Leucoagaricus Locq. ex Singer is a genus of Agaricaceae and comprises approximately 135 species (He et al., 2019). Representatives of the genus are saprotrophic and grow in soil, mostly in tropical and subtropical regions (Singer, 1986; Vellinga, 2004a). The genus is characterized morphologically by an entire or slightly striate pileus margin that is never plicate, free lamellae, metachromatic basidiospores in Cresyl blue, and absence of clamp connections and pseudoparaphyses (Singer, 1948, 1986).

Several molecular studies have demonstrated that the genus is heterogeneous and may not be monophyletic since species of Leucoagaricus and Leucocoprinus Pat. were recovered intermixed in a single clade (Johnson & Vilgalys, 1998, Johnson, 1999; Vellinga, 2004b; Vellinga et al., 2011). Because of the large number of species in the genus, the small number of taxa included in molecular studies and the low support in inferred phylogenies, the circumscription of Leucoagaricus (and conversely Leucocoprinus) remains unclear (Vellinga, 2004b; Vellinga et al., 2011). Neotropical taxa are poorly represented in these studies and most sequenced samples are of European and North American species (Johnson, 1999; Vellinga, 2004b; Vellinga et al., 2011). However, recent papers have examined phylogenetic relationships among New World species, describing new taxa from Brazil (Heisecke et al., 2021) and the Dominican Republic (Justo et al., 2015, 2021). The ant-fungus mutualism between La. gongylophorus (Möller) Singer and the leaf-cutter ants has also been studied with phylogenetic approaches (Chapela et al., 1994; Mueller et al., 1998, 2001; Pagnocca et al., 2001; Silva-Pinhati et al., 2004; Ješovnik et al., 2017).

Knowledge of Brazilian fungal diversity has improved in the last decade (Maia & Carvalho-Jr., 2010; Forzza et al.,2010, 2012; Maia et al.,2015). As has the quantity of DNA sequences from Brazilian fungal samples deposited in GenBank (Menolli & Sánchez-Garcia, 2019). Even so, Leucogaricus remains understudied and its current taxonomic understanding in Brazil is based on regional treatments or species lists (Menolli & Sánchez-Garcia 2019). Twenty species of Leucoagaricus have been reported from Brazil (Spegazzini, 1889; Möller, 1893; Rick, 1920, 1937, 1961; Singer, 1949, 1953, 1989; Grandi et al., 1984; Batista, 1957; Muchovej, et al., 1991; Pegler, 1997; Spielmann & Putzke, 1998; Maia et al., 2002; Sobestiansky, 2005; De Meijer, 2006; Albuquerque et al., 2007; Rother & Silveira, 2008, 2009; Wartchow et al., 2008; Rosa & Capelari, 2009; Ferreira & Cortez, 2012; Magnago et al., 2013; Heisecke et al., 2021). Among them, seven species (Leucoagaricus abbreviated below as La.) have been described from specimens collected in Brazilian territory: La. confusus (Rick) Singer (Rick, 1937, 1961; Singer, 1949, 1953), La. gongylophorus (Möller) Singer (Möller, 1893; Spielmann & Putzke, 1998), La. nzumbae C. Heisecke, A.A. Carvalho & M.A. Neves (Heisecke et al., 2021), La. olivaceomamillatus Singer (Rick, 1920, 1961; Singer, 1949, 1953), La. tricolor Singer (Singer, 1989), La. imperialis (Speg.) Pegler (Spegazzini, 1889; Pegler, 1997), and La. weberi Muchovej, Della Lucia & R. Muchovej (Muchovej et al., 1991).

Here we describe Leucoagaricus taniae, a new species from Santa Catarina State, Brazil, based on morphological and molecular data. This is the first species of the genus known to grow in sand in the coastal restinga ecosystem in Brazil (Araujo et al., 1998). This is unusual because species of Leucoagaricus are reported to need a higher availability of nutrients than other Lepiotaceous fungi (Vellinga, 2004b), and restinga ecosystem soils are chemically poor with the main sources of nutrients being salt spray deposition (Leão & Dominguez, 2000; Scarano, 2002; Barcelos et al., 2012) and flooding (Magnago et al., 2010). Only four species of Leucoagaricus have been reported from sandy coastal environments to date: La. gaillardii Bon & Boiffard from France (Bon & Boiffard, 1974; Gennari et al., 1995), La. menieri (Sacc.) Singer also from France (Saccardo, 1891), La. singeri (Bon ex Contu & Signor.) Consiglio & Contu from Italy and Uruguay (Singer, 1968; Bon, 1993; Consiglio & Contu, 2004), and La. subvolvatus (Malençon & Bertault) Bon from Italy, Morocco, and Spain (Gennari et al., 1995). Another species that could be related to this sand inhabiting group of species is Amanita cystidiosa O.K. Mill. & Lodge, which also was recorded for sand dunes in Puerto Rico (Miller et al., 2000), and was cited by Vellinga (2010) as a Lepiotaceous fungus. However, it was not formally transferred to Leucoagaricus.

Material and methods

Throughout this manuscript the abbreviation L. is used for Lepiota (Pers.: Fr.) S. F. Gray, La. for Leucoagaricus and Lc. for Leucocoprinus.

Field Sampling and Morphological Studies

A total of 5 collections of La. taniae were made at Armação beach on the south of Santa Catarina Island. Additionally, 11 other collections of Lepiota s.l. from Santa Catarina State were included in this study (Table 1). The basidiomes were collected and characterized as follows. A small portion of the basidiome was preserved in silica gel for DNA extraction. Macromorphological characters were studied from fresh material following Lodge et al. (2004) and Largent (1986). Color codes (e.g., 1A1) were assigned according to Kornerup and Wanscher (1978). Micromorphological studies were made from hand cut sections of dried specimens mounted in water, or 3% KOH, or 3% KOH plus Congo red, Cresyl blue, or Melzer’s reagent (Largent, 1977; Singer, 1986). At least 20 of each structure were measured for each collection. The length-width ratio of the basidiospores is indicated by Q, and its average by avQ (Largent et al., 1977; Vellinga, 1988). Descriptive terminology follows Vellinga (1988) and Vellinga and Noordeloos (2001).

Table 1 Specimens and sequences used for the molecular analyses. The sequences generated in this work are in bold.
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DNA Extraction, Amplification and Sequencing

DNA was extracted using the CTAB method (Dentinger et al., 2010). PCR amplifications of the nuclear rITS1–5.8S-ITS2 (ITS) loci were made with the primers ITS8F and ITS6R, following the procedures described by Dentinger et al. (2010). PCR products were purified with Polyethylene glycol (PEG) 20%. Sequencing was undertaken at Fundação Oswaldo Cruz (Fiocruz), Minas Gerais State, Brazil. The generated sequences and their respective chromatograms were manually checked and edited with Geneious 6.1.8 (Kearse et al., 2012), and then verified with BLAST searches. The sequences were deposited in GenBank (accession numbers MT952873–MT952884).

Taxon Sampling, Alignment and Phylogenetic Analyses

Sequences of the internal transcribed spacer (ITS) from 57 specimens were used for molecular analyses and are detailed in Table 1. Twelve new sequences of ITS were generated for this work, one corresponds to an accession of new species and the others to accessions of Chlorophyllum (1), Leucoagaricus (5) and Leucocoprinus (5) from Santa Catarina, Brazil (see Table 1). The ingroup includes representatives of Leucoagaricus and Leucocoprinus. The sequences chosen for comparison were selected based on results of a BLAST search for the new species sequence; only returned sequences with Expect (e) values of 0.0 were chosen. In addition, sequences of Leucoagaricus species that exhibit drops on the pileus surface and other putatively related species were included (Vellinga & Balsley, 2010, Yuan et al., 2014; Dutta et al., 2021); as were Leucoagaricus species that occur on sand (Gennari et al., 1995; Vellinga et al., 2011; Munoz et al., 2015); whitish and fleshy species of the genus (Vellinga, 2004b; Vellinga & Davis, 2006); and La. nzumbae, recently described from Brazil (Heisecke et al., 2021). Sequences of Lepiota atrodisca Zeller, L. besseyi H.V. Sm. & N.S. Weber, L. subclypeolaria (Berk. & M.A. Curtis) Sacc. and L. roseolivida Murrill were included in the ingroup because they fall into the Leucoagaricus clade even though the new combinations transferring them into Leucoagaricus have not yet been made. Three sequences of Chlorophyllum were chosen as outgroups (Vellinga et al., 2011).

Sequences were aligned using the MAFFT 7 (Katoh, 2013) plugin in the Geneious software package with the E-INS-I strategy. The aligned matrix is available in Suppl. Material 1. Phylogenetic analyses using Maximum Likelihood (ML) and Bayesian Inference (BI) were performed. The aligned matrix was divided in three partitions corresponding to ITS1, 5.8S and ITS2. ML analysis was carried out in RAxML 8.2 (Stamatakis, 2014) using the rapid bootstrap algorithm with 1000 replicates associated with a search of the best-scoring ML tree, the GTRGAMMA model, and other default parameters. The best nucleotide substitution model for each partition was estimated using jModelTest 2.1 (Darriba et al., 2012) and selected based on the Akaike Information Criterion (AIC). The best models were GTR+G+I for ITS1 and ITS2, and GTR+I for 5.8S. BI analysis was performed using MyBayes 3.2 (Ronquist et al., 2011) with two independent analyses, each with four Monte Carlo Markov Chains, 20,000,000 generations, sampling frequency of 1000, and burn-in of 10%. The best nucleotide substitution models as described above were applied for each partition. Results from BI were examined in Tracer 1.6 (Rambaut et al., 2014) to ensure convergence of the analyses and sufficient sample size of each parameter (>200). The results were visualized in FigTree 1.4 (http://tree.bio.ed.ac.uk/software/figtree). All analyses were done using the CIPRES Science Gateway 3.3 (Miller et al., 2010).

Results

Phylogeny

The ITS matrix was 858 characters in length; 54% of the characters were variable and 43.6% were parsimony informative. The ML and BI analyses recovered trees with congruent topologies. Only the ML tree with both BS and BPP values is shown here (Fig. 1). ITS sequences from representatives of Leucoagaricus and Leucocoprinus were mixed together in a single well-supported clade (PP 1.0, BS 99%), suggesting, as reported previously by Johnson and Vilgalys (1998), Johnson (1999), Vellinga (2004b), and Vellinga et al. (2011), that the two genera are not reciprocally monophyletic. Leucoagaricus taniae was placed in a strongly supported clade (PP 1.0, BS 83%), together with two other undetermined Brazilian species of Leucocoprinus (CHC101/MT952882 and CHC102/MT952883).

Fig. 1
figure 1

Phylogenetic tree based on Maximum Likelihood analysis of ITS sequences showing relationships among Leucoagaricus taniae and related species. Posterior probability (PP) (above 0.7) and bootstrap (BS) (above 50%) support values are shown above branches (PP/BS). The sequences generated in this work are in bold. GenBank accession numbers are given for each terminal followed by the country of origin of the specimen (2-letter codes given by the ISO). Chlorophyllum species were used as the outgroup.

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Taxonomic Treatment

Leucoagaricus taniae C.Heisecke & M.A.Neves sp. nov. MycoBank: MB838221.—Type: Brazil: Santa Catarina, Florianópolis, Área de Preservação Permanente Praia da Armação, 27°44′39”S, 48°30′28.3”W, 3 June 2018, Neves MAN1266 (holotype: FLOR [!]; isotype: BR [!]). (Figs. 2 and 3.)

Fig. 2
figure 2

Leucoagaricus taniae basidiomes in situ. A. MAN1188 with droplets at the pileus. B & C. MAN1266 (holotype). [Scale bar = 10 mm. Photos by M.A. Neves.]

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Fig. 3
figure 3

Leucoagaricus taniae (MAN1266 Holotype). A. Basidiospores. B. Basidia. C. Cheilocystidia. D. Pileus covering. [Scale bars = 10 μm. Illustration by C. Heisecke.]

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Diagnosis—Most similar to Leucoagaricus amanitoides R.M.Davis & Vellinga, but distinguished by the presence of yellow-brown drops on the pileus when fresh (versus the absence of any exudate drops in La. amanitoides), a relatively narrower stipe that can reach approximately 11 mm wide in the center and 22 mm wide at the bulbous base (vs. 20 mm wide in the center and ca. 30–35 mm wide in the turbinated bulbous base in La. amanitoides), and cheilocystidia that are broadly ellipsoidal to subglobose (vs. narrowly clavate and often slightly capitate in La. amanitoides).

Description.—Pileus 27–50 mm diameter, hemispherical when young, later expanding to plano-convex or applanate, sometimes slightly depressed at the center; pileus surface dry, smooth to slightly radially fibrillose, white to yellowish white (1A1, 1A2) or light orange to yellowish brown (5A4–5, 5D5) when fresh, producing liquid exudates; margin often exceeding the lamellae, straight, entire; context 5 mm broad, white, with no change in color. Lamellae, crowded, free, ventricose, 3–5 mm wide, white becoming cream to light yellow in older basidiomes; edges entire to slightly serrulate. Stipe 65–80 × 10–11 mm, central, cylindrical with a bulbous base 17–22 mm wide; context fistulose; surface fibrillose. Annulus inferior, white, membranous, fragile, easily detachable, and ascending. Basidiospore print pale yellow (1A3). Smell and taste not recorded.

Basidiospores [375, 10, 5] 7.2–10 × 5.3–7.3 μm, on average 8.5 × 6 μm, Q = 1.38–1.64, avQ = 1.37, ellipsoid to broadly ellipsoid to obovoid, aguttulate or uniguttulate, yellowish in water, pale to hyaline in KOH, dextrinoid, metachromatic, thick-walled, without germ pore but an inconspicuous germ pore is visible in Melzer. Basidia 22.2–38.4 × 9.6–12.8 μm, clavate, 4-spored. Lamella edges sterile. Cheilocystidia 23.6–31.3 × 10.3–17 μm, ellipsoidal, broadly ellipsoidal to subglobose, generally short-pedunculate, hyaline, slightly thick-walled. Pleurocystidia absent. Hymenophoral trama subregular, subhymenium cellular. Pileus covering a cutis made up of interwoven, cylindrical to slightly articulated repent hyphae, up to 13 μm diameter, pale yellowish in water and paler in KOH, thick-walled. Stipe covering a cutis made of repent cylindrical hyphae, hyaline in water and KOH, thin-walled. Scattered oleiferous hyphae present in all parts of the basidiome, most numerous in the pileus context. Clamp connections absent.

Habitat and distribution.—Scattered or in small groups over a large area of the beach. It can be difficult to spot because it is almost completely buried in the sand with only part of the pileus above the sand layer, among bushes of Canavalia rosea and Cyperus pedunculatus. Even though both plant species have a wide distribution along the coast of Brazil, they are restricted to beach habitats (Brazil Flora Group, 2021), and the coarse sand found in the Armação beach is uncommon in other areas of the coast. In the last ten years, several mycologists have undertaken mycological surveys at other sites where the same plants occur, but this new species has not been seen. Armação beach is subjected to tidal fluctuations, and almost every year the ocean invades the sand area, disturbing the vegetation that grows there and creating a novel set of conditions (Neves, personal observation).

Etymology.—In honor of Tania Cuturi, whose name was suggested by one of the Synchronicity Earth supporters, a UK conservation charity, which donated financial support that helped this research.

Common name.—Seashell mushroom (cogumelo-concha-da-praia) because it looks like a seashell, and it grows on the sand at the beach, among seashells.

Conservation status.—As discussed above, Leucoagaricus taniae is only known from one small strip of beach in Southern Brazil, on Santa Catarina Island, Florianópolis, in the Área de Preservação Permanente Praia da Armação. The authors know of no other sites in Santa Catarina or Southern Brazil with similar characteristics. So, even though La. taniae is difficult to find as its basidiomes grow buried in the sand, its very restricted distribution is likely due to habitat specificity and not low detectability. The beach is approximately 3.2 km long and 5–20 m wide, and the species has been recollected at this site over several years. The species is assessed as Critically Endangered under the IUCN Red List D criterion (IUCN, 2012; IUCN Standards and Petitions Committee, 2019), given that (1) the number of sites with suitable habitat is small, (2) survey work along the coast of Southern Brazil has been ongoing for over ten years, and (3) the total population size of the species is estimated to be less than 50 based on recommendations for inferring population size by Dahlberg & Mueller (2011). It does not meet criterion B as there is no evidence of continuing decline in population size. While the species appears resilient to tidal fluctuations, increased tourism, ongoing illegal development, and rising sea level due to climate change are threats (Barcelos et al., 2012, Clark et al., 2015). As with almost all fungi, continued fieldwork to confirm its range and better understand its ecological specificity is needed.

Additional specimens examined.—BRAZIL. Santa Catarina: Florianópolis: Área de Preservação Permanente Praia da Armação, 27°44′39”S, 48°30′28.3”W, 07 May 2016, Smith & Drewinski MAN1206 (FLOR, RB); 22 May 2016, Neves & Smith MAN1187 (FLOR); 26 May 2016, Neves & Caddah MAN1188 (FLOR); 03 June 2018, Neves & Smith MAN1267 (FLOR).

Notes.—Leucoagaricus taniae is characterized macromorphologically by fleshy basidiomes, a smooth and white to yellowish pileus with yellow-brown drops on the surface when fresh, and a bulbous stipe. Micromorphogically it is characterized by broad basidiospores, broadly ellipsoidal to subglobose cheilocystidia, and a pileus covering composed of pale to hyaline repent hyphae. Morphologically, the most similar species is La. amanitoides described from the vast Central Valley of California, U.S.A. Both species have whitish pilei, bulbous stipes and cuticular pileus coverings (Vellinga & Davis, 2006). Leucoagaricus amanitoides was not reported as producing drops on its pilei. It also differs by the wider stipe that can reach approximately 20 mm wide in the center and around 30–35 mm in the turbinated bulbous base, and narrowly clavate to narrowly lageniform cheilocystidia (Vellinga & Davis, 2006).

Leucoagaricus menieri, La. subvolvatus, and La. volvatus Bon & A.Caball. (Bon, 1993; Vellinga, 2001) from Europe also have some similarities to La. taniae. These three European species have fleshy and whitish basidiomes with a bulbous stipe and cutis-like pileus covering, but they can be easily distinguished by their cheilocystidia with crystals on the apex (Bon, 1993; Gennari et al., 1995; Vellinga, 2001). Leucoagaricus gaillardii resembles La. taniae as it also grows in the sand, has a bulbous stipe and basidiospores of the same size as La. taniae, however the pileus is pinkish, and the cheilocystidia are narrowly lageniform to cylindrical and wavy (Bon & Boiffard, 1974; Gennari et al., 1995).

Amanita cystidiosa from Puerto Rico (Miller et al., 2000) and La. singeri reported from Uruguay (Singer, 1968; Bon, 1993; Consiglio & Contu, 2004) are sand inhabiting species and have similar basidiomes as La. taniae. However, A. cystidiosa can be distinguished by its white basidiospore print, cylindrical to flexuose cheilocystidia and inamyloid basidiospore (Miller et al., 2000). While La. singeri has variable basidiospores, pseudoparaphyses are present and cheilocystidia were not observed (Singer, 1968; Bon, 1993; Consiglio & Contu, 2004).

Other fleshy white or whitish species with widespread distributions that have been reported from Brazil are: Leucoagaricus barssii (Zeller) Vellinga, which was described from the U.S.A. and is also known from Europe, and La. leucothites (Vittad.) Wasser, which although it was described from Italy is also known from the Southern Hemisphere (Rick, 1937, 1961; Grandi et al., 1984; De Meijer, 2006; Rosa & Capelari, 2009). Leucoagaricus barssii differs from La. taniae by the whitish to greyish fibrillose pileus surface, stipe tapering downward, narrower basidiospores, and narrower cheilocystidia with greyish content (Zeller, 1934; Vellinga, 2000, 2001). Leucoagaricus leucothites has a cylindrical stipe, sometimes with a small bulbous base, cylindrical and narrowly clavate to irregular cheilocystidia and trichodermal pileus covering (Bon, 1993; Vellinga, 2001).

The presence of exudate drops is considered an important diagnostic character for the taxonomy of Lepiotaceous fungi (Kumar & Manimohan, 2004; Vellinga & Balsley, 2010; Yuan et al., 2014). However, the pigmentation and portion of the basidiome where the exudate is produced varies greatly in Lepiota s.l. (Vellinga & Balsley, 2010; Yuan et al., 2014). The chemical composition of the drops and factors involved in their production are still unclear (Vellinga & Balsley, 2010; Yuan et al., 2014). In La. taniae, the drops are golden and were observed only on the pileus surface of some fresh basidiomes. They were not observed on the stipe or annulus.

Other Leucoagaricus species that exhibit drops on their basidiomes are La. brunneodiscus A.K.Dutta & K.Acharya from India, La. dacrytus Vellinga from the U.S.A., La. tangerinus Y.Yuan & J. F.Liang from China and La. tener (P.D.Orton) Bon from Great Britain (Vellinga & Balsley, 2010; Yuan et al., 2014; Orton, 1960; Bon, 1993; Dutta et al., 2021). These four species are closely related to each other and were placed by the phylogenetic analysis in a clade, together with L. cf. atrodisca (ecv237 and ecv3265), which also forms droplets on the pileus, but this clade was relatively far removed from that containing La. taniae (Fig.1; Vellinga & Balsley, 2010; Yuan et al., 2014; Dutta et al., 2021). Morphologically, those species differ from La. taniae by the smaller and more delicate basidiomes, stipe without a bulbous base, darker pileus surface, and narrower basidiospores (Vellinga & Balsley, 2010; Yuan et al., 2014; Orton, 1960; Bon, 1993; Dutta et al., 2021).

Our phylogenetical analyses (Fig.1) recovered an unexpected relationship among La. taniae and related species within the Leucoagaricus/Leucocoprinus clade. Based on morphology, it was expected to be closely related to La amanitoides, La. leucothites or La. subvolvatus and allies. Instead, La. taniae was recovered in a group with Leucocoprinus species with delicate and fragile basidiomes and dark fibrils on the pileus. However, to better elucidate relationships in this group, more taxonomic and phylogenetic studies that include other similar species and more markers are needed.