﻿Elaphomycescastilloi (Elaphomycetaceae, Ascomycota) and Entolomasecotioides (Entolomataceae, Basidiomycota), two new sequestrate fungi from tropical montane cloud forest from south Mexico

﻿Abstract Two new species of sequestrate fungi are described from south Mexico based on morphological and molecular evidences. Here we describe Elaphomycescastilloi characterized by the yellowish mycelial mat, dull blue gleba and ascospores of 9.7–11.5 µm; Entolomasecotioides is characterized by the secotioid basidiomata, sulcate, pale cream pileus, and basidiospores of 7–13 × 5–9 µm. Both species grow in montane cloud forest under Quercus sp. in the state of Chiapas, Mexico. Descriptions, photographs, and multilocus phylogeny for both species are presented.


Introduction
Sequestrate fungi are characterized by producing hypogeous sporome, protected by a thick peridium to avoid desiccation, changes in temperature, and humidity (Thiers 1984). Due to this morphological modification, these species are not capable of dispersing their spores through the air, so they use other strategies such as producing aromas to attract animals that consume and disperse them through their feces (Castellano et al. 1986;Caiafa et al. 2021). Many species are ectomycorrhizal and are associated with the roots of angiosperms and gymnosperms, mainly trees and shrubs of the genera Abies, Coccoloba, Dycimbe, Eucalyptus, Quercus, and Pinus (Trappe et al. 2009). Some saprobic species are also known, mainly from tropical forests (de la Fuente et al. 2021). This diversity in the tropics is just beginning to be discovered and, according to Sulzbacher et al. (2016), there are more than 1,500 described species in the world with more being regularly added.
Hypogeous fungi have been studied in Mexico since the 1970's and the studies of García-Romero et al. (1970) and Trappe and Guzmán (1971), although the first record of a hypogeous fungus was Melanogaster umbriniglebus Trappe & Guzmán, recorded in Chihuahua by Lumbholz in the late 1800's (Cázares et al. 2008). Since the 2000's, species have been regularly described, mainly species from temperate forests. Approximately 100 species of hypogeous fungi are currently known, mainly from northeastern Mexico and the Neovolcanic axis, areas where more studies have been carried out (Trappe and Guzmán 1971;Cázares et al. 1992;Guevara-Guerrero et al. 2014;Gómez-Reyes et al. 2018);however, many Mexican states and different types of vegetation have been under-explored.
The state of Chiapas is located in southern Mexico and is an important reservoir of montane cloud forest (Williams-Linera 1991). This type of vegetation represents less than 1% of the national coverage and is declining alarmingly (SEMARNAT 2010). This kind of forest is located between 1500 to 2500 m above sea level. Annual precipitation exceeds 1500 mm. The most common trees in this forest type include Pinus, Quercus, Lyquidambar and Magnolia (González-Espinoza et al. 2012). Due to several species of fungi growth associated with the roots of trees, it is possible to carry out successful reforestation experiments with native species. Hence knowing the native fungi of the forest becomes a priority in their conservation (Martínez-Reyes et al. 2012). In this study, two new species of hypogeous fungi are described: Elaphomyces castilloi, characterized by the blackish ascoma covered with yellow mycelial mat and bluish gleba, and Entoloma secotioides, characterized by its pale-colored secotioid basidiome. Both species grow in montane cloud forest under Quercus species. Photographs, descriptions and multiloci phylogeny are presented for both species.

Sampling data
Mycological explorations were carried out in the state of Chiapas, southern Mexico (Fig. 1). The dominant vegetation in the sampling site corresponds to a tropical montane cloud forest. For the collection of the specimens, the protocols proposed by Castellano et al. (1986) were followed. The specimens were registered and herborized. A color chart was used for color terminology (Kornerup and Wanscher 1978). Hand cuts were made on dried specimens and temporal preparation was mounted in order to observe microstructures. KOH 5% and Melzer's reagent were used to observe amyloid reactions. At least 30 spores and other microstructures were measured using an optical microscope (Motic ba310, San Antonio, USA) to obtain the average length (L), average width (W) and Q ratio (Q). The scanning electron microscope (Hitachi Su 1510, Hitachi, Japan) of IB-UNAM (Mexico City, Mexico) was utilized to observe spore ornamentation. The collected specimens were deposited in ITCV.

DNA extraction, amplification, and sequencing
The DNA was obtained from herbarium specimens (Tables 1, 2). The CTAB protocol of Martínez-González et al. (2017) was used to extract genomic DNA. The DNA was quantified with a Nanodrop 2000c (Thermo ScientificTM, Wilmington, USA). We prepared dilutions from each sample at 20 ng/µL to amplify the Internal Transcribed Spacer rDNA-ITS1 5.8S rDNA-ITS2 (ITS), the nuclear large subunit ribosomal DNA (LSU) and the second largest subunit of the RNA polymerase II gene (rpb2). The reaction mixture for PCRs was performed on a final volume of 15 µL containing 1× buffer, 0.8 mM dNTPs mix, 20 pmol of each primer, 2 units of GoTaq DNA (Promega, 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, 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, USA), following the manufacturer's instructions. They were quantified and prepared for the sequence reaction using a BigDye Terminator v.3.1 (Applied Biosystems, USA). These products were sequenced in both directions with an Applied Biosystem model 3730XL (Applied BioSystems, Foster City, 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,  Table 2. GenbBank accession numbers corresponding to the sequences used in the phylogenetic analyses for Entoloma secotioides. The accessions of the new species are in bold.

Species name Isolate/Voucher/strain GenBank Accessions
edited and assembled using the BioEdit v. 7.0.5 (Hall 1999) to generate a consensus sequence which compared with those deposited in GenBank using the tool BLASTN v. 2.2.9 (Zhang et al. 2000).

Phylogenetic analyses
To explore the phylogenetic relationships of the new species of Elaphomyces, an alignment was made based on the taxonomic sampling employed by Paz et al. (2017). Outgroup was selected according to Paz et al. (2017). Each gene region was independently aligned using the online version of MAFFT v. 7 (Katoh et al. 2002(Katoh et al. , 2017Katoh and Standley 2013). Alignment was reviewed in PhyDE v.10.0 (Müller et al. 2005), followed by minor manual adjustments to ensure character homology between taxa. The matrix was formed for ITS by 24 taxa (697 characters), while LSU by 19 taxa (845 characters). The aligned matrices were concatenated into a single matrix (32 taxa, 1542 characters). Two partitioning schemes were established: one for the ITS and one for the LSU, which were established using the option to minimize the stop codon with Mesquite v3.70 (Maddison and Maddison 2017).
To explore the phylogenetic relationships of the new species of Entoloma, an alignment was made based on the taxonomic sampling employed by Elliott et al. (2020). The outgroup was selected according to Elliott et al. (2020). Each gene region was independently aligned using the online version of MAFFT v. 7 (Katoh et al. 2002(Katoh et al. , 2017Katoh and Standley 2013). Alignment was reviewed in PhyDE v.10.0 (Müller et al. 2005), followed by minor manual adjustments to ensure character homology between taxa. The matrix was formed for ITS by 45 taxa (700 characters), for LSU by 31 taxa (831 characters), while rpb2 consisted of 17 taxa (670 characters). The aligned matrices were concatenated into a single matrix (47 taxa, 2201 characters). Five partitioning schemes were established: one for the ITS, one for the LSU and three for rpb2 gene region, which were established using the option to minimize the stop codon with Mesquite v3.70 (Maddison and Maddison 2017).
Phylogenetic inferences were estimated with maximum likelihood (ML) in RAxML v. 8.2.10 (Stamatakis 2014) with a GTR + G model of nucleotide substitution. To assess branch support, 10,000 nonparametric rapid bootstrap pseudoreplicates were run with the GTRCAT model. For Bayesian posterior probability (PP), the best evolutionary model for alignment was sought using Partition Finder (Frandsen et al. 2015;Lanfear et al. 2014Lanfear et al. , 2017. Phylogeny analyses was performed using MrBayes v. 3.2.6 ×64 (Huelsenbeck and Ronquist 2001). 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 samples were discarded as burn-in, and stationarity was checked in Tracer v. 1.6 (Rambaut et al. 2014). Trees were visualized and optimized in FigTree v. 1.4.4 (Rambaut 2014), and then edited in Adobe Illustrator vCS4 (Adobe Systems, Inc., San Jose, CA).

Phylogenetic analyses
The ITS and LSU sequences obtained from Elaphomyces castilloi and ITS, LSU and rpb2 from Entoloma secotioides were deposited in GenBank. The two simultaneous Bayesian runs continued until the convergence parameters were met, and the standard deviation fell below 0.001 after 10 million generations for Elaphomyces castilloi and 0.002 for Entoloma secotioides. No significant changes in tree topology trace or cumulative split frequencies of selected nodes were observed after about 0.33 million generations for E. castilloi and 0.45 million generations for E. secotioides, so the first 2,500,000 sampled trees (25%) were discarded as burn-in. Both the Bayesian analyses and Maximum Likelihood (Figs 2, 3) recovered Elaphomyces castilloi supporting the

Diagnosis.
Elaphomyces castilloi differs from other species of the genus by the following combination of characteristics: ascomata embedded in a yellow mycelial mat, dull blue powdery gleba, and globose reticulate ascospores (9.7-11.5 µm). Etymology. The species was named castilloi in honor of José Castillo Tovar (ad memoriam), a Mexican pioneer mycologist dedicated to studying the fungi from northeast Mexico.
Distribution. Known only from the Mexican state of Chiapas, growing scattered, and hypogeous under Quercus sp. in montane cloud forest.
Notes. Elaphomyces castilloi is phylogenetically close to Elaphomyces aculeatus Vittad. from Italy, the last one with similar ascospore color and ornamentation. It was previously reported from Mexico by Gómez-Reyes et al. (2012). Elaphomyces aculeatus has a reddish peridium and dark-brown gleba; meanwhile, E. castilloi has dark peridium and bluish gleba. The yellow mycelial mat and the small ascospores resemble those of Elaphomyces citrinus Vittad. (Section Malacodermei). However, it differs by the smaller ascocarp (less than 10 mm), the brownish peridium in young specimens, and by its geographic distribution (Europe) (Pegler et al. 1993). Although the morphological features of the new species are typical in the Malacodermei, these are also seldom observed in the Ceratogaster (Paz et al. 2017). Diagnosis. Entoloma secotioides is characterized by cream colored, sulcate, secotioid basidiomata, not anastomosed gills, and angular basidiospores (7-13 × 5-9 µm).
Distribution. Known only from the state of Chiapas, growing sub hypogeous under Quercus sp. and Pinus sp. in montane cloud forest.

Discussion
Hypogeous fungi in Mexico have been scarcely studied compared to epigeous fungi; however, from the 2000s, new species have been regularly described, mainly from temperate forests (Guevara-Guerrero et al. 2014;Gómez-Reyes et al. 2018). In the case of Elaphomyces, it is one of the best represented genera in the country because it is associated with a large number of hosts, mainly Pinus and Quercus (Trappe and Guzmán 1971;Cázares et al. 1992;Castellano et al. 2012;Gómez-Reyes et al. 2012). Some species of Elaphomyces have even been described as culturally important. Elaphomyces muricatus has been reported for ritual or medicinal use (Trappe et al. 1979). For Entoloma secotioides, this is the first record of a sequestrate Entoloma in Mexico, these being mostly of the previously recorded species pileate-stipitate. Although the species has been described growing under Quercus species, there are no data on its ecological habits and these are presented here as putatively associated with Quercus.
Chiapas is one of the states with the greatest biological richness in Mexico, only surpassed by Oaxaca (López-Guzmán et al. 2017). The diversity of fungi in this state spans approximately 850 species. Recent research suggests a fungal diversity between 20,000 and 49,000 species (Chanona-Gómez et al. 2007;Ruan-Soto et al. 2013;Kong et al. 2018). Efforts are currently being made to document this diversity, which is threatened by land use change. Elaphomyces castilloi represents the first record of the genus Elaphomyces in Chiapas and represents the southernmost distribution of the genus in Mexico. Another species reported in southern Mexico is E. maculatus, which has been reported in north Oaxaca in oak forests (Trappe et al. 1979).
The sequestrate fungi have been studied mostly in the temperate regions of the north and center of the country; Elaphomyces castilloi and Entoloma secotioides are new contributions that represent the first findings of sequestrate fungi from the montane cloud forest in Chiapas, more than 50%; of which have unfortunately disappeared; montane cloud forest constitutes less than 1% of the Mexican territory. However, it is vital to carry out samplings that include taxa from this ecosystem considering that its losses are so high. Some localities are deemed critical for conservation of this ecosystem which is considered "endangered" under the definition of the Official Mexican Law (SEMAR-NAT 2010). The degradation of the montane cloud forests in Chiapas is high, therefore the level of threat to the habitat of the new two species is also high. So far, they are only known from the type locality and it is necessary to increase the sampling to assess the current status of the species. Keeping the taxonomical studies about the fungi from the montane cloud forest could help to encourage its conservation and management.