additionally, there are several examples of new fungal species being described based on single or a few collections. Tulostoma portoricense was described based on two collections, while T. irregulireticulatum was described using a single collection [12, 22]. T. loonbanglaense was described based on two collections, but both from exactly the same location [11].
Our phylogenetic analyses included six specimens with reticulated spores, where one specimen had a verrucose exoperidium, and five specimens were morphologically close to T. exasperatum—two from the Neotropics (UFRN-Fungos 1908 and RB 845594—Brazil) and three from Southeast Asia (Singapore, MA-Fungi 83796; and Thailand, SDBR-CMUNK1815 and SDBR-CMUNK1819). The specimen MA-Fungi 83796 included in our phylogenetic analyses was identified by Calonge et al. [57] as T. exasperatum. However, based on its geographic locality (Singapore), which is close to the type locality of T. ridleyi (Perak, Malaysia) [59], and based on illustrations and morphological description, we believe this specimen is in fact T. ridleyi described by Massee; therefore, we propose to preserve the species status of this taxon. The results of phylogenetic analysis supported this decision, as the species groups in a clade distinct from other reticulated spore species, with high support values (T. paratyense, T. aff. exasperatum UFRN-Fungos 1908, T. exasperatum from Thailand, and T. mucugeense; PP = 1, BP = 100), which have other differences (see Taxonomy section). In addition, a search for T. exasperatum at the Global Biodiversity Information Facility (GBIF) [60] and iNaturalist (https://www.inaturalist.org) limited to Southeast Asia returned five observations with associated photos, from which we observed a pattern of macrocharacters including large spines on the exoperidium and a cespitose growth habit. However, a detailed morphological analysis of microcharacters should be performed, especially for spore characterization. As suggested by Wright [1], we also believe this species may be endemic to this area, and it is important to include the type specimen in future phylogenetic analyses.
Specimens morphologically close to T. exasperatum grouped together in a clade with high support values (T. paratyense and T. aff. exasperatum UFRN-Fungos 1908, PP = 1, BP = 100). However, the specimens are morphologically different from each other. Tulostoma aff. exasperatum (UFRN-Fungos 1908) has smaller spores (up to 6.3 μm) with spiny ornamentation on light microscopy, the walls of alveoli on the spores are not continuous under SEM (Fig 5c), and the basidiomata have a cespitose growth habit [61]. For T. paratyense, the spores are up to 10 μm and have a halo formed mainly by the continuous walls of alveoli, and the basidiomata have a gregarious growth habit. Tulostoma aff. exasperatum (UFRN-Fungos 1908) is morphologically close to T. exasperatum, mostly in spore size and shape (spiny in light microscopy) and cespitose growth habit, but other specimens from this locality (Serra das Confusões, Piauí, Brazil) must be analyzed to determine the affinity to T. exasperatum.
Tulostoma exasperatum was originally described from Cuba. There are 89 records with this name in the GBIF database, which are from Africa (n = 6), Asia (n = 6), North America (n = 10), South America (n = 65), and two are not assigned to a continent. However, it is recognized that few fungal species are truly cosmopolitan, suggesting some level of endemism at regional scales [62]. Several examples have been published, and T. exasperatum may be such a case of a species complex and/or cryptic speciation, as we found morphological and phylogenetic differences between at least four T. exasperatum-like specimens, sufficient to delimit and define two species (T. paratyense and T. ridleyi). Additionally, specimens from Thailand included in analyses grouped in a different clade within the reticulated-spore group, with high support values (PP = 1, BP = 100). Although it was identified by the authors as T. exasperatum [29], our analyses show that it may be a putative species, since it is phylogenetically quite different from other T. exasperatum-like specimens (genetic distances between 8% to 10% based on ITS sequences). This is another reason to understand this species as a species complex or a case of cryptic speciation. Jeppson et al. [7] found a similar situation when studying European species, finding possible evidence of cryptic speciation and species complexes in T. fimbriatum Fr. and T. cretaceum Long, with strong geographical signals. Only a few descriptions of T. exasperatum from different localities are publicly available, especially from the type locality. The size and shape of spores, the presence or absence of the halo, and the size and distribution of the scales on the stipe may differentiate species similar to T. exasperatum.
Tulostoma mucugeense was collected in a rocky field from Chapada Diamantina, which is part of the Espinhaço Range in Brazil, the largest area of rupestrian grasslands, and an area considered to be an old, climatically buffered, and infertile landscape (OCBIL) [63]. The particularities of these areas favored the generation and maintenance of species endemism and microendemism, as observed for plants, amphibians, and birds [64–66]. These environments act simultaneously as ancient refugia and areas of recent speciation, which explains the observed high levels of diversity and endemism [63]. The plant diversity is mainly driven by the altitudinal and latitudinal range, the influence of the adjacent biomes (Cerrado, Caatinga, and the AF), habitat heterogeneity, and isolation between vegetation islands [63]. However, studies on fungal diversity have only recently been performed in Brazilian rupestrian grasslands, with the discovery of rare and new species [67–70]—and here we now include T. mucugeense—suggesting that these areas may also be species-diverse for fungal taxa. Nonetheless, further studies are necessary to assess this diversity and to understand the factors driving fungal diversity in rupestrian grasslands.
The rupestrian grasslands and AF have tropical semihumid and humid climates, respectively, and both new species found in these environments have reticulated spores. Although there is a tendency for species to develop smooth spores in dry areas and reticulated spores in humid areas, we show here that there may be species with reticulated spores in semihumid climates. In addition, at least two species with smooth spores have been reported from humid areas of Brazil, T. cretaceum Long and Tulostoma obesum Cooke & Ellis, both recorded from the AF in northeast Brazil [14]. Further studies are needed to test the hypothesis that species with ornamented spores are likely to occur only in humid areas, at least when extended to other neotropical areas.
Moreover, the two collection areas are known as biodiversity hotspots. The AF has been subject to severe threats, which have mainly caused habitat fragmentation; similarly, rupestrian grasslands are considered to be naturally vulnerable to disturbance, and would be severely affected by climate change [36, 65]. The finding of new species, especially understudied species, in these areas highlights the importance of planning and implementing conservation strategies. A good example is the “Fundação SOS Mata Atlântica”, a Brazilian environmental nongovernmental organization that has been monitoring and helping forest restoration in more than 500 parks and reserve forests [71]. Similar to SOS Mata Atlântica initiative, the Parque Municipal de Mucugê is a conservation area that aims to preserve ecosystems, promotes sustainable activities through environmental education and sustainable tourism. Tulostoma mucugeense was collected from Parque Municipal de Mucugê, and T. paratyense was collected from Fazenda Bananal, a reference company in the Atlantic Forest for it combines forest restoration, agroforestry and sustainable cultivar production and receives visitors. The preservation of these areas has allowed the maintenance of biodiversity, and therefore new scientific findings. This illustrates the need to plan and develop conservation strategies in the face of global climate change and other anthropogenic threats.
Supporting information
S1 Table. Specimens’ vouchers and locality, and Genbank accession numbers.
The specimens obtained in the present study are in bold.
https://doi.org/10.1371/journal.pone.0294672.s001
(DOCX)
S1 Fig. Consensus phylogenetic tree of genus Tulostoma, obtained by Maximum Likelihood inference with ITS, nucLSU and Tef1-α.
Numbers on nodes are bootstrap support values.
https://doi.org/10.1371/journal.pone.0294672.s002
(PDF)
S2 Fig. Heat map for pairwise genetic distance between species.
Shorter distances are indicated by darker colours.
https://doi.org/10.1371/journal.pone.0294672.s003
(PDF)
S1 Data. Genetic p-distances values calculated between species and between reticulated-spore group and other Tulostoma species.
https://doi.org/10.1371/journal.pone.0294672.s004
(XLSX)
S2 Data. bPTP results with MOTUs and support values.
Sheet 1 is results from simple heuristic search (bayesian) and sheet 2 results from maximum likelihood.
https://doi.org/10.1371/journal.pone.0294672.s005
(XLSX)
Acknowledgments
The authors thank the staff of Parque Municipal de Mucugê and Fazenda Bananal for support in sample collection. We thank Doriane Picanço Rodrigues for support in DNA molecular analyses, Rafael E. de Freitas for photographs of T. paratyense, the Instituto Nacional de Pesquisas da Amazônia (INPA), Universidade Estadual do Amazonas (UEA), and Universidade Federal da Bahia (UFBA) for the infrastructure provided for morphological analyses.
References
- 1.
Wright JE. The genus Tulostoma (Gasteromycetes): a world monograph. Berlin, Stuttgart; J. Cramer; 1987.
- 2. Persoon CH. Neuer Versuch einer systematischen Eintheilung der Schwämme. Neues Magazin für die Botanik. 1794;1:63–128.
- 3.
Persoon CH. Synopsis Methodica Fungorum. Gottingae. Göttingen: Henricus Dieterich.; 1801.
- 4. Robert V, Vu D, Ben A, Amor H, van de Wiele N, Brouwer C, et al. MycoBank gearing up for new horizons. IMA Fungus. 2013;4:371–379. pmid:24563843
- 5. Wright JE. Evaluation of Specific Characters in the Genus Tulostoma Pers. Papers of the Michigan Academy of Science, Arts and Letters. 1955;40:79–87.
- 6.
Pouzar Z. Tulostoma. In: Pilát A, editor. Flora CSR: Gasteromycetes. Praha: Prace Ceskolovenske Akademie Ved.; 1958. pp. 589–613.
- 7. Jeppson M, Altés A, Moreno G, Nilsson RH, Loarce Y, De Bustos A, et al. Unexpected high species diversity among European stalked puffballs—A contribution to the phylogeny and taxonomy of the genus Tulostoma (Agaricales). MycoKeys. 2017;21:33–88.
- 8. Hussain S, Yousaf N, Afshan NUS, Niazi AR, Ahmad H, Khalid AN. Tulostoma ahmadii sp. nov. and T. squamosum from Pakistan. Turk J Botany. 2016;40:218–225.
- 9. Hernández-Navarro E, Gutiérrez A, Vargas G, Esqueda M. Nuevos registros de Tulostoma (Agaricales: Agaricaceae) de México. Rev Mex Biodivers. 2017;88:36–40.
- 10. Rusevska K, Calonge F de D, Karadelev M, Martín MP. Fungal DNA barcode (ITS nrDNA) reveals more diversity than expected in Tulostoma from Macedonia. Turk J Botany. 2019;43:102–115.
- 11. Niazi AR, Ghafoor A, Afshan N ul S, Moreno G. Tulostoma loonbanglaense: A new species from Azad Jammu and Kashmir using light and scanning electron microscopy and DNA barcoding technique. Microsc Res Tech. 2022;85:3720–3725. pmid:36214670
- 12. Esqueda-Valle M, Pérez-Silva E, Herrera T, Altés A, Moreno G. Tulostoma portoricense (Tulostomatales, Gasteromycetes) from Mexico. Mycotaxon. 1998;499–503.
- 13. Baseia IG, Galvão TCO. Some interesting Gasteromycetes (Basidiomycota) in dry areas from Northeastern Brazil. Acta Bot Brasilica. 2002;16:1–8.
- 14. Silva BDB, Calonge FD, Baseia IG. Studies on Tulostoma (Gasteromycetes) in the Neotropics. Some Brazilian species. Mycotaxon. 2007;47–54.
- 15. Cortez VG, Baseia IG, Silveira RMB. Gasteroid mycobiota of Rio Grande do Sul, Brazil: Tulostomataceae. Mycotaxon. 2009;108:365–384.
- 16. Trierveiler-pereira L, Baltazar JM, Mara R, Silveira BDA. Clarifying the typification of Tulostoma dumeticola (Agaricaceae, Basidiomycota). 2017;296:292–294.
- 17. Lima AA, Baseia IG. Tulostoma reticulatum (Agaricales, Basidiomycota), a rare stalked puffball reported from the Brazilian Atlantic rainforest. Stud Fungi. 2018;3:121–125.
- 18. Lima A de A, Melanda GC de S, Marinho P, Accioly T, Martín MP, Baseia IG. Two new species of Tulostoma (Agaricales, Basidiomycota) from the Neotropics. Turk J Botany. 2023;47:399–407.
- 19. Kreisel H, Al-Fatimi M. Further Basidiomycetes from Yemen. Feddes Repert. 2008;119:463–483.
- 20. Kuhar F., Castiglia V, Zamora JC, Papinutti L. New records and notes on gasteroid fungi of arid regions in Argentina. Sydowia. 2012;64:233–244.
- 21. Baseia IG, Milanez AI. Tulostoma Persoon (Gasteromycetes) from the cerrado region. Acta Bot Brasilica. 2002;16:9–14.
- 22. Barbosa KD, Oliveira R de L, de A de Lima A, Baseia IG, da Cruz RHSF. Tulostoma irregulireticulatum (Agaricaceae, Basidiomycota): a new species from Cerrado Biome of northeastern Brazil. N Z J Bot. 2023.
- 23. Long WH, Ahmad S. The genus Tylostoma in India. Farlowia. 1947;3:225–267.
- 24. Lee SML. Macro-fungal diversity of Bukit Timah Nature Reserve, Singapore. Gardens’ Bulletin Singapore. 2019;71:135–144.
- 25.
Dennis RWG. Fungus Flora of Venezuela and Adjacent Countries. London: H. M. Stationary Off.; 1970.
- 26. Chabrol J, Poumarat S, Courtecuisse R. Trois espèces de Tulostoma récoltés en Guadeloupe. Bulletin de la Société Mycologique de France. 2008;124:55–72.
- 27. Spegazzini C. Gasteromicetas Argentinas. Revista Soc Argent Cienc Nat. 1927;8:421–435.
- 28. Demoulin V, Dring DM. Gasteromycetes of Kivu (Zaire), Rwanda and Burundi. 1975;45:339–372.
- 29. Paloi S, Suwannarach N, Kumla J, Phonrob W, Karunarathna SC, Lumyong S. An Update on Species Diversity, Distribution and Sequence Data of Tulostoma in Asia with the Addition of Tulostoma exasperatum, A New Record for Thailand. Chiang Mai Journal of Science. 2023;50:1–14.
- 30.
Choong A. Tulostoma exasperatum var. ridleyi (Massee) J.E. Wright. In: http://iucn.ekoo.se/iucn/species_view/135331/. 2019.
- 31. Sousa JO, Suz LM, García MA, Alfredo DS, Conrado LM, Marinho P, et al. More than one fungus in the pepper pot: Integrative taxonomy unmasks hidden species within Myriostoma coliforme (Geastraceae, Basidiomycota). PLoS One. 2017;12:e0177873. pmid:28591150
- 32. Accioly T, Sousa JO, Moreau P-A, Lécuru C, Silva BDB, Roy M, et al. Hidden fungal diversity from the Neotropics: Geastrum hirsutum, G. schweinitzii (Basidiomycota, Geastrales) and their allies. PLoS One. 2019;14:e0211388. pmid:30726262
- 33. Cabral TS, Silva BDB, Martín MP, Clement CR, Hosaka K, Baseia IG. Behind the veil—Exploring the diversity in Phallus indusiatus s.l. (Phallomycetidae, Basidiomycota). MycoKeys. 2019;58:123–127. pmid:31616207
- 34. Melanda GCS, Accioly T, Ferreira RJ, Rodrigues ACM, Cabral TS, Coelho G, et al. Diversity trapped in cages: Revision of Blumenavia Möller (Clathraceae, Basidiomycota) reveals three hidden species. PLoS One. 2020;15:e0232467. pmid:32357194
- 35. Ranta P, Blom T, Niemela J, Joensuu E, Siitonen M. The fragmented Atlantic rain forest of Brazil: size, shape and distribution of forest fragments. Biodivers Conserv. 1998;7: 385–403.
- 36. Matos DMS, Bovi MLA. Understanding the threats to biological diversity in southeastern Brazil. Biodivers Conserv. 2002;11:1747–1758.
- 37. Conceição AA, Pirani JR. Diversidade em quatro áreas de campos rupestres na Chapada Diamantina, Bahia, Brasil: espécies distintas, mas riquezas similares. Rodriguésia. 2007;58:193–206.
- 38.
Stradmann MTS. Plano de manejo do Parque Municipal de Mucugê. Mucugê: Prefeitura Municipal; 1998.
- 39.
Vargas-Isla R, Cabral TS, Ishikawa NK. Instruções de coleta de macrofungos: Agaricales e gasteroides. Manaus: Editora INPA; 2014.
- 40.
Küppers H. Atlas de los colores. Barcelona: Editorial Blume; 1979.
- 41. Yafetto L. The structure of mycelial cords and rhizomorphs of fungi: A mini-review. Mycosphere. 2018;9(5): 984–998.
- 42. Bas C. Morphology and subdivision of Amanita and a monograph of its section Lepidella. Persoonia—Molecular Phylogeny and Evolution of Fungi. 1969;5:285–573.
- 43. Silva BDB, Sousa J de O, Baseia IG. Discovery of Geastrum xerophilum from the Neotropics. Mycotaxon. 2011;118:355–359.
- 44. Cabral TS, da Silva BDB. New species of Tulostoma (Basidiomycota) with reticulated spores—Additional data. Zenodo. 2023.
- 45.
White TJ, Bruns TD, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Sninsky DH, White TJ, editors. PCR protocols: A Guide to Methods and Applications. New York: Academic Press Inc.; 1990. p. 315–322.
- 46. Vilgalys R, Hester M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptoccocus species. J Bacteriol. 1990;172:4238–4246. 0021-9193/90/084238-09$02.00/0
- 47. Rehner SA, Buckley E. A Beauveria phylogeny inferred from nuclear ITS and EF1-sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia. 2005;97:84–98.
- 48. Larsson A. AliView: A fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014;30:3276–3278. pmid:25095880
- 49.
Nylander JAA. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University; 2004.
- 50. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19:1572–1574. pmid:12912839
- 51. Stamatakis A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–1313. pmid:24451623
- 52.
Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees. Proceedings of the Gateway Computing Environments Workshop (GCE). New Orleans; 2010. pp. 1–8.
- 53. Zhang J, Kapli P, Pavlidis P, Stamatakis A. A general species delimitation method with applications to phylogenetic placements. Bioinformatics. 2013;29:2869–2876. pmid:23990417
- 54. Paradis E, Schliep K. Ape 5.0: An environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019;35:526–528. pmid:30016406
- 55. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–2739. pmid:21546353
- 56. Brown SDJ, Collins RA, Boyer S, Lefort MC, Malumbres-Olarte J, Vink CJ, et al. Spider: An R package for the analysis of species identity and evolution, with particular reference to DNA barcoding. Mol Ecol Resour. 2012;12:562–565. pmid:22243808
- 57. Calonge FD, Vidal JM, Altés A. Tulostoma exasperatum Mont., from Singapore. Bolletín de la Sociedad Micologica de Madrid. 2013;37:59–63.
- 58. Aime MC, Miller AN, Aoki T, Bensch K, Cai L, Crous PW, et al. How to publish a new fungal species, or name, version 3.0. IMA Fungus. 2021;12:1–15.
- 59.
Massee G. Fungi Exotici, II. Bulletin of miscellaneous information (Royal Gardens, Kew). 1899;659[153–154]:164–185.
- 60.
GBIF.org. GBIF Occurrence. 30 Jan 2023. https://doi.org/10.15468/dl.setnsp
- 61.
Silva BDB. Estudos sobre fungos gasteroides (Basidiomycota) no Nordeste brasileiro [thesis]. Recife (PE): Universidade Federal de Pernambuco; 2013.
- 62. Tedersoo L, Mikryukov V, Zizka A, Bahram M, Hagh-Doust N, Anslan S, et al. Global patterns in endemicity and vulnerability of soil fungi. Glob Chang Biol. 2022;28:6696–6710. pmid:36056462
- 63. Silveira FAO, Negreiros D, Barbosa NPU, Buisson E, Carmo FF, Carstensen DW, et al. Ecology and evolution of plant diversity in the endangered campo rupestre: a neglected conservation priority. Plant Soil. 2016;403:129–152.
- 64. Chaves A V., Freitas GHS, Vasconcelos MF, Santos FR. Biogeographic patterns, origin and speciation of the endemic birds from eastern Brazilian mountaintops: A review. Syst Biodivers. 2015;13:1–16.
- 65. Bitencourt C, Rapini A, Santos Damascena L, de Marco Junior P. The worrying future of the endemic flora of a tropical mountain range under climate change. Flora: Morphology, Distribution, Functional Ecology of Plants. 2016;218:1–10.
- 66. Leal F, Leite FSF, Costa WPDA, Nascimento LB, Lourenço LB, Garcia PCA. Amphibians from serra do cipó, minas gerais, Brasil. VI: A new species of the Physalemus deimaticus group (Anura, Leptodactylidae). Zootaxa. 2020;4766:306–330. pmid:33056598
- 67. Silva CR, Gusmão LFP, Castañeda-Ruiz RF. Two new conidial fungi from Chapada Diamantina, Brazil. Mycotaxon. 2015;130:437–443.
- 68. Wartchow F. Amanita viridissima (Amanitaceae, Basidiomycota), a striking new species from highlands of the semiarid region of Bahia, Brazil. Plant Ecol Evol. 2016;149:241–248.
- 69. Vieira LC, da Silva DKA, da Silva IR, Gonçalves CM, de Assis DMA, Oehl F, et al. Ecological aspects of arbuscular mycorrhizal fungal communities in different habitat types of a Brazilian mountainous area. Ecol Res. 2019;34:182–192.
- 70. Miranda L, Pereira MC, Veloso TGR, Tartarine N, de Carvalho SBG, Kasuya MCM. Endophytic fungi in roots of native orchids of rupestrian grasslands (campos rupestres) in Serra do Cipó, Brazil. Iheringia—Serie Botanica. 2021;76:1–12.
- 71.
sosma.org.br. SOS Mata Atlântica. [cited 8 Oct 2023]. https://www.sosma.org.br/