Novelties in Macrofungi of the Tropical Montane Cloud Forest in Mexico

The tropical montane cloud forest in Mexico is the most diverse and threatened ecosystem. Mexican macrofungi numbers more than 1408 species. This study described four new species of Agaricomycetes (Bondarzewia, Gymnopilus, Serpula, Sparassis) based on molecular and morphological characteristics. Our results support that Mexico is among the most biodiverse countries in terms of macrofungi in the Neotropics.


Introduction
In Mexico, the tropical montane cloud forest (TMCF), also known as a cloud forest or "bosque mesófilo de montaña", groups together a set of physiognomically heterogeneous plant communities. The canopy of these forests is usually composed of evergreen trees, but the medium and low-strata trees are deciduous. The high abundance of epiphytes and ferns gives the forest an exuberant appearance [1,2]. The TMCF features a mixture of Holarctic affinities in the upper arboreal stratum and meridional affinities in the medium, low, shrub, and herbaceous strata [3][4][5]. These forests inhabit mountainous areas, mainly between 600 (1200) and 2500 (3200) m above sea level, with seasonal persistence of high relative humidity and rainfall that ranges from 2000 to 4000 (5300) mm per year. The soils are mainly acidic and prosper in temperate climates [6][7][8]. One of the most outstanding characteristics of these forests is that the arboreal canopy intercepts and condenses the fog, which precipitates and contributes about 50% of the total local precipitation [9].
Mexican TMCF presents a great β diversity, showing a high turnover of species [10]. In Mexico, this ecosystem has the highest number of species per unit area (in Oaxaca, up to 75 spp/0.1 ha: div. α). Mexico has 2500 almost exclusive vascular plant species in less than 1% of the territory [1,11]. The forest contains approximately 10% of the total flora of Mexico, of which 30% of the species are endemic [1].
TMCF is one of the most threatened ecosystems in the country [3,12,13] (Figure 1). The demographic explosion, clandestine logging, coffee cultivation, cattle grazing, and semi-nomadic agriculture have caused a drastic decrease in its extension in recent decades. The area occupied by this forest was reduced to less than a tenth in twenty years [13]. The extensive use of these forests began after the conquest, with the displacement of indigenous In the last 70 years in Mexico, various studies have been carried out on the TMCF [1,5,6,[16][17][18][19][20][21][22][23][24][25][26][27]. The floristic associations in the TMCF are unique and maintain a variable degree of relationships. This variation in the TMCF of Mexico increases when it is considered on the regional scale [28]. The Mexican states with the highest forest coverage are Oaxaca, Chiapas, and Hidalgo [13].
The diversity of fungi is estimated at 1.5-3.8 million species worldwide, of which 120,000 have been described [29]; macrofungi represent 18% of this diversity. Fungi have not been studied thoroughly, and it has been estimated that there could be as many as 53,000 to 110,000 more species [30]. This number may vary if we consider cryptic and species complexes that have been clarified from polyphasic or comprehensive studies [31].
The study of macrofungi in Mexico is still incipient. The groups best studied are Pezizales and Xylariales for Ascomycota, while Agaricales, Boletales, Dacrymycetales, Hymenochaetales, Polyporales, and Russulales are the most recorded in Basidiomycota. Monographic studies have only been undertaken for 60 genera [32]. In the Comisión In the last 70 years in Mexico, various studies have been carried out on the TMCF [1,5,6,[16][17][18][19][20][21][22][23][24][25][26][27]. The floristic associations in the TMCF are unique and maintain a variable degree of relationships. This variation in the TMCF of Mexico increases when it is considered on the regional scale [28]. The Mexican states with the highest forest coverage are Oaxaca, Chiapas, and Hidalgo [13].
The diversity of fungi is estimated at 1.5-3.8 million species worldwide, of which 120,000 have been described [29]; macrofungi represent 18% of this diversity. Fungi have not been studied thoroughly, and it has been estimated that there could be as many as 53,000 to 110,000 more species [30]. This number may vary if we consider cryptic and species complexes that have been clarified from polyphasic or comprehensive studies [31].
The study of macrofungi in Mexico is still incipient. The groups best studied are Pezizales and Xylariales for Ascomycota, while Agaricales, Boletales, Dacrymycetales, Hymenochaetales, Polyporales, and Russulales are the most recorded in Basidiomycota. Monographic studies have only been undertaken for 60 genera [32]. In the Comisión Nacional para el Estudio y Conocimiento de la Biodiversidad (CONABIO) catalog [33], Cifuentes (2008) [34] [35] analyzed 6349 records of fungi ascribed to 2962 species from the tropical montane cloud forests (Mexico, Guatemala, Belize, Brazil, Colombia, Costa Rica, Panama, Venezuela), of which 220 taxa were described initially from this ecosystem. These authors indicated that Mexico presents 36% of these records (1274 species).
The Agaricales or euagarics is the largest clade of Agaricomycetes, including more than half of all known species. Most are agaricoid fungi, including also clavarioid and gasteroid fungi [38]. The order contains 38 families, 508 genera, and 17,291 species. The family Hymenogastraceae and the genus Gymnopilus have been recognized phylogenetically based on well-supported PP values [37]. The family Hymenogastraceae includes nine agaricoid genera and one gasteroid genus with ornamented or smooth, brown spores. There are no morphological synapomorphies that distinguish the members of this group.
The saprotrophs species (or wood decay fungi) of Boletales developed a unique mode of brown rot called "Coniophoraceae-rot". Serpulaceae comprises three genera: two of them are ectomycorrhizal fungi and Serpula produces brown rot [39]. Given the diversity of fruiting body forms, we assume that there has been extensive homoplasy in the evolution of Boletales. However, no apparent morphological character distinguishes the group, which is only diagnosed by molecular sequences.
The Polyporales include various basidiocarp types and hymenophore configurations, including bracket-shaped, effused resupinate, stipitate with poroid, lamellate, or smooth hymenophores. Few species produce shelf-like or flabellate clusters of overlapping basidiocarps [40]. The order contains 18 families, 285 genera, and 2544 species. The family Sparassidaceae and the genus Sparassis have been recognized phylogenetically with wellsupported PP values; analyzed the combination of rpb1 and ribosomal RNA genes [37,41]; these authors discovered a robust resolution of many clades, including 18 families. However, these authors mentioned that some nodes remain weakly supported; perhaps because numerous taxa have not been sampled yet. The researchers [41] found that macroscopic and microscopic characters are variable and are present in several families of Polyporales [41]. Variations and transitions among basidiocarp types exist, and no morphological synapomorphy unites the Polyporales [40]. The most common "polyporoid" basidiocarp type also has convergently evolved in at least 11 additional orders of Agaricomycetes [39].
In Mexico, particular emphasis has been placed on studying the macrofungi of this type of forest. Many have been classified as in danger of extinction due to the high anthropogenic action [1,4]. The main objective of this study is (1) to describe new species and (2) to recognize and publicize the Mexican TMCF fungal richness. The present contribution aims to describe species distributed in the Mexican TMCF, an ecosystem that is in danger of extinction.
Microscopic observations were taken from tissues rehydrated in 5% aqueous KOH and Melzer's reagent; basidiospore dimensions include the ornamentation. Macroscopic features were photographed with a Nikon D7000 camera, (Nikon Corporation, Tokyo, Japan) and the micrographs were with a Sony DSCWX350 camera (Tokyo, Japan). Additionally, scanning electron microscopy (SEM; Hitachi SU1510, Hitachi, Japan) was used to observe the detail of the spore wall. The meanings of taxonomic terms are based on [44].

Extraction, Amplification, and Sequencing
We obtained the DNA from herbaria material. The CTAB protocol of [45] was used to extract genomic DNA. The DNA was quantified with a Nanodrop 2000c (Thermo Scientific TM , Wilmington, CA, USA). Then, we prepared dilutions from each sample at 20 ng/µL to amplify the following regions: the internal transcribed spacer rDNA-ITS1 5.8S rDNA-ITS2 (ITS), the larger nuclear subunit ribosomal DNA (nLSU), the second largest subunit of the RNA polymerase II gene (rpb2), the region of the small mitochondrial subunit (SSU), the subunit (atp6), and the translation elongation factor 1-α (tef1) ( Table 1). The sequences used for each species are described in the corresponding section. The reaction mixture for PCRs was performed on a final volume of 15 µL containing 1x buffer, 0.8 mM dNTPs mix, 20 pmol of each primer, 2 units of GoTaq DNA (Promega Corporation, Madison, WI, USA), and 100 ng of template DNA. The PCR products were verified by agarose gel electrophoresis. The gels were run for 1 h at 95 V cm −3 in 1.5% agarose and 1× TAE buffer (Tris Acetate-EDTA). The gel was stained with GelRed (Biotium, Fremont, CA, USA), and the bands were visualized in an Infinity 3000 transilluminator (Vilber Lourmat, Eberhardzell, Germany). The amplified products were purified with the ExoSAP Purification kit (Affymetrix Inc., Santa Clara, CA, USA), following the manufacturer's instructions. Then, they were quantified and prepared for the sequence reaction using a BigDye Terminator v.3.1 (Applied Biosystems, Foster City, CA, USA). These products were sequenced in both directions with an Applied Biosystem model 3730XL (Applied BioSystems, Foster City, CA, USA) at the Instituto de Biología of the Universidad Nacional Autónoma de México (UNAM). The sequences obtained were compared with the original chromatograms to detect and correct possible reading errors. The sequences of both strands of each of the genes were analyzed, edited, and assembled using BioEdit v. 7.0.5 [46] to generate a consensus sequence and then compared with those deposited in GenBank (2020) using the tool BLASTN v. 2.2.9 [47][48][49][50][51].

Phylogenetic Analysis
The alignment obtained to explore the phylogenetic relationships of the new species of Gymnopilus was based on the taxonomic sampling employed by [52,53] (Table 2). The ITS region was aligned using the online version of MAFFT v. 7 [54][55][56]. Alignments were reviewed in PhyDE v.10.0 [57], followed by minor manual adjustments to ensure character homology between taxa. The matrix was composed of 88 taxa (697 characters). In the case of the new species of Serpula, we followed the taxonomic sampling of [58] (Table 3). First, the ITS region was aligned using the online version of MAFFT v. 7 [54][55][56]. Next, alignment was reviewed in PhyDE v.10.0 [57], followed by minor manual adjustments to ensure character homology between taxa. The matrix was composed of 46 taxa (700 characters).  To analyze the new species of Sparassis, we followed the taxonomic sampling of [59] (Table 4). Each gene region was independently aligned using the online version of MAFFT v. 7 [54][55][56]. Alignment was reviewed in PhyDE v.10.0 [57], followed by minor manual adjustments to ensure character homology between taxa. The matrix was formed for ITS by 20 taxa (690 characters) and LSU by 19 taxa (831 characters). We established two partitioning schemes, one for the ITS and one for the LSU, using the option to minimize the stop codon with Mesquite v 3.70 [60].
In the case of the new species of Bondarzewia, an alignment was made based on the taxonomic sampling employed by [61] (Table 5). Each gene region was independently aligned using the online version of MAFFT v. 7 [54][55][56]. The alignment was reviewed in PhyDE v.10.0 [57], followed by minor manual adjustments to ensure the character homology among taxa. The matrix was formed for ITS by 30 taxa (690 characters), for LSU by 26 taxa (831 characters), and for mtSSU by 21 taxa (640 characters), while translation elongation factor 1-α (tef1) was formed by 23 taxa (670 characters). Finally, the aligned matrices were concatenated into a single matrix (30 taxa, 2831 characters). Six partitioning schemes were established: one for the ITS, one for the nLSU, one SSU, and three for the tef1 gene region, using the option to minimize the stop codon with Mesquite v3.70 [60]. Phylogenetic inferences were estimated with maximum likelihood in RAxML v. 8.2.10 [62] with a GTR + G model of nucleotide substitution. In addition, 10,000 non-parametric rapid bootstrap pseudoreplicates assessed the branch support that was run with the GTRGAMMA model. For Bayesian posterior probability, the best evolutionary model for alignment was sought using the Partition Finder [63][64][65]. Phylogeny analyses were performed using MrBayes v. 3.2.6 × 64 [66]. The information block for the matrix includes two simultaneous runs, four Montecarlo chains, temperature set to 0.2, and sampling 10 million generations (standard deviation ≤ 0.1) with trees sampled every 1000 generations. The first 25% of the samples were discarded as burn-in, and stationarity was checked in Tracer v. 1.6 [67]. Trees were visualized and optimized in FigTree v. 1.4.4 [67] and edited in Adobe Illustrator vCS4 (Adobe Systems, Inc., San Jose, CA, USA).   Diagnosis: This species is different from other large annulate Gymnopilus species its basidiomata growing in caespitose clusters on the ground of TMCF with fibrillo squamulose, yellowish-orange to orange-red pileus and microscopically, basidiospores 9 × 5.5-7 µm, broadly ellipsoid, subreticulate to coarsely roughened with irregular lar warts and ridges.

Results Taxonomy
Type   Diagnosis: This species is different from other large annulate Gymnopilus species by its basidiomata growing in caespitose clusters on the ground of TMCF with fibrillosesquamulose, yellowish-orange to orange-red pileus and microscopically, basidiospores 7-9 × 5.5-7 µm, broadly ellipsoid, subreticulate to coarsely roughened with irregular large warts and ridges.
Habitat: Gregarious to caespitose in TMCF soil. Taxonomical notes: Gymnopilus guzmanii is characterized by its large annulate basidiomes growing in caespitose clusters on the soil of the tropical montane cloud forest, with fibrillose-squamulose, yellowish orange to orange-red pileus, margin appendiculate, eroded and recurved. Microscopically, basidiospores differ with subreticulate to coarsely roughened ornamentation with irregular large warts and ridges. Morphological and molecular characters locate the species in the G. junonius (Fr.) P.D. Orton group [68]. Phylogenetically, G. guzmanii is well-supported based on ITS sequence data. It is close to G. subspectabilis Hesler and G. ventricosus (Earle) Hesler. The first species grows on hardwoods, while the second grows on the wood of conifers. Other characteristics that separate these species are the shape and ornamentation of spores in G. subspectabilis, ellipsoidal to amygdaliform, with acutely conical apices and conspicuous suprahylar depressions, moderately roughened with irregular warts and short ridges. In G. ventricosus, the spores are amygdaloid, with conical apices, finely to coarsely roughened with irregular warts and ridges.
Habitat: This species grows at the base of living Quercus trees, attached to underground roots in the TMCF.
Additional Taxonomical notes: Sparassis isis is characterized by its basidiomata composed of a few layers of loosely arranged flabellae, very wavy and darkening margins, trama of flabellae composed of hyphae embedded in a gelatinous or mucilaginous matrix, cystidioles present, and for its size and shape of basidiospores. This species is separated from other Sparassis by morphological, ecological, and molecular characters and phylogenetically is well supported based on ITS, LSU, rpb2, and atp6 sequences [63][64][65][79][80][81]. Sparassis isis belongs to the North American clade with S. americana R. H. Petersen, S. radicata Weir, and an unidentified species. S. americana differs by its upper basidiome coarsely and irregularly branched to produce expanded petaloid crisped flabellae, top margins drying cartilaginous when fresh appearing waxy, retaining this appearance when dried; basidiospores 4.5-6 (7.0) × (3.0) 3.5-4.0 (4.5) µm, broadly ellipsoid and flattened axially and associated with Pinus sp., perhaps as root parasite [59,66]. S. radicata is separated by its subterranean pseudosclerotial stipe, an above-ground fertile structure composed of complex, often anastomosed lacunose branches, with ultimate flabellae thin, parchmentlike in well-dried specimens, curled or crisped, basidiospores 5-6.5 × 3.5-4 (5) µm, broadly ellipsoid to ellipsoid and flattened axially. It is associated with the roots of conifers in North American temperate forests [59].
Russulales  Diagnosis: It differs from other Bondarzewia species in having pileus in several tones, e.g., orange, yellow ochre, brownish yellow, light brown, yellowish brown or rust brown, tomentose to hirsute and towards the margin tomentose adpressed, margin white, yellow to orange, most extended basidiospore ridges, up to 2 µm; basidiomata grows on the soil.
Habitat Taxonomical notes: This species is characterized by the color of basidiomata with several tones in the pileus (orange, yellow ochre, brownish yellow, light brown, yellowish brown, rust brown), margin (white, yellow to orange), and stipe (white, yellow, yellow ochre, light brown to yellowish brown), the texture of pileus (velvety, tomentose, hirsute and tomentose adpressed) and basidiospores ornamentation with ridges blunt, up to 1 µm high and up to 2 µm long. Bondarzewia mesofila can be separated by morphological, ecological, and molecular characteristics [67,82]. Phylogenetically is well supported based on ITS, LSU, SSU, and tef 1 sequences. This species is close to B. berkeleyi (Fr.) Bondartsev and Singer, B. dickinsii (Berk.) Jia J. Chen, B.K. Cui, and Y.C. Dai, and to B. occidentalis Jia J. Chen, B.K. Cui, and Y.C. Dai, all of them belonging to a sister clade. Bondarzewia berkeleyi is separated by growing on the wood of several species of Fagaceae, basidiomata developing underground sclerotia, and larger basidiospores 7-9 × 6-8 µm [82,83]. Basidiomata of Bondarzewia dickinsii grow on fallen trunks of Quercus and roots of Castanea; the pileus color is white to brownish, basidiospores with sharp ridges, up to 2 µm long [82]. Bondarzewia occidentalis grows on gymnosperm wood, with a pileus surface characteristically from yellowish brown to orange-brown, concentrically zonate and glabrous, basidiospores with ridges up to 1 µm long [83].

Conclusions
Based on this study and other research performed by us and other colleagues, the Mexican TMCF is the most diverse ecosystem for fungi. Databases from different organisms, e.g., plants [84] and birds [85], are available for this ecosystem but not for fungi. Unfortunately, there are no extensive studies of fungi because of the lack of specialists, so their representation in the herbaria is poor. So far, 1407 species of Macromycetes have been registered for Mexico, of which 220 species have been found in this ecosystem. In addition, this study described four new species found in this ecosystem. Similar worldwide studies at the same latitude, e.g., in Yunnan, China [86,87], an area also considered as a biodiversity hotspot [88], recently recorded 314 macrofungi. Based on this, the Mexican TMCF could harbor more than 100 fungi species than those registered in this study.
The characterization of fungal diversity in TMCF is relevant for forest conservation. Fungi provide different environmental services and are sources of bioactive secondary metabolites [35]. Altogether, 1106 species of Agaricomycetes are registered from the TMCF [35]. This study contributes with four new species.
Mexico represents one of the world's most diverse areas for fungi diversity, so it is essential to record and describe the fungal species of this type of vegetation. TMCFs are the most threatened terrestrial ecosystems at the national level and are classified as "habitat in danger of extinction". In addition, a meta-analysis recently revealed that Mexico is a hotspot for oak species and their ectomycorrhizal mycobionts [89]. These last authors considered the Mexican oak forest essential for maintaining biodiversity due to its high richness and endemism of fungi, mainly those associated with Fagaceae.
The loss of the TMCF is due to its transformation into grazing land for livestock and agriculture, mainly for avocado and citric. The fungal wealth is strongly affected by the