A new species and a new record of Laccaria (Fungi, Basidiomycota) found in a relict forest of the endangered Fagus grandifolia var. mexicana

Abstract Two species of Laccaria discovered in relicts of Fagus grandifolia var. mexicana forests in eastern Mexico are described based on the macro- and micromorphological features, and their identity supported by molecular analysis of the internal transcribed spacer (ITS) and large subunit (LSU) of the ribosomal RNA gene. The phylogeny obtained here showed that one of the Mexican species is nested in an exclusive clade which in combination with its striking morphological features, infers that it represents a new species, while the other species is placed as a member in the Laccaria trichodermophora clade. This is the first report in Mexico of Laccaria with Fagus grandifolia var. mexicana trees, with which the reported species may form ectomycorrhizal association. Descriptions are accompanied with illustrations of macro- and micromorphological characters and a discussion of related taxa are presented.


Introduction
It has long been recognized that Laccaria species are important ectomycorrhizal associates of ectotrophic plants worldwide (Mueller 1992). They are known to form interactions, for example with members of the Pinaceae, Dipterocarpaceae, Fagaceae, Betulaceae, Myrtaceae, Tiliaceae andSalicaceae (Kropp andMueller 1999, Wilson et al. 2013). Some species as Laccaria laccata and L. bicolor have been considered host-generalists, and inclusive, have been subject of a lot of in vitro experimentation worldwide. However, recent studies developed based on molecular systematics showed that under those names, complexes of species are included (Taylor et al. 2006, Jargeat et al. 2010, Vincenot et al. 2012, Sheedy et al. 2013, Popa et al. 2014. A wide ectomycorrhizal host range has also been attributed to L. amethystina, but in this case it has some support for its generalist abilities at the population genetics level by Roy et al. (2008), while consideration for cryptic biological species was discarded, at least among the populations sampled in France.
In the monographic work of Laccaria by Mueller (1992), 19 species are recognized from North America, and 40 worldwide. New or potential undescribed species from different regions, based on morphological and molecular characteristics of fructifications, or on DNA identifications of environmental samples, have been discovered recently (Wang et al. 2004, Osmundson et al. 2005, Sheedy et al. 2013, Wilson et al. 2013, Montoya et al. 2015, Luo et al. 2016, Popa et al. 2014. Nowadays, MycoBank recognizes 112 records in this group of fungi, and additionally, Wilson et al. (2017) inferred 116 phylogenetic species from 30 countries covering the known geographic range of Laccaria. During the advances on the systematics of the group, a small number of morphological (macro-and microscopic) features had been found taxonomically informative (McNabb 1972, Mueller 1992, which may be the cause of false interpretations, leading to conceptual misunderstandings. In fact, since early taxonomic studies on the group, the need to revise the species of Laccaria commonly treated under names widely cited in the literature was considered as an important task, due to the existence of different, even undescribed species, confused under apparently wellknown ones, such as in the groups of L. laccata (Scop.) Cooke and L. proxima (Boud.) Pat. (Singer 1967, Mueller and Sundberg 1981, Irving et al. 1985. For example, the study by Sheedy et al. (2013) based on DNA multigene sequences, even noted that cryptic phylogenetic species were not nested as sister taxa. Thus, strict species identifications and achieving phylogenetic inferences with stronger resolution in Laccaria, will aid in building a robust data set, dealing with each species ectomycorrhizal host range.
In Mexico, the reports of the diversity of the genus Laccaria include about 17 species (Aguirre-Acosta and Pérez-Silva 1978, Bandala et al. 1988, Montoya et al. 1987, Cifuentes et al. 1990, Pérez-Silva et al. 2006, Garibay-Orijel et al. 2009). The edibility and use of some species as food has been documented (e.g. Montoya et al. 1987, Montoya-Esquivel et al. 2002, Lampman 2007, Pérez-Moreno et al. 2008) and ectomycorrhizae formed under in vitro culture conditions, isolated from native specimens have also been achieved (Santiago-Martínez et al. 2003, Carrasco-Hernández et al. 2010, Galindo-Flores et al. 2015. Molecular studies on most of those records are needed not only to support their identifications but for being included in phylogenetic studies. Laccaria roseoalbescens T. J. Baroni, Montoya and Bandala, described as new (Montoya et al. 2015) from the mesophytic forest in Veracruz, was recognized under morphological features and confirmed through phylogenetic DNA sequence analyses and recently incorporated by Luo et al. (2016) in their mo-lecular phylogeny to confirm the distinction of the new L. rubroalba X. Luo, L. Ye, Mortimer & K.D. Hyde from China.
We have under research the fungal community associated to the two southernmost relicts of mesophytic forests dominated by Fagus grandifolia var. mexicana in the American Continent. This tree species is currently in danger of extinction and in the Red list of Mexican cloud forest trees, inhabiting a narrow range of nearly 145 hm 2 in Mexico (Rodríguez-Ramírez et al. 2013, Montoya et al. 2017. Taking into account its current status, we consider important to document the associated fungal species with particular focus to the ectomycorrhizal forming species. During our study, we found two species of Laccaria which after their morpho-and molecular analyses we concluded that with strong support can be recognized, one as L. trichodermophora G.M. Mueller and the other, as a distinct undescribed species close to L. angustilamella Zhu L., Yang & L. Wang from China. As both are part of the unknown potential mycobionts of this endangered ectotrophic tree species, we were motivated to document them.

Sampling and morphological study of basidiomes
Random visits were conducted during August-September 2005 and 2007, in two stands of Fagus grandifolia var. mexicana from Veracruz, Mexico, one in Acatlán Volcano, Acatlán (19°40'43.9"N; 96°51'9.8"W, 1840 m) and the other in Mesa de la Yerba, Acajete (19°33'37.2"N; 97°01'9.8"W, 1900 m). Basidiomes of Laccaria growing close to Fagus were gathered. Macromorphological characters and color were recorded, alphanumeric color codes in descriptions refer to Kornerup and Wanscher (1967). Measurements and colors of micromorphological structures were recorded in 3% KOH. Basidiospores were studied in Melzer's reagent. Methods to determine spore ranges are those used by Montoya and Bandala (2003), with 45-50 spores measured per collection (length and width of the sporoid excluding the ornamentation) and given as a range with the symbol X ̅ representing mean values. Q ̅ represents the basidiospore length/width ratio and is given as range of mean values. Line drawings were made with a drawing tube. The examined specimens studied are deposited in XAL herbarium (acronym from B. Thiers, continuously updated; Index Herbariorum: http://sweetgum.nybg.org/ih/). The SEM images were obtained after critical point drying of pieces of lamellae previously rehydrated in ammonia, fixed in glutaraldehyde and dehydrated in an ethanol series (Bandala and Montoya 2000).

DNA extraction, PCR amplification, and sequencing
Genomic DNA of the Mexican specimen was extracted according to Montoya et al. (2014). PCR was performed to amplify the ITS (Internal Transcribed Spacer) and LSU (Large Subunit) regions of the nuclear rDNA, using primers ITS1F, ITS5/ITS4, LR0R/LR21, LR7 (Vilgalys and Hester 1990, White et al. 1990, Gardes and Bruns 1993. PCR conditions, as well as procedures for the purification of amplified PCR products, cycle sequencing reactions and their purification were done according to Montoya et al. (2014). Once sequences were assembled and edited, they were deposited at GenBank database (Benson et al. 2017) (Table 1).

Phylogenetic methods
The phylogenetic analysis was performed with the sequences obtained in this study, as well as some retrieved from GenBank (http://www.ncbi.nlm.nih.gov/) derived from the Blast analysis (only those that best match), and complemented with related sequences used by Osmundson et al. (2005), Montoya et al. (2015) and Wilson et al. (2017) (Table 1). For this purpose, we constructed a dataset (ITS+LSU) using PhyDE v.0.9971 (Müller et al. 2010), also with MEGA 6.06 (Tamura et al. 2013) calculated the best evolutionary model and constructed the phylogenetic tree under the method of Maximum Likelihood (ML) with 500 bootstrap replications, and finally with MrBayes v 3.2.6 (Ronquist et al. 2012) constructed the phylogenetic tree (as Montoya et al. 2014) under the method of Bayesian Inference (BI). The phylogenies from ML and BI analyses were displayed using Mega 6.06 and FigTree v1.4.3 (Rambaut 2016) respectively.

Results
A total of 13 new ITS and 28S sequences for Laccaria were generated in this study (Table 1 and alignment in TreeBASE S21413). They were obtained from Laccaria samples proceeding from the two stands of Fagus grandifolia var. mexicana in the subtropical cloud forest in central Veracruz (sample AR24 comes from a conifers forest in Veracruz) (Table 1). Only bootstrap values of ≥70% and posterior probabilities (ML/ PP) of ≥0.90 were considered and indicated on the tree branches. The phylogeny displayed (Fig. 1)  Diagnosis. Differs from other species by having medium sized basidiomes, with pinkish to pale brownish-orange colors, smooth to finely squarrose surfaces, especially on the stipe, basal mycelium with whitish to pale brownish with pinkish tinges, and globose, echinulate basidiospores, 7-10 (-11.5) × 7-10.5 µm, with the echinulae 0.5-1.4 in length, 0.45-0.9 µm in width at base, subcylindrical to contorted cystidia and pileipellis arranged in a cutis with mounds of intermixed and irregularly projected hyphae.

Discussion
In the phylogeny presented here that is based on sequences used in the worldwide survey of Laccaria by Wilson et al. (2017) and complemented with some from GenBank ( Fig. 1) and sequences of L. squarrosa, described here, this new taxon is clearly shown to be phylogenetically isolated from other Laccaria species. Laccaria squarrosa is dis- tinct by possessing typical medium sized basidiomes with scaly surfaces, more obvious especially on the stipe and by having the basal stipe mycelium whitish to pale brownish with pinkish tinges. Microscopically it differs by globose, echinulate basidiospores, cylindrical cystidia and pileipellis arranged in a cutis with mounds of intermixed and irregularly projected hyphae. In Fig. 1, L. squarrosa is shown to be phylogenetically close to L. angustilamella Zhu L., Yang & L. Wang from China. This later species is characterized, however, by having a marasmioid to mycenoid habit, with a short basidiome size (pileus 20-30 mm diam), narrow (2 mm length) and subdistant lamellae, non-scaly stipe, with more ellipsoid basidiospores (Q up to 1.18) and larger echinulae (2.0-) 2.5-5.0 µm long and up to 2.5 µm wide at base (Wang et al. 2004).
In the phylogeny obtained (Fig. 1), the sampled sequences of this species appear in three clades. One of them, with collections from North America, included the type specimen (DQ149868) and 21 specimens from Mexico. Our collections clustered in this later lineage interestingly with one sample from Costa Rica too. The other two clades are composed of specimens from Texas, one of them sister to the type clade, and the third clearly separated, probably representing an undescribed species. A specimen (KP128033) labeled as L. trichodermophora in the GenBank, clustered in L. alba group from Asia in our analysis. This sample lacks geographic information and could well be a misidentified collection.
There are no previous reports of Laccaria trichodermophora being associated with Fagus grandifolia var. mexicana. This report serves as the first documentation of this association. According to the reports of L. trichodermophora, it shows a wide ecological range. Mueller (1992) observed that all collections of this Laccaria species from the southeastern United States appeared to be associated with Pinus. He also collected it, in Costa Rica, beneath Neotropical species of Quercus. In central Mexico, in the states of Tlaxcala and Michoacán, it has been recorded associated to mixed Pinus-Alnus and Pinus-Abies forests (Montoya et al. 1987, Montoya-Esquivel et al. 2004). In the eastern part of Mexico, in Veracruz, it has been found (as L. farinacea sensu Singer) in monodominant Pinus and mixed Pinus-Abies forests (Montoya et al. 1987). In this later country, it is interesting to note that, basidiomes of this species, specially from conifers, are sold in local markets as edible fungi (Montoya et al. 1987, Montoya-Esquivel et al. 2004). Based on the available ecological information of the samples in the phylogenetic tree (Fig. 1), a wide host range for L. trichodermophora type specimen clade can be inferred. Among the potential hosts, it can be recognized as occurring with Fagus grandifolia, Pinus elliottii, P. palustris and Quercus sp. in Texas, as well as P. patula, other species of Pinaceae and Quercus spp. in both US and in Mexico, and the endangered F. grandifolia var. mexicana as confirmed here. Abies religiosa represents another host also, as proved by data from two sequences (MF669964 and MF669970) ( Table 1, Fig. 1) obtained here, from the sample AR24, from an A. religiosa forest at Cofre de Perote National Park in Veracruz, Mexico.