Geastrum tupiense: a new earthstar fungi species from the Brazilian Atlantic Forest and a new insertion in the Velutina subsection (Geastrales, Basidiomycota)

Abstract Geastrum tupiense is a new species belonging to subsect. Velutina (Sect. Myceliostroma). The fundamental diagnostic characteristics are reddish immature basidiomata and rhizomorphs with bipyramidal crystals. It was found in two areas of the Atlantic Forest biome, considered one of the main hotspots in the world for its high level of endemism and biodiversity. Delimitation of this new species was based on morphological characters and phylogenetic analyses of ITS/LSU nrDNA. Descriptions, phylogenetic tree, photographs, and taxonomic discussion are provided.

Rodriguésia 74: e00482022.2023 In the genus Geastrum some species complexes have already been proven, such as: Geastrum hirsutum Baseia & Calonge, G. schweinitzii Zeller and G. triplex Jungh.based on molecular techniques, mainly with data from the ITS and LSU region that provide support on identification in this fungal group (Kasuya et al. 2012;Accioly et al. 2019), besides showing that several other species await formal names and descriptions (Zamora et al. 2015).
The Atlantic Forest is an important Brazilian Biome that runs along the coast of the Country from northeast to south, composed mainly of two types of vegetation: coastal forest and Atlantic semideciduous forest (Morellato & Haddad 2000).Despite the efforts of researchers and conservation programs created by nongovernmental organization (such as "SOS Mata Atlântica") in the last few decades to alert and halt the deforestation of this biome, less than 8% of its original vegetation cover remains intact (Faria et al. 2006).This fact is alarming, since the Atlantic Forest was ranked 4 th in the level of importance of global hotspots by Myers et al. (2000).Due to the various ecosystems associated with this vegetation domain, the diversity of species may be even greater than what has been observed so far in the Amazon rainforest (Morellato & Haddad 2000).
The Atlantic Forest is also representatively important for the Brazilian mycobiota.Even with numerous non-inventoried areas (most of them occurring only in areas of environmental protection), only the genus Geastrum has 50 species catalogued for this biome, including the recently described new species: G. pusillipilosum J.O. Sousa, Alfredo, R.J.  (Accioly et al. 2019); and two of the five species already mentioned as new to Brazil after Accioly et al. (2019): G. lanuginosum (Crous et al. 2019); and G. calycicoriaceum (Crous et al. 2020).Thus, this study contributes to the growth of knowledge about Geastrum in Brazil with the description of a new species endemic to the Atlantic Forest.Besides that, it provides new data for the phylogeny of the group, which is essential for modern taxonomic studies that are not based only on the specimen's morphology.

Collection site
The specimens were collected in two Atlantic Rainforest regions of Brazil (Fig. 1): the Mata do Pau Ferro State Park is located in the municipality of Areia, microregion of Paraiba Marsh (Northeastern Brazil) with an area of about 607 ha.Due to the orographic effect on precipitation and temperature reduction, this forest fragment is considered "Brejo de Altitude" and is part of the Atlantic Forest biome (Freitas et al. 2020).The second area, Serra de Santa Rita Mitzi Brandão Biological Reserve, is an important region of water supply in the state of Minas Gerais (Southeastern Brazil), located in the municipality of Santa Rita do Sapucaí.It belongs to an area classified as a transition between the Semideciduous Seasonal Forest and the Mixed Ombrophylous Forest and has an average annual rainfall of 1,500 mm (Alfredo et al. 2016).

Morphological analyses
The materials were analyzed in the Laboratório de Biologia de Fungos (LBF), Departamento de Botânica e Zoologia (DBZ) -Universidade Federal do Rio Grande do Norte (UFRN) using as reference Sunhede (1989), Baseia et al. (2014), Sousa et al. (2014) and Zamora et al. (2014) for the methodological procedures and morphological analyses of the specimens.Analyses of macroscopic characters were done based on detailed observations with the naked eye and with the aid of a stereo microscope.The color chart of Kornerup & Wanscher (1978) was used to determine and describe the colors.For observation of the microstructures of the specimens, small portions of the basidiomata were removed by hand, under a stereo microscope, and the layers were separated with the aid of two histological tweezers and placed on separate microscopic slides containing 5% potassium hydroxide (KOH).The basidia were analyzed using Congo red dye for better visualization.All the microstructures were separated and observed under a Nikon Eclipse Ni (LM) optical microscope with Nikon DS-Ri1 camera attached, usually using 40x or 100x magnification objectives (Baseia et al. 2014).At least 30 basidiospores (including ornamentations) and 20 measurements of the other microstructures were randomly chosen and measured on the microscopic slide.All measurements and image captures were performed using the software NIS -Elements AR v.4.51.00.Statistical measurements such as the mean of the basidiospore diameter and height, standard deviation (x ± SD, respectively), and the ratio between mean width and height (Q m ) followed the methodology proposed by Sousa et al. (2017).Geastrum tupiense differs from other Geastrum species mainly by reddish unexpanded basidiomata with coriaceous surface and wrinkled regions; mycelial layer surface coriaceous with hyphae inflated at the apex (until 11.2 μm diam.) and rhizomorph hyphae covered by bipyramidal crystal aggregates.

Molecular analyses
Unexpanded basidiomata epigeous, reddish when fresh (7C8), dark blond (5D4) to bronze (5E5) when dried, subglobose with umbo when   In honor of the Tupinambá indigenous people who inhabited the Brazilian coast, where the new species was collected.
The specimens were found in the Atlantic Rainforest of the states of Paraiba and Minas Gerais, Brazil.Growing on wood and leaf litter, with gregarious habit.Until now, the distribution of G. tupiense is restricted to Brazilian Atlantic Rainforest.
As shown by our phylogenetic analyses that section Exareolata De Toni and sect.Myceliostroma (Henn.)P.Ponce de León , subsect.Velutina J.C. Zamora form well delimited phylogenetic clusters with a high level of confidence (100% posterior probabilities).Geastrum fornicatum (Huds.)Hook.was used as an outgroup because it presented molecular and morphological characters distinct from the species included in the analyses.(ITS and LSU concatenated, Fig. 3).Geastrum tupiense is inserted in sect.Myceliostroma, subsect.Velutina.Inside this subsection, the species G. velutinum Morgan and G. calycicoriaceum (Crous et al. 2020) has some features in common with G. tupiense (mycelial layer with coriaceous surface, delimited peristome, presence of subiculum and rhizomorph).Geastrum velutinum differs from G. tupiense by the fibrillose peristome, velutinous tufts in the mycelial layer, brownish to violet endoperidium, and basidiospores less than 4 μm (Dissing & Lange 1962).Geastrum calycicoriaceum is distinguished by the dark color in the unexpanded badisiomata, mycelial layer predominantly ephemeral, peelingoff forming a cup under the fruit body and composed of thin hyphae (up to 5 μm), smaller basidiospores (up to 4.1 μm) with smaller warts (up to 0.5 μm), as well as rhizomorph with acicular crystals (Crous et al. 2020).

Discussion
The identification of fungi through classical taxonomy using only morphological aspects can aggravate taxonomic problems, especially when the objects of study are organisms with identical morphology or that present inconspicuous differences in their phenotype, known as cryptic or semi-cryptic species (Vizzini et al. 2013).This is mainly because taxonomists assign different weights to morphological characteristics, in addition to the subjectivity relating to some Rodriguésia 74: e00482022.2023 characters, generating a barrier to the establishment of widely accepted classifications (Stielow et al. 2011).Integrative taxonomy using at least two identification perspectives (such as morphological and molecular analysis) is on its way to becoming an excellent alternative in solving problems like these.
The problem generated by identification based only on morphological characters can create a range of hidden species known as species complexes: these organisms are named and attributed to existing species, masking their real diversity (Accioly et al. 2019).Geastrum calycicoriaceum and G. tupiense are phylogenetically close and are inserted in the subsect.Velutina.This subsection is characterized by humicolous species with a well-developed rhizomorph system covered by narrow prism-shaped crystals, and basidiomata not caespitose (Zamora et al. 2014).Both species are from South American (Neotropical) and they were previously identified as G. javanicum, despite having as type locality Java Island (Southeast Asia), a distinct ecoregion (Indo-Malay) (Olson et al. 2001).Furthermore, G. javanicum presents uncertainties regarding its nomenclature and systematic position.According to Ponce de Léon (1968), using only morphological data, G. javanicum and G. velutinum are considered synonyms.These species (G. calycicoriaceum, G. tupiense, G. velutinum and G. javanicum) have almost similar phenotypic characteristics and may be a new species complex in the genus Geastrum.From the revision of the genus proposed by Zamora et al. (2014), using both morphology and molecular tools, the type collection of the Geastrum javanicum was not recovered; thus, it was considered "enigmatic".Therefore, we hope that our work can contribute to more robust phylogenetic studies involving the subsection Velutina that can elucidate the incongruities related to the species in this subsection.
The molecular procedures of DNA extraction, amplification, and purification were performed at the Laboratório de Genética Molecular de Plantas (LGMP), Departamento de Biologia Celular e Genética -UFRN.Fragments of the gleba were used for DNA extraction.The extraction protocol used was an adaptation of the QIAGEN DNeasy Plant Mini Kit protocol (Qiagen, Valencia, California, U.S.A.), based on Martin & Winka (2000) for fungal studies.The amplification was performed by polymerase chain reaction (PCR), having as target regions: the Internal Transcribed Spacer (ITS) and Large Subunit region (LSU) of nuclear ribosomal DNA.Amplification, purification and sequencing protocols, and the phylogenetic analyses of ITS/LSU nrDNA were based onAccioly et  al. 2019, deposited in protocols.io(<dx.doi.org/10.17504/protocols.io.wpdfdi6>).The ITS and LSU sequences of the new species were compared with homologous sequences from GenBank (Tab.1), from collections already published with both sequences (ITS and LSU).ResultsTaxonomyGeastrum tupiense J.O.Sousa, sp.nov.Mycobank: 839424.

Figure 1 -
Figure 1 -Map showing the collection sites, Mata do Pau Ferro State Park (PB, Brazil) and Serra de Santa Rita Mitzi Brandão Biological Reserve (MG, Brazil).

Figure 3 -
Figure 3 -Phylogenetic tree based on combined dataset (ITS/LSU).Section clades are colored and named on the right.Tree topology based on Bayesian analyses.Geastrum fornicatum was used as out-group.Numbers on branches are Parsimony and Maximum likelihood bootstrap, as well as Posterior Probabilities from Bayesian analysis.Asterisk (*) represents the fully supported branches in Parsimony, Maximum likelihood and Bayesian analysis.The new Geastrum species is indicated in bold.

Table 1 -
Specimens and sequences used to recover the phylogenetic trees.The access numbers of the sequences of the new species Geastrum tupiense are indicated with asterisks.