Stephanosporamayana (Stephanosporaceae, Russulales), a new sequestrate fungus from Yucatán Peninsula, Mexico

Abstract Stephanosporamayana is presented as a new species from the Yucatán Peninsula, Mexico. This species is distinguished by the yellowish pileus, basidiospores with a small corona (4–6 × 1–2.5 µm), and variable size (8.0–17.0 × 6.0–11.0), thin pileus (21–40 µm) and the ecological association to lowland forest with Haematoxylumcampechianum, Gymnopodiumfloribundum, Coccolobadiversifolia, Metopiumbrownei and Pinuscaribaea. It differs from the American species of Stephanospora, like S.michoacanensis and S.chilensis, by its larger basidiospores. Descriptions, photographs and discussions are presented.


Introduction
The species within Stephanospora Pat. were previously accommodated in Hymenogastraceae Vittad by Cunningham (1979) as Octaviania Vittad. and also in Octavianiaceae Locq. ex Pegler & T.W.K. Young by Pegler and Young (1979), due to the spiny basidiospores. They also included Hydnangium Wallr., Sclerogaster R. Hesse and Wakefieldia Corner & Hawker. Nevertheless, Overwinkler and Horak (1979) placed these species in the family Stephanosporaceae Overwinkler & Horak along with Lindtneria Pilát. due to the spines around the basidiospore base, forming what is called a corona. Actually, Stephanosporaceae includes both sequestrate and resupinate species with or without a corona (Martín et al. 2004;Vidal 2004;Castellano et al. 2007;Lebel et al. 2015). Stephanospora is a genus with sequestrate species characterized by the subhypogeous habit, spiny or crested basidiospore ornamentation, and the conspicuous corona at the basidiospore base. Most of the species have a yellowish to orange pileus, pale-orange, olive-grey to pale-brown hymenophore and lack a stipe (Castellano et al. 1986;Pegler et al. 1993;Montecchi and Sarasini 2000;Vidal 2004). According to Lebel et al. (2015), 15 species are recognized worldwide.
Most Stephanospora species grow in association with broadleaf trees in Oceania (Cunningham 1979;Bougher and Lebel 2001;Lebel et al. 2015) or temperate forest in Europe (Palacios and Lakisbar 1991;Pegler et al. 1993;Vidal 2004;Fraiture and Novello 2013) and America (Vidal 2004;Guevara-Guerrero et al. 2015). Some additional undescribed species and genetic sequences were mentioned by Lebel et al. (2015) from Belize, Costa Rica and the Caribbean. In the USA, no species have been described from fruiting bodies, but DNA sequences have been included in a couple of analyses (Edwards and Zak 2010;Lebel et al. 2015). Most species can be found growing under mycorrhizal trees species such as Podocarpus, Eucalyptus, Quercus or Pinus, but no evidence of ectomycorrhizal associations has been observed (Tedersoo et al. 2010). The genus is represented in Mexico so far by a single species, S. michoacanensis Guevara & Castellano from central Mexico (Guevara-Guerrero et al. 2015).
In recent mycological exploration conducted by us on the Yucatán Peninsula in southern Mexico, some interesting sequestrate fungi were found, collected and identified as Stephanospora. The specimens were collected under Haematoxylum campechianum L., Gymnopodium floribundum Rolfe, Metopium brownei (Jacq.) Urband, and Pinus caribaea Morelet in lowland forest and pine savanna. Due to the basidiospore size, small corona, the association to lowland forest and pine savanna, and a molecular analyses of DNA we conclude that it is a novel species and we propose it as S. mayana de la Fuente, García-Jiménez, Guevara-Guerrero & Oros-Ortega.

Sampling data
Basidiomata were collected at Calakmul municipality in the state of Campeche and Othón P.  describing sequestrate fungi were used (Castellano et al. 1986). Hand cuts sections were made from dried specimens mounted in KOH 5% and Meltzer reagent for microscopic description. Colour terminology was according to the Handbook of Colour (Kornerup and Wanscher 1978). All the specimens were curated and deposited at the mycological herbarium José Castillo Tovar of Instituto Tecnológico de Ciudad Victoria (ITCV).

Molecular analysis
For DNA extraction from basidiomata tissue we used the protocol reported by Cordova et al. (2014). Briefly, 0.1 g of the tissue was pulverized in liquid nitrogen and 1 ml extraction buffer of CTAB (20 mM EDTA pH 8.0, 100 mM Tris-HCl pH 8.0, 2% CTAB, 1.4 M NaCl, and 2-mercaptoethano) was add and incubated for 20-30 min at 65 °C and then vigorously mixed with a solution of phenol-chloroform isoamyl alcohol. After centrifugation, the supernatant was precipitated using cold isopropanol and sodium acetate and then incubated at -20 °C for 1 h. The DNA was pelleted by centrifugation and dried at room temperature. Finally, the DNA was resuspended in 100 µL of nuclease-free ultrapure water. Quantity and quality of the DNA was estimated with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
The ITS region of the ribosomal DNA was amplified using the primers ITS1F/ ITS4B reported by Gardes and Bruns (1993). The final concentration of the PCR reaction was: 1× of MyTaq reaction buffer, 0.4 µM of primer, 40 ng of DNA and 1.5 Unit of MyTaq DNA polymerase (Bioline, USA Inc.). The PCR conditions used for amplification were according to Gardes and Bruns (1993). The PCR products were observed on a 1.5 % agarose gel stained with ethidium bromide and visualized by UV transillumination in a Gel-DOC (Bio-Rad) equipment. Bands amplified were removed and purified with the QIA quick gel extraction kit (QIAGEN). Purified PCR products were sequenced using automated equipment in Davis Inc., CA, USA. Both sides of the cloned inserts were sequenced. Sequences were aligned with MUSCLE (Edgar 2004). Alignments were manually checked and ambiguous regions were excluded. Sequences produced in this study are deposited in GenBank under accession number MK033630. A search of GenBank nucleotide databank (NCBI) for homologous sequences was performed by BLAST analyses.
Phylogenetic analyses was performed from sequences obtained from basidiomata. References sequences (Lebel et al. 2015) and consensus sequence were aligned using BioEdit version 7.0.4.1 (Hall 1999). The tree was built in MEGA X (Kumar et al. 2018) using maximum likelihood analyses and the Kimura 2-parameter model (Kimura 1980) of nucleotide substitution with bootstrap values based on 1000 runs. Piloderma fallax and Athelia arachnoidea were used as outgroups (Lebel et al. 2015).

Molecular analyses
A total of 48 sequences of Stephanospora species, including the new species, were analyzed (Fig. 2). The sequence consensus from the holotype clustered in the Stephanospora Clade III Subclade A (i) from Lebel et al. (2015). The designation of S. mayana as a new species is supported by ITS rDNA analyses and morphological features.
Etymology. Named mayana in reference to the Mayan zone where this species was found.

Discussion
This species belongs to the Stephanospora clade III A (i) following Lebel et al. (2015). All the species in this clade are characterized by having basidiospores with ornamentation that does not project more than 2.5 µm, basidia with sterigma up to 7 µm long, and a small corona that never surpasses 7 µm in width (Lebel et al. 2015). However, the Mexican material has larger basidiospores than any other species in this clade (up to 17 µm), unlike S. poropingao T. Lebel & Castellano,S. papua T. Lebel & Castellano,Castellano & K. Hosaka,and S. cribbae T. Lebel & Castellano (up to 14 µm). Stephanospora kanuka T. Lebel & Castellano has similar pileus colour, sweet odour, and basidiospore length, but it has fine spines, an orange to yellowish hymenophore, and a fibrillose pileus (Lebel et al. 2015). Stephanospora cribbae T. Lebel & Castellano is similar to S. mayana in its yellowish pileus, corona size, and greyish hymenophore but differs in the smaller basidiospore size, the fibrillose pileus, and the coconut odour (Lebel et al. 2015). Stephanospora michoacanensis differs from S. mayana in having smaller basidiospores, the fruit odour absent, a cream colour pileus, and its association with oak-pine forest (Guevara-Guerrero et al. 2015). Stephanospora chilensis (E. Horak) J.M. Vidal differs in having an orange pileus and hymenophore, as well as smaller basidiospores (Vidal 2004).
Stephanospora mayana is in an unsupported clade with undescribed species from Belize (KM086881) and close to another unsupported clade with undescribed taxa from the USA and Spain (Lebel et al. 2015). Further collections and descriptions of taxa are required for Belizean and US material to better place this new species. Table 1. Comparative morphology of Stephanospora species in the clade IIIA (i) according to Lebel et al. (2015).