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.

Ectomycorrhizal fungi, ITS, Neotropical fungi, nLSU, Tricholomatales

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 and Salicaceae (Kropp and Mueller 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, 2017, Montoya et al. 2015, Luo et al. 2016, Popa et al. 2014, 2016). 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 well-known 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, 2015, 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, 2003, 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 molecular 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 hm² 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.

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) inferred the Mexican samples clustered in two distinct clades. A group clearly related to Laccaria trichodermophora and another, in a separate clade, representing an undescribed species.

Figure 1. 

Phylogenetic relationships within Laccaria species inferred from the combined ITS and LSU sequence data by maximum likelihood method. Tree with the highest log likelihood (–4163.7219), the percentage of trees in which the associated taxa clustered together (only values ≥ 70% are considered) is shown next to the branches, followed by the posterior probabilities (only values ≥ 0.90 are indicated) obtained after Bayesian inference. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

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 distinct 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).

Color features of the basidiomes and whitish mycelia relate Laccaria squarrosa to metasection Laccaria (Mueller 1992), where it superficially resembles L. proxima (Boudier) Patouillard. This later species, however, can be distinguished based on the longitudinally striate stipe, with a fibrillose surface only, ellipsoid basidiospores [9–11.5 × 6.7–8 (-8.8) µm, Q = 1.25–1.35 (-1.4)], having shorter echinulae (0.5–1 µm length), pleurocystidia absence and larger cheilocystidia [19–66.5(-92) × 2–8.5(-16.5) µm] (Mueller 1992). Among the species in the genus, Laccaria nobilis A.H. Smith, L. amethysteo-occidentalis G.M. Muell., L. trichodermophora and L. ochropurpurea (Berk.) Peck also produce fibrillose to somewhat scaly pileus surfaces. Laccaria ochropurpurea even can have recurved scales on the stipe surface. However, all those taxa clearly differ from Laccaria squarrosa by basidiomes and mycelia with violaceous colors, besides other macro and microscopical features (Mueller 1992).

Laccaria trichodermophora G.M. Mueller (Figs 6–7) was previously reported from Mexico (as L. farinacea sensu Singer) by Montoya et al. (1987) from conifer forests of Cofre de Perote National Park areas. The collections from Fagus grandifolia var. mexicana forest here studied, were collected in the locality of Mesa de la Yerba (Veracruz), on Aug 04 2005, Montoya 4393, Montoya 4394; Aug 28 2007, Bandala 4282 (XAL). Excepting by narrower hyphae disposed in the pileipellis mounds and the basidiospores including broadly ellipsoid to ellipsoid shapes, exhibit a similar morphological variation as those described by Mueller (1984, 1992) and other collections reported before from Mexico. A summary of the main morphological features that characterize the studied materials are: pileus 15–55 mm diam, fibrillose to fibrillose-minutely scaly, brownish-orange (6C6–C7), light brown or pale pinkish-brown or pale brownish towards the margin (6D6, 7C5–C4), hygrophanous. Lamellae 2‒6 mm in length, adnate to sinuate, close to subdistant, pinkish or incarnate (6A2‒B3). Stipe 20–75 × 2–8 mm, cylindrical, attenuated towards the apex, striate, fibrillose and fistulous. hygrophanous, concolorous to pileus but more pinkish-violaceous (13CD2) towards the base. Basal mycelium violaceous becoming white. Basidiospores 6–9 (-10) × 6–8.5 (-10) µm, X̅= 6.9–8.18 × 6.84–7.9 µm, Q̅= 1.00–1.05, globose, hyaline, echinulate, under SEM the echinulae appear 0.8–1.13 × 0.6–0.8 µm. Basidia 27–65 (-80) × 7–13 µm, clavate, tetra or at times tri-sporic, hyaline, thin walled, clamped. Cheilocystidia 12–49 × 2–6 µm cylindrical to narrowly clavate, at times somewhat utriform, hyaline, thin walled, frequently clamped. Pileipellis composed of periclinally oriented hypahe 3–10 µm diam, in a more or less cutis arrangement but with frequent mounds of intermixed or erect hyphae, with terminal elements 25–125 × 10–13 µm, cylindrical to clavate other somewhat utriform 20–65 × 5–17 µm. Clamps present.

Figure 6. 

Laccaria trichodermophora, a basidiospores b pleurocystidia c basidia d pileipellis e cheilocystidia a–e Montoya 4393. Scale bars: 5 µm (a); 10 µm (b–c, e); 25 µm (d).

Figure 7. 

Laccaria trichodermophora, a–b basidiospores under SEM c–d pileipellis a, c–d Montoya 4393 b Bandala 4282. Scale bars: 1 µm (a–b); 50 µm (c); 25 µm (d).

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.

We appreciate the assistance in the field and in the laboratory by Biol. D. Ramos (at Instituto de Ecología, A.C.), Biól. J.C. Corona and Ing. Alicia García. We recognize the support given by CONACYT (CB 169172) to study the EcM fungi associated with Fagus grandifolia var. mexicana in the montane cloud forest of Central Veracruz and by the postdoctoral grant to A. Ramos. We acknowledge the support given by CONACYT (225382) to the Laboratorio de Presecuenciación, Red Biodiversidad y Sistemática, INECOL. M.Sc. Bertha Pérez at this lab aid in some molecular procedures.