New Ascomycetes from the Mexican Tropical Montane Cloud Forest

The tropical montane cloud forest is the most diverse and threatened vegetation type in Mexico. In the last decade, the number of described Ascomycetes species has notably increased, reaching more than 1300 species. This study describes six new species based on their molecular and morphological characteristics. Our results suggest that Mexico has the highest number of described species in the Neotropics. However, many other Mexican lineages still need to be described.


Introduction
The fungi of the phylum Ascomycota, such as endophytes, mycorrhiza, phytopathogens, and saprobes, have various symbiotic ecological functions, with the latter producing many enzymes that degrade complex polymers such as starch, cellulose, chitin, keratin, and lignin [1].These functions serve to balance the ecosystem.In tropical regions, species diversity is due to the structural complexity of microclimates and microhabitats.In this sense, the Mexican tropical montane cloud forest (TMCF) has been cataloged as the most diverse per area unit [2,3], which is also reflected by the Ascomycetes group; a significant number of species of this group have been described recently.
Mexico comprises a wide variety of vegetation types.One of the most diverse ecosystems, which is under significant threat, is the TMCF, also known as bosque mesófilo de montaña or bosque nuboso (cloud forest), which is characterized mainly by the presence of clouds at the vegetation level.The TMCF is characterized by high levels of atmospheric humidity, 1500-3000 mm of rainfall, and temperatures of 12-23 • C. The vegetation types in this ecosystem develop in rugged reliefs with a discontinuous distribution pattern, analogous to an archipelago of islands, and in ravines or slopes in the Sierra Madre Occidental to the north of Sinaloa, Nayarit, Jalisco, Colima, and Michoacán; in the Sierra Madre Oriental, from southwestern Tamaulipas to northern Oaxaca, including portions of San Luis Potosí, Hidalgo, Puebla, and Veracruz; and in the Sierra Madre south of Guerrero and Oaxaca.In addition, TMCF is also located in some areas of the Trans-Mexican Volcanic Belt.The

Amplification and Sequencing
DNA was obtained from herborized exemplars.Genomic DNA was extracted using the CTAB method [13].The DNA was quantified with a NanoDrop 2000c (Thermo, Waltham, MA, USA).Dilutions were prepared from each sample at 20 ng/µL to amplify 4 regions: internal transcribed spacer rDNA-ITS1 5.8S rDNA-ITS2 (ITS), large nuclear subunit ribosomal DNA (nLSU), the second largest subunit of the RNA polymerase II gene (rpb2), and the region of the small mitochondrial subunit (mtSSU).The reaction mixture for PCR was prepared at a final volume of 15 µL and contained 1× buffer, 0.8 mM dNTPs mix, 20 pmol of each primer, 2 units of GoTaq DNA (Promega, 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× Tris acetate-EDTA (TAE) buffer.The gels were 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 an ExoSAP purification kit (Affymetrix, Santa Clara, CA, USA), following the manufacturer's instructions.They were quantified and prepared for 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 Biosystems 3730XL DNA analyzer (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 gene were analyzed, edited, and assembled using BioEdit v. 7.0.5 [14] to generate a consensus sequence, which was compared with those deposited in GenBank [15] using BLASTN v. 2.2.9 [16].

Phylogenetic Analysis
Alignment was carried out based on the taxonomic sampling method employed by Pem et al. [17] to explore the phylogenetic relationships of the new species of Holmiella (Table 1).Each gene region was independently aligned using the online version of MAFFT v. 7 [18][19][20].The alignment was reviewed in PhyDE v. 10.0 [21], followed by minor manual adjustments to ensure character homology between taxa.A matrix was formed for ITS with 10 taxa (690 characters) for ITS, 23 taxa (831 characters) for LSU, and 14 taxa (640 characters) for mtSSU.The aligned matrices were concatenated into a single matrix (24 taxa, 2161 characters).Three partitioning schemes were established, one each for the ITS, nLSU, and mtSSU, using the option to minimize the stop codon with Mesquite v3.70 [22].
Alignment was carried out based on the taxonomic sampling method employed by Sun et al. [23] to explore the phylogenetic relationships of the new species of Kirschsteiniothelia (Table 2).Each gene region was independently aligned using the online version of MAFFT v. 7 [18][19][20].The alignment was reviewed in PhyDE v.10.0 [21], followed by minor manual adjustments to ensure character homology between taxa.A matrix was formed with 23 taxa (695 characters) for ITS and 37 taxa (836 characters) for LSU.The aligned matrices were concatenated into a single matrix (37 taxa, 1534 characters).Two partitioning schemes were established, one each for the ITS and LSU, using the option to minimize the stop codon with Mesquite v3.70 [22].
Alignment was carried out based on the taxonomic sampling method employed by Healy et al. [24] to explore the phylogenetic relationships of the new species of Microglossum (Table 3).Each gene region was independently aligned using the online version of MAFFT v. 7 [18][19][20].The alignment was reviewed in PhyDE v.10.0 [21], followed by minor manual adjustments to ensure character homology between taxa.A matrix was formed with 61 taxa (690 characters) for ITS, 23 taxa (831 characters) for LSU, and 22 taxa (670 characters) for the second largest subunit of the RNA polymerase II gene (rpb2).The aligned matrices were concatenated into a single matrix (61 taxa, 2191 characters).Five partitioning schemes were established, one each for the ITS and nLSU and three for the rpb2 gene region, using the option to minimize the stop codon with Mesquite v3.70 [22].
Alignment was carried out based on the taxonomic sampling method employed by [25] to explore the phylogenetic relationships of the new species of Claussenomyces (Table 4).First, the ITS region was aligned using the online version of MAFFT v. 7 [18][19][20].Next, the alignment was reviewed in PhyDE v.10.0 [21], followed by minor manual adjustments to ensure character homology between taxa.The matrix was composed of 22 taxa (700 characters).
Alignment was carried out based on the taxonomic sampling method employed by Argnello et al. [26] and Healy et al. [24] to explore the phylogenetic relationships of the new species of Wolfina (Table 5).The ITS region was aligned using the online version of MAFFT v.7 [18][19][20].The alignment was reviewed in PhyDE v.10.0 [21], followed by minor manual adjustments to ensure character homology between taxa.The matrix was composed of 11 taxa (700 characters).
Alignment was carried out to resolve the phylogenetic relationships of the new species of Dematophora based on the taxonomic sampling method employed by Wittstein et al. [27] (Table 6).Each gene region was independently aligned using the online version of MAFFT v. 7 [18][19][20].The alignment was reviewed in PhyDE v.10.0 [21], followed by minor manual adjustments to ensure character homology between taxa.A matrix was formed with 30 taxa (699 characters) for ITS and 18 taxa (836 characters) for LSU.The aligned matrices were concatenated into a single matrix (30 taxa, 1535 characters).Two partitioning schemes were established, one each for the ITS and LSU, using the option to minimize the stop codon with Mesquite v3.70 [22].
Habitat: Grows on branches of angiosperms.GenBank: ITS: OQ877258; nrLSU:OQ889487.Etymology: The name refers to the state of Oaxaca, where this species was found.

Conclusions
The Mexican tropical montane cloud forest (Figure 14) is one of the most diverse ecosystems for fungi.However, databases of other organisms, e.g., plants [45] and birds [46], but not fungi, are available for this ecosystem type.Unfortunately, they have not been

Conclusions
The Mexican tropical montane cloud forest (Figure 14) is one of the most diverse ecosystems for fungi.However, databases of other organisms, e.g., plants [45] and birds [46], but not fungi, are available for this ecosystem type.Unfortunately, they have not been extensively studied because of the lack of specialists; so, their representation in herbaria is poor.This study phylogenetically and morphologically describes six new species found in the Mexican TMCF.
Characterizing fungal diversity in TMCFs is relevant for forest conservation.These forests provide environmental services such as terrestrial biomass and water degradation and are the source of bioactive secondary metabolites [9].
In 2017, Del Olmo et al. [9] reported 954 Ascomycota species from the Mexican TMCF, and other recent studies added different species to the Mexican TMCF mycobiota.For example, Raymundo et al. [47] described Marthamyces coronadoae, Raymundo et al. [48] described seven species of Hypocreales, Arias et al. [49] registered the asexual phases of 355 species, Medel-Ortiz et al. [50] found seven new records for the TMCF, and Raymundo et al. [41] recorded 10 new species in Mexico.Other studies that recorded new taxa are as follows (in chronological order): Sánchez-Flores et al. [51] described Hymenoscyphus herrerae from Puebla and registered six new species in the country; Raymundo et al. [43] recorded 17 new species from different TMCF localities; and Cobos-Villagrán et al. [52] registered Rhytidhysteron esperanzae and R. mesophila from Oaxaca and Hidalgo, respectively.In Puebla, three studies are relevant: Barbosa-Reséndiz et al. [53] described Daldinia rehmii, Raymundo et al. [54] recorded Unguiculariopsis ravenelii, and Sánchez-Flores et al. [55] described Ionomidotis mesophile.Then, Raymundo et al. [56] described Smardaea isoldae from Hidalgo, and Valenzuela et al. [57] added 10 new records for the TMCF in Oaxaca.In Veracruz, Chacón-Zapata and Gonzalez [58] described Euacanthe renispora, Guzmán-Guillermo et al. [59] described Paruephaedria heimerlii, and Chacón-Zapata and Ramirez-Guillén [60] listed 11 new records of Coronophorales.Finally, de la Fuente et al. [61] described Elaphomyces castilloi from Chiapas.The above information allows us to assume the existence of at least 1389 species inhabiting the Mexican TMCF.As González et al. [5] suggested, the precise number of species is difficult to establish due to nomenclature changes and the imprecision of Ascomycetes species identification.
extensively studied because of the lack of specialists; so, their representation in herbaria is poor.This study phylogenetically and morphologically describes six new species found in the Mexican TMCF.Characterizing fungal diversity in TMCFs is relevant for forest conservation.These forests provide environmental services such as terrestrial biomass and water degradation and are the source of bioactive secondary metabolites [9].
In 2017, Del Olmo et al. [9] reported 954 Ascomycota species from the Mexican TMCF, and other recent studies added different species to the Mexican TMCF mycobiota.For example, Raymundo et al. [47] described Marthamyces coronadoae, Raymundo et al. [48] described seven species of Hypocreales, Arias et al. [49] registered the asexual phases of Among the six new species described in this study, three species are distributed in Hidalgo, Sierra Madre Oriental, a mountainous area characterized by its abrupt topography and high beta diversity.Two were re-collected in Sierra de Juárez (Sierra Norte de Oaxaca), and one in Lagunas de Montebello, Altos de Chiapas, on the southern border with Guatemala.It is worth mentioning that the genera Holmiella and Wolfina are cited for the first time in the country.
Mexico is one of the world's most diverse areas for fungi; so, it is essential to inventory and describe the fungal species in this type of ecosystem.TMCFs are the most threatened terrestrial ecosystems at the national level and are classified as "habitats in danger of extinction" [62].In addition, a meta-analysis recently revealed that Mexico is a hotspot for oak species and their ectomycorrhizal mycobionts [63].Those authors considered that the Mexican oak forests are essential for maintaining biodiversity due to the 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 avocados and coffee.The fungal abundance is strongly affected by the loss of this ecosystem type.The effects of global warming have not yet been evaluated in the case of these fungi.

Figure 2 .
Figure 2. Bayesian inference phylogram of ITS, LSU, and SSU sequence data.Posterior probability (left of slash) from Bayesian analysis and bootstrap support (right of slash) are given above the nodes.New species Holmiella hidalgoensis is shown in bold.

Figure 4 .
Figure 4. Bayesian inference phylogram of ITS, LSU, and rpb2 sequence data.Posterior probability (left of slash) and bootstrap support values (right of slash) Bayesian analysis are given above the nodes.New species Kirschsteiniothelia esperanzae is shown in bold. .

Figure 6 .
Figure 6.Bayesian inference phylogram of ITS, LSU, and rpb2 sequence data.Posterior probability (left of slash) from Bayesian analysis and bootstrap support (right of slash) are given above the nodes.New species Microglossum flavoviride is shown in bold.

Figure 12 .
Figure 12.Bayesian inference phylogram of ITS, LSU, and rpb2 sequence data.Posterior (left of slash) from Bayesian analysis and bootstrap support (right of slash) given abov New species Dematophora oaxacana is shown in bold.

Figure 12 .
Figure 12.Bayesian inference phylogram of ITS, LSU, and rpb2 sequence data.Posterior probability (left of slash) from Bayesian analysis and bootstrap support (right of slash) given above the nodes.New species Dematophora oaxacana is shown in bold.Taxonomical notes: Ascospores measure 20-29.6 × 9.6-12 µm, without germline and double cell appendage.Phylogenetically, this species is close to Dematophora buxi (Fabre) C. Lamb., Wittstein & M. Stadler, differing from the latter in its macro and microscopic characteristics, as a more persistent subicula, with narrower ascospores 19.8-30.1 × 6-8.9 µm, fusoid, with straight germline and rounded apices.It is also similar to D. francisiae (L.E.Petrini) C. Lamb., Wittstein & M. Stadler; however, the latter has a persistent and felted subicula, 29-35 × 8-13 µm, longer ascospores, with a straight germline and rounded apices.Some species of Dematophora were earlier considered under the genus Rosellinia[44].The distribution of the described new species is shown in Figure13.

Figure 13 .
Figure 13.Distribution of new species.

Figure 13 .
Figure 13.Distribution of new species.

Table 1 .
GenBank accession numbers corresponding to sequences used in phylogenetic analyses of Holmiella hidalgoensis sp.nov.Accessions of new species indicated in bold.

Table 4 .
GenBank accession numbers corresponding to sequences used in phylogenetic analyses of Claussenomyces paulinae sp.nov.Accessions of new species indicated in bold.

Table 5 .
GenBank accession numbers corresponding to sequences used in phylogenetic analyses of Wolfina molangoensis sp.nov.Accessions of new species indicated in bold.

Table 6 .
GenBank accession numbers corresponding to sequences used in phylogenetic analyses of Dematophora oaxacana sp.nov.Accessions of new species indicated in bold.