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Botanical Sciences

versão On-line ISSN 2007-4476versão impressa ISSN 2007-4298

Bot. sci vol.100 no.2 México Abr./Jun. 2022  Epub 22-Mar-2022 

Structural botany

Morphoanatomical and phylogenetic characterization of the ectomycorrhiza between Laccaria squarrosa with Pinus pseudostrobus and its relevance for reforestation programs

Caracterización morfoanatómica y filogenética de la ectomicorriza de Laccaria squarrosa con Pinus pseudostrobus y su relevancia para programas de reforestación

Mariana Herrera1

Fu-Qiang Yu1

David Ramos-Rendón2 

Magdalena Martínez-Reyes3

Faustino Hernández-Santiago3  4

Caspar C.C. Chater5

Jesús Pérez-Moreno3  *

1Germplasm Bank of Wild Species, Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China.

2 Investigador independiente, Xalapa, Veracruz, México.

3 Colegio de Postgraduados, Edafología, Campus Montecillo, Texcoco, México.

4 Universidad Intercultural del Estado de México, Campus Tepetlixpa, Tepetlixpa, México.

5 Department of Natural Capital and Plant Health, Royal Botanic Gardens, Kew, Richmond, UK.



Pinus (Coniferophyta) and Laccaria (Basidiomycota) establish ectomycorrhizal symbioses in natural forests. However, their detailed morphoanatomical and phylogenetic characterization have received little attention. Accurate identification of native host symbionts is of paramount relevance to the production of mycorrhized seedlings for successful reforestation programs.


We aimed to determine if L. squarrosa is able to establish ectomycorrhizal symbiosis with gymnosperms, thereby widening its host range and highlighting its relevance as a potential inoculant for pine seedlings. Currently, L. squarrosa is only known from its type collection associated with the angiosperm Fagus grandifolia var. mexicana.

Studied species:

The fungus L. squarrosa and Pinus pseudostrobus, a tree endemic to Mexico.

Study site and dates:

A Pinus-Quercus forest in Piedra Canteada, Nanacamilpa, Tlaxcala; 2018-2020.


L. squarrosa basidiomata were identified and ectomycorrhizal roots were collected and morphoanatomically characterized. For molecular identification, DNA was extracted, PCR was performed targeting the nuclear ribosomal internal transcribed spacer region (nucrDNA ITS) for the mycobiont identification and the chloroplastic single-locus trnL region for the phytobiont.


In the phylogenetic analyses, our sequences from basidiomata and ectomycorrhizae clustered together with L. squarrosa with high values of supporting identity. Meanwhile, P. pseudostrobus was molecularly identified as the phytobiont.


This is one of the few worldwide characterizations of Laccaria ectomycorrhiza under field conditions and contributes to the understanding of the ecology, distribution, and economic relevance of the symbiotic association. Our data suggest that L. squarrosa has potential for use as a native inoculant for P. pseudostrobus tree production.

Keywords: Ectomycorrhizal symbiosis; edible wild mushrooms; inoculants; Neotropics; pines



Las especies de Pinus (Coniferophyta) y Laccaria (Basidiomycota) establecen simbiosis ectomicorrízicas en bosques naturales. Sin embargo, su caracterización detallada morfológica y molecular ha recibido poca atención, a pesar de que la identificación precisa de fitobiontes nativos es fundamental para la producción exitosa de plantas micorrizadas con fines de reforestación.


Conocer si Laccaria squarrosa establece ectomicorrizas con gimnospermas, ampliando su rango de hospederos y relevancia como inoculante ectomicorrízico potencial para la producción de plántulas de pino. Hasta ahora L. squarrosa solo se conocía de su colección tipo asociada con Fagus grandifolia var. mexicana.

Especies estudiadas:

L. squarrosa y Pinus pseudostrobus, árbol endémico de México.

Sitio y años de estudio:

Bosque de Pinus-Quercus, Piedra-Canteada, Tlaxcala; 2018-2020.


Se identificaron basidiomas de L. squarrosa y se recolectaron y caracterizaron morfoanatómicamente raíces ectomicorrizadas. Para la identificación molecular, se extrajo ADN, se realizaron PCR de la región espaciadora transcrita interna del ADN ribosomal (nuc-rDNA ITS) para la identificación del micobionte y de la región cloroplástica trnL para el fitobionte.


En los análisis filogenéticos, las secuencias de basidiomas y ectomicorrizas se agruparon junto con L. squarrosa con altos valores de similitud. Mientras tanto, P. pseudostrobus se identificó molecularmente como el fitobionte.


Esta es una de las pocas caracterizaciones mundiales de ectomicorrizas de Laccaria en campo; y se contribuye a la comprensión de la ecología y distribución de esta asociación simbiótica económicamente relevante. Se demuestra que L. squarrosa podría usarse potencialmente como inoculante nativo para la producción de árboles de P. pseudostrobus.

Palabras claves: hongos silvestres comestibles; inoculantes; Neotrópico; pinos; simbiosis ectomicorrízica

Mexico is losing an average of 318,000 hectares of forest annually and is one of the countries with the highest deforestation rates, resulting in rapid ecosystem loss and decreasing native biodiversity (including plants and mushrooms) (Pérez-Moreno et al. 2020). Mexico is also the center of diversity of Quercus and Pinus species (Perry 1991, Valencia & Flores-Franco 2006) which are both widely known as ectomycorrhizal (ECM) trees. As Mexico harbors a great diversity of ECM trees, a high number of ECM mushrooms are expected. These ectomycorrhizal associations occur in all forest ecosystems from the tropics to the subpolar zones (Nara 2015). The ECM trees depend on the ECM fungi for obtaining greater access to mineral nutrients and water, while the trees provide a supply of carbohydrates to the symbiotic fungi (Smith & Read 2008).

Laccaria species associate with many ECM trees (Trappe 1962, Mueller 1992, Kropp & Mueller 1999, Wilson et al. 2017, DEEMY 2021). It is estimated that 116 species of Laccaria are distributed across temperate and tropical regions of the world (Wilson et al. 2017). The diversity and taxonomy of the genus Laccaria have been well documented (Mueller 1984) including with phylogenetic and molecular studies (Osmundson et al. 2005, Vincenot et al. 2012, Sheedy et al. 2013, Wilson et al. 2013, Popa et al. 2014, 2016, Montoya et al. 2015, Cho et al. 2018). More recently, biogeographic (Wilson et al. 2017) and genomic studies have used some Laccaria taxa (e.g.,Martin et al. 2008, Kang et al. 2020, Li et al. 2020) as model species in the understanding of the physiology, ecology, and evolution of ectomycorrhizal symbiosis.

In general, Laccaria species are considered early-stage colonizers (Nara et al. 2003, Reverchon et al. 2012) and therefore play a paramount ecological role in the establishment of ECM host plants (Kropp & Mueller 1999, Nara et al. 2003, Ashkannejhad & Horton 2005). They can grow vegetatively and are relatively easy to manipulate under experimental conditions. Several species of the genus, including for example Laccaria bicolor (Maire) P.D. Orton, L. laccata (Scop.) Cooke, and L. proxima (Boud.) Pat., have successfully been used in the production of ECM inoculants for forest management purposes (Molina 1980, Molina & Chamard 1983, Chakravarty & Hwang 1991, Duponnois & Garbaye 1991, Werner & Zadworny 2003, Pérez-Moreno et al. 2020). Laccaria species such as Laccaria amethystina Cooke. and L. laccata (Scop.) Cooke also have great cultural, social, and economic value and are harvested for income and as food in different parts of the world (McKnight et al. 1998, Pérez-Moreno et al. 2008, Kalita et al. 62016, Wu et al. 2019). Laccaria squarrosa Bandala, Montoya & A. Ramos, however, is a recently described species. It is only known from its type locality, where it associates with endangered Fagus grandifolia var. mexicana (Martínez) E. Murray trees in eastern Mexico (Ramos et al. 2017), and from discovery at a market in western Mexico, where it was identified as a rare species by Farfán-Heredia et al. (2018) without stating the vegetation type or location from where the specimens were gathered.

Pinus pseudostrobus Lindl. has a wide geographical distribution in Mexico and Central America with a broad altitudinal range between 1,600-3,300 m above sea level (Delgado et al. 2007). The species usually establishes pure stands or occurs with some other species of Pinus, Abies, Arbutus, Juniperus, or Quercus (Perry 1991). In Mexico, P. pseudostrobus has been used in reforestation programs especially on degraded soils, and its resins and wood are highly commercialized (Gómez-Romero et al. 2013).

During some expeditions to forest located in Piedra Canteada, Nanacamilpa, Tlaxcala, in Central Mexico, to document the diversity of wild edible mushrooms consumed by the local people, we found and collected specimens of L. squarrosa (a legitimate species included in the Mycobank database with the number 823034). The study site is in a natural reserve called the “Santuario de las Luciernagas” (The Sanctuary of the Fireflies) famous for the displays of fireflies that inhabit its Pinus-Quercus forest. The local people make an income from this activity during the summer, motivating them to conserve the forest and manage its resources. Basidiomata of L. squarrosa are also harvested as a source of food by members of the Nahua ethnic groups in this area.

In this study, we aim to determine for the first time whether Laccaria squarrosa establishes ectomycorrhizal symbiosis with the gymnosperm Pinus pseudostrobus in natural conditions, confirming the taxonomic identity of both symbionts with molecular analyses. The confirmation of this association would provide evidence to promote the use of L. squarrosa as a potential native inoculum in the production of ectomycorrhized P. pseudostrobus seedlings in successful reforestation programs in the studied area.

Materials and methods

Study area. The study area is in the state of Tlaxcala, Mexico, on the Trans-Mexican Volcanic Belt (TMVB; Figure 1). This ecoregion connects the Sierra Madre Oriental and the Sierra Madre Occidental, and serves as a center of biodiversity. The sampling site is in Piedra Canteada, Nanacamilpa municipality (Figure 1). The dominant climate is temperate subhumid with rains in summer and annual temperatures of 12-18 °C. Mean annual precipitation is < 500 mm (INEGI 1997). The type of vegetation in the study area is dominated by Pinus-Quercus (pine-oak) forest and Abies (fir) forest. The most representative species in the Pinus-Quercus Forest include Pinus montezumae Lamb, P. pseudostrobus, P. teocote Schiede ex Schltdl. & Cham., which usually co-occur with Quercus spp. such as Quercus crassipes Bonlp., Q. laurina Bonpl., and Q. rugosa Née. The Abies forest is composed of Abies religiosa (Kunth) Schltdl. & Cham., Alnus jorullensis Kunth., Salix paradoxa Kunth., and Arbustus xalepensis Kunth., all coexisting in the same area.

Figure 1 Map of the sampling site in Piedra Canteada, Nanacamilpa (brown), Tlaxcala (green), Mexico. Range of the Trans-Mexican Volcanic Belt (red). 

Sampling and description. L. squarrosa basidiomata were collected in a Pinus-Quercus forest area which is known by local mushrooms pickers as a fruiting area for this species of Laccaria. The collection was made in a small area where Pinus pseudostrobus dominates (Figure 2). The basidiomata were identified in the field as L. squarrosa due to the squarrose surfaces on the stipe which is a diagnostic macromorphological feature of this species. We compared the macroscopic and microscopic characters of our samples with those described for the L. squarrosa type specimen (Ramos et al. 2017), and as additional evidence we extracted DNA and amplified the internal transcribed spacer (ITS) region (see the following section) from the basidiomata collected in the study area to confirm their identity. Soil samples were collected below the basidiomata following Agerer (1991) and Gardes & Bruns (1996). A stereomicroscope (Leica S8APO) was used to separate fine roots from the soil samples to select, photograph, and describe the morphology of the ectomycorrhizae that were found (Agerer 1987-2002). From each ectomycorrhiza, hand-made sections were made (cross and longitudinal), mounted in 3 % KOH and observed under a compound light microscope (Leica DM2500) for microscopic characterization (Agerer 1987-2002, DEEMY 2021). The ectomycorrhizae were preserved in 75 % ethanol and tips were stored at -20 °C for DNA extraction to identify the symbionts.

Figure 2 Pinus pseudostrobus forest located in the study area. 

DNA extraction, PCR, and phylogenetic analyses. Total genomic DNA from both basidiomata and ectomycorrhizae was obtained using a modified cetyltrimethylammonium bromide (CTAB) procedure of Gardes & Bruns (1993). The polymerase chain reaction was performed targeting the nuclear ribosomal ITS regions using the primers ITS1F and ITS4 (White et al. 1990). PCR conditions were performed as follows: 94 °C for 5 min; then 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; followed by a final elongation step of 72 °C for 7 min. We constructed a dataset of aligned sequences obtained from root tips and basidiomata using the Phylogenetic Data Editor (PhyDE, v.0.997, Muenster, Germany, Genbank sequences that showed highest similarity scores (> 97) using the BLAST tool (Altschul et al. 1997) and sequences used in previous studies (Osmundson et al. 2005, Wilson et al. 2013) were included in the analyses. Cortinarius violaceus (L.) Gray (DQ486695) and Psathyrella rhodospora G. Weaver & A.H. Sm. (DQ486695) were included as outgroups (Table 1). To identify the host tree species at the nucleotide level, the plastid/chloroplastic single-locus trnL region was amplified from DNA isolated from the root tip. We amplified the trnL region using trnC/trnD primer pairs (Tedersoo et al. 2008). A second dataset was constructed including the new trnL sequences and 10 sequences of Pinus species retrieved from BLASTN results in GenBank (Altschul et al.1997) and one of Pseudolarix amabilis (NC030631) included as an outgroup. Both datasets (ITS-trnL) were separately aligned using the MUSCLE program (Edgar 2004). The best DNA model was selected using Mega 6.06 (Tamura et al. 2013). For the ITS dataset, Maximum likelihood (ML) and Bayesian inference (BI) were used. ML analysis was performed using MEGA 6.06 (Tamura et al. 2013) with 500 bootstrap replicates, while the BI analysis was estimated using MrBayes on XSEDE (3.2.6) using the CIPRES portal (Miller et al. 2010), employing two independent runs of 1,000,000 generations. For each run, four chains were employed, and one tree was sampled every 1,000 generations. respectively. Only Bootstrap values (BS) higher than 75 and Bayesian posterior probabilities (BPPs) above 0.85 were considered significant. As with the ITS dataset, ML analysis of the trnL dataset was performed using MEGA 6.06, and only BS values higher than 65 were considered significant.

Table 1 Laccaria species included in this study: voucher specimen, location, and sequence accession numbers. Black asterisk (*) indicates new sequences generated in this study. 

Taxon Voucher Specimen Location Genbank ITS
Laccaria sp. ALB183 Tibet, China JX504092
Laccaria sp. GMM6019 Costa Rica KU685757
Laccaria sp. GMM6012 Costa Rica KU685758
L. alba Zhu L. Yang & Lan Wang AWW438 Yunnan, China JX504094
L. alba F1120750 China JX504126
L. amethystea (Bull.) Murrill FP-98556 Vorpommern, Germany DQ499640
L. amethystea TUB 011464 Germany AF539737
L. amethysteo-occidentalis G.M. Muell. AWW556 California, USA JX504107
L. amethysteo-occidentalis AWW590 Oregon, USA JX504112
L. amethystina Cooke GMM7041 Caucasus, Russia KU685654
L. amethystina GMM7621 France JX504150
L. angustilamella Zhu L. Yang & L. Wang BAP226 Yunnan, China JX504118
L. angustilamella HKAS58714 Yunnan, China JX504168
L. aurantia Popa, Rexer, Donges, Zhu L. Yang & G. Kos KUN-F78557 Type Yunnan, China JQ670895
L. aurantia MB-FB-101109 Yunnan, China JQ681209
L. bicolor (Maire) P.D. Orton AWW539 Illinois, USA KM067817
L. bicolor AWW537 Illinois, USA JX504105
L. nobilis A.H. Sm. F1091206 Michigan, USA KU685636
L. ochropurpurea (Berk.) Peck JMP0038 Wisconsin, USA EU819479
L. ochropurpurea PRL4777 Illinois, USA KU685733
L. ochropurpurea KH_LA06_016 Louisiana, USA KU685721
L. ochropurpurea PRL3777 Illinois, USA KU685732
L. proxima (Boud.) Pat. F1133825 Mississippi USA KU685642
L. roseoalbescens T.J. Baroni, Montoya & Bandala LM5042 Veracruz, Mexico KJ874327
L. roseoalbescens LM5099 Type Veracruz, Mexico KJ874328
L. salmonicolor A.W. Wilson & G.M. Muell. GMM7596 Type Tibet, China JX504143
L. squarrosa Bandala, Montoya & A. Ramos DM63 Type Veracruz, Mexico MF669958
L. squarrosa DM93 Veracruz, Mexico MF669959
L. squarrosa DM121 Veracruz, Mexico MF669960
L. squarrosa* Hernández-Santiago 67 Tlaxcala, Mexico MT026967
L. squarrosa* Hernández-Santiago 65 Tlaxcala, Mexico MT026968
L. squarrosa* Martínez-Reyes 273 Tlaxcala, Mexico MT026969
L. squarrosa* Martínez-Reyes 274 Tlaxcala, Mexico MT026970
L. trichodermophora G.M. Muell. GMM7697 Texas, USA KM067863
L. trichodermophora GMM7698 Texas, USA KM067864
L. trichodermophora GMM7703 Texas, USA KM067865
L. trichodermophora GMM7712 Texas, USA KM067866
L. trichodermophora GMM7714 Texas, USA KM067867
L. trichodermophora GMM7716 Texas, USA KM067869
L. trichodermophora TENN42523 Type Texas, USA DQ149868
L. trichodermophora Montoya 4394 Veracruz, Mexico MF669962
L. trichodermophora Bandala 4282 Veracruz, Mexico MF669963
L. trichodermophora F1111951 Costa Rica KU685640
L. trichodermophora KH_LA06_013 Lousiana, USA KM067881
L. trichodermophora GMM7733 Texas, USA JX504157
Psathyrella rhodospora M.G.Weaver & A.H. Sm. MP133 MN ------- DQ267129
Cortinarius violaceus (L.) Gray MTS 4854 (WTU) Washington, USA DQ486695


Four soil samples were collected beneath basidiomata of Laccaria squarrosa (MT026967 and MT026968) (Figure 3) from different sites in a pure Pinus pseudostrobus stand in Nanacamilpa, Tlaxcala, Mexico. A total of eight morphotypes were found in the soil samples and photographed, described, and sequenced. After BLAST analysis (Genbank), the samples labeled as Martínez-Reyes 273 ECM (MT026969) and Martínez-Reyes 274 ECM (MT026970), were confirmed as L. squarrosa. In our phylogenetic analysis, these samples clustered together (with high bootstrap and posterior probabilities values, BS = 99, BPPs = 1) with the sequence from the basidiomata of L. squarrosa collected in the study area and the sequence of the type specimen (Figure 4). In the case of the host tree the ML analyses clustered together our samples (Martínez Reyes 273, MW082603; Martínez Reyes 274, MW082604) with Pinus pseudostrobus sequences with a moderate but significant support value (BS = 65; Figure 5).

Figure 3 Laccaria squarrosa. A-B Mature basidiomata in their natural environment in Piedra Canteada, Nanacamilpa, Tlaxcala, Mexico (Hernández-Santiago 65); C-D Scanning electron micrograph of echinulate spores (Hernández-Santiago 65). 

Figure 4 Phylogenetic relationships of Laccaria squarrosa based on ITS sequences from basidiomata and ECM root tips and inferred from the maximum likelihood tree, including bootstrap values (≥ 70) and Bayesian posterior probabilities (≥ 0.95). New sequences obtained here are indicated in bold. Specimen vouchers, accession numbers of ITS sequences, and country of origin are indicated for each branch. 

Figure 5 Phylogenetic relationships of Pinus species based on trnL sequences and inferred from the maximum likelihood tree, including bootstrap values (≥ 65). New sequences obtained here are indicated in bold. 

Description of the ectomycorrhiza. Laccaria squarrosa (Bandala, Montoya & A. Ramos) + Pinus pseudostrobus Linl. (Figure 6). Ectomycorrhizal systems 1.83 mm in length and 3.15 mm wide, dichotomous, with short branches in two levels of ramification, branches 0.54-0.80 mm in length × 0.38-0.41 mm wide, straight to sinuous, brownish to pale brownish, dark-brownish to the ends, pale at the tip; smooth with some white appressed fibrils that give a glistening aspect, emanating hyphae scattered and loose; tips cylindrical to rounded; mantle opaque at distal end, hydrophilic, hyaline; rhizomorphs and sclerotia absent (Figure 6A-B). Longitudinal section: Mantle 15-35 μm thick, two layered (Figure 6C). Outer mantle 14 μm wide, loose, plectenchymatous, not gelatinized, with cylindrical cells up 2.5-3.5 μm, thin-walled, smooth. Inner mantle 18 μm broad, plectenchymatous, compact, hyphae up to 5 μm wide, with cylindrical cells 2.5-4 µm hyaline, thin-walled, tannin cells present, parallel to root axis. Hartig net palmetti, paraepidermal, penetrating the second layer of cortical root cells (Figure 6C-D), the hyphae between the root cells up to 3.5 μm wide, rounded; cortical cells rectangular, cylindrical 55-75 × 45-60 μm (in longitudinal section) and 27-65 × 27-51 μm (in cross-section) (Figure 6C-D). Clamp connections absent. Cross section: Mantle disposed in a plectenchymatous arrangement (Figure 6D), two layers discernible, hypha disposed in parallel and prostrate, some loose hyphae in the external mantle and compact in the inner mantle, tannin cells cylindrical, cortical cells 3.5 μm wide rectangular to rounded, hyphae between cortical cells cylindrical. Plan view: Outer mantle in a plectenchymatous layer, hyphae in an irregular arrangement composed of cylindrical cells 3-4 μm wide, sinuous, compact layer thin-walled, smooth hyphae (Figure 6E). Inner mantle in a plectenchymatous layer, in an irregular arrangement composed of cylindrical 2.5-5 (8) μm wide sinuous, tortuous; compact layer, thin-walled, smooth hyphae (Figure 6F).

Figure 6 Laccaria squarrosa + Pinus pseudostrobus ectomycorrhiza. A) Ectomycorrhizal (ECM) system; B) Details of the pruinose surface and tips of ECM roots; C) Tangential section showing mantle (M), Tannin cells (TC), and Hartig net (HN); D) Ectomycorrhiza in cross-section showing Mantle (M), Hartig net (HN), Tannin cells (TC), and hyphae between cortical cells (HCC); E) Plan view of the outer mantle; F) Plan view of the inner mantle. Bars 1 mm in A; 0.5 mm in B; 50 μm in C) and D); and 20 μm in E) and F). 


For the first time, we describe the ectomycorrhiza of Laccaria squarrosa + Pinus pseudostrobus in natural conditions and extend the distribution and host associations of L. squarrosa. L. squarrosa was previously known only from the type locality (in Veracruz state located in eastern Mexico) and associated only with Fagus grandifolia var. mexicana, and from a traditional Purépecha market (in Michoacán, located in western Mexico) without stating the associated host trees. Our new data show that the species is distributed along the Trans-Mexican volcanic belt (TCMV; Figure 1). Detecting the ECM association in the field (through the morphological and molecular characterization of the mycobiont and host tree) is the first step toward the identification of suitable hosts for forest management and reforestation programs. This is the second record of L. squarrosa in Mexico and confirms that this species is distributed along the Trans-Mexican-Volcanic-Belt in association with two different ECM host tree species. Laccaria squarrosa can therefore be used not only as native inoculum for reforestation programs in the study area (Nanacamilpa, Tlaxcala), but also in the type locality of the species (Acatlán, Veracruz, Mexico), where the Fagus grandifolia var. mexicana forest is endangered. Further sampling should be performed in Mexico to identify additional ECM host tree species associated with this ECM fungus.

The ectomycorrhiza here described displays some similarities with the previously documented ectomycorrhizae of other Laccaria species associated with different host trees (Table 2). They display similar colors (except those formed by L. amethystina Cooke + Quercus sp. and L. trichodemorphora G.M. Muell. + Pinus montezumae), present emanating hyphae (however, abundance differs among the species), lack of rhizomorphs (except those formed by L. amethystina Cooke + Quercus sp. and L. amethystina Cooke + Betula sp.) and has a plectenchymatous outer mantle. Features such as the ramification type, mycorrhizal system length, and mantle thickness vary among all the species. However, more detailed descriptions are needed for further discussion about Laccaria ectomycorrhizae morphotypes.

Table 2 Diagnostic morphological characteristics of ectomycorrhizas established between species of Laccaria associated with different host trees. RT: Ramification type; MSL: Mycorrhizal system length; MC: Mycorrhiza color; MS: mantle surface; EHP: Emanating hyphae presence; Rhiz: Rhizomorphs; MT: Mantle thickness; MO: Mantle organization; D: Dichotomous; M-pin: Monopodial-pinnate; M-pyr: monopodial pyramidal; C: Coralloid; Pl: Plectenchymatous; Ps: pseudoparenchymatous; Nd: No data; N: natural conditions, A: artificial conditions.  

Species + host RT
MO Reference Origin
Laccaria squarrosa + Pinus pseudostrobus D 1.83 Brownish to pale brownish, dark-brownish White fibrils glistening aspect Scattered and loose Absent 15-35 Pl *This study N
L. amethystina + Betula pendula M-pin 1-2 Ochre, yellowish brown, or orange or red Loosely woolly Concentrated distally Present 36-93 Pl Cuvelier 1991 N
L. amethystina + Fagus sp. M-pin M-pyr 1- 6 Brown, ochre, yellowish brown of grey Loosely woolly Concentrated distally Absent 25-70 Pl DEEMY 2021 N
L. amethystina + Quercus robur. M-pyr Nd Violet, dark reddish blue or white Loosely cottony Abundant Present Nd Pl DEEMY 2021 N
L. bicolor + Pinus montezumae D 2.5-3.66 Brown to pale brownish and orange brownish Metallic aspect Abundant Absent 23.5 -31.4 Pl Santiago-Martínez et al. 2003 A
L. bicolor + Pinus montezumae D Nd Reddish-brown Cottony Scattered Nd Nd Ps Rodríguez-Gutiérrez et al. 2019 A
L. bicolor + P. patula D 1-4.5 Brown to orange, reddish brown Glistening aspect Nd Absent Nd Pl Carrasco-Hernández et al. 2010 A
L. laccata + Pinus patula D 5.5 Whitish orange to greyish brown Pubescent Present Absent 10-50 Pl Mohan et al. 1993 A
L. laccata var. laccata + P. montezumae D Nd Golden yellow Cottony Scattered Nd Pl Rodríguez-Gutiérrez et al. 2019 A
L. proxima. + Betula spp. + Picea sitchensis +Pinus spp. M-pin M-pyr 1-8 Brown to white Shiny Present Absent 15-40 Pl Ingleby et al. 1990 A
L. proxima + P. pseudostrobus D 1-8 Pale Brown to dark brown Nd Nd Nd Nd Pl Carrasco-Hernández et al. 2010 A
L. tortilis + Betula pendula and Picea sitchensis M-pin M-pyr 7 Brown to white Loosely or cottony Present Absent 10-30 Pl Ingleby et al. 1990 A
L. trichodemorphora + Pinus montezumae D C 0.4-2.9 White to pale white Loosely cottony Abundant Absent 14.4 -81.5 Pl Galindo-Flores et al. 2015 A
L. trichodermophora + P. montezumae D Nd Orange, yellowish brown, bright orange Cottony Nd Nd Nd Pl Rodríguez-Gutiérrez et al. 2019 A
L. vinaceobrunnea + P. montezumae D Nd Orange to brownish yellow Cottony Nd Nd Nd Ps Rodríguez-Gutiérrez et al. 2019 A

Both the previous record of the holotype of Laccaria squarrosa under a pure stand of Fagus grandifolia var. mexicana and our results suggest that L. squarrosa is a generalist species that can be associated either with Angiosperms or Gymnosperms. This record of L. squarrosa with both Fagus and Pinus supports the hypothesis of Wilson et al. (2017) who suggest that Laccaria´s association with Fagaceae aided their dispersal into the northern hemisphere and its eventual association with Pinus. Although in our sampling area there were other ECM host trees (Abies religiosa, Pinus teocote and P. montezumae, and Quercus spp.) we only found basidiomata of L. squarrosa associated with P. pseudostrobus. This result was confirmed by our sequencing and phylogenetic analyses with a good support (BS = 65; Figure 5). Further fieldwork sampling of root tips in the study area would provide valuable ecological information of its possible symbiotic associations with other host species.

Despite the great diversity of Laccaria species and their associated host trees in both northern and southern hemispheres, only thirteen descriptions of ectomycorrhizae of six species (L. amethystina, L. bicolor, L. proxima, L. tortillis, L. squarrosa, and L. trichodermophora) have been described (Table 2). Most of the records are ectomycorrhizae synthesized under artificial conditions and only four of them have described the morphoanatomical features of Laccaria ectomycorrhizae collected in natural conditions (Ingleby et al. 1990, Cuvelier 1991, DEEMY 2021). Therefore, the ectomycorrhiza here described represent one of the few morphotype descriptions of the ecologically and economically relevant Laccaria genus in natural conditions in the world.

Laccaria squarrosa was recorded as an edible wild mushroom representing an important food source for local gatherers in the study area, who have traditionally considered the forests as a source of ecosystem services and food. Our robust confirmation of the establishment of ectomycorrhizal symbiosis between L. squarrosa and P. pseudostrobus, with morphoanatomical, molecular, and phylogenetic evidence, open the way to future biotechnological research to enhance sustainable forest management in the study area. Production of bioinoculants using this pioneer fungal mycobiont in P. pseudostrobus plant production (either as mycelial inoculum, dry pilei, or spore slurries) is a real prospect, as it has been previously demonstrated for other phylogenetically-related Neotropical Laccaria species (Pérez-Moreno et al. 2020). In doing so, it is hoped that we can protect endangered Mexican forest species and ecosystems by promoting efficient plant production and successful reforestation.


The first author thanks the China-Latin America Young Scientists Exchange Program (2017) from the Ministry of Science and Technology of China. Financial support from the Mexican Council of Science and Technology (CONACyT)-PRONACES Food Sovereignty "FOP07-2021-03 Project 316198” is also gratefully acknowledged. The authors also deeply acknowledge the valuable collaboration of Aurelio Hernández-López, Ascension Guzmán García, Eliseo Guzmán Brindis, Sixto Guzmán Rivera, Juan José Morales Pérez y Lucia García Guzmán (members of the Social Solidarity Society “Piedra Canteada” from San Felipe Hidalgo community in Nanacamilpa, Tlaxcala) during the fieldwork and sampling of this research. We are also grateful to two anonymous reviewers for a revision and helpful comments that improved the manuscript.

Literature cited

Agerer R. 1991. Characterization of ectomycorrhiza. Methods in Microbiology 23: 25-73. DOI: [ Links ]

Agerer R. 1987. Colour atlas of Ectomycorrhizae, Schwäbisch-Gmünd, Germany: Einhorn-Verlag Eduard Dietenberger. ISBN 3921703778 [ Links ]

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-Blast: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402. DOI: [ Links ]

Ashkannejhad S, Horton TR. 2005. Ectomycorrhizal ecology under primary succession on coastal sand dunes: interactions involving Pinus contorta, suilloid fungi and deer. New Phytologist 169: 345-354. DOI: [ Links ]

Carrasco-Hernández V, Pérez-Moreno J, Espinosa-Hernández V, Almaraz-Suárez JJ, Quintero-Lizaola R, Torres-Aquino M. 2010. Caracterización de micorrizas establecidas entre dos hongos comestibles silvestres y pinos nativos de México. Revista Mexicana de Ciencias Agrícolas 1: 567-577. [ Links ]

Chakravarty P, Hwang SF. 1991. Effect of an ectomycorrhizal fungus, Laccaria laccata, on Fusarium damping-off on Pinus banksiana seedlings. European Journal of Forest Pathology 21: 97-106. DOI: [ Links ]

Cho HJ, Park MS, Lee H, Oh S-Y, Wilson AW, Mueller GM, Lim YM. 2018. A systematic revision of the ectomycorrhizal genus Laccaria from Korea. Mycologia 110: 948-961. DOI: [ Links ]

Cuvelier JJ. 1991. Characterization of birch ectomycorrhizae (II): Laccaria amethystea and Russula ochroleuca. Belgian Journal of Botany 124: 195-203. [ Links ]

DEEMY. 2021. An information system for characterization and determination of ectomycorrhizae. Ludwig Maximilians, Universität München and Botanische Staatssammlung München. Munich. (accessed June 20, 2021). [ Links ]

Delgado P, Salas-Lizana R, Vázquez-Lobo A, Wegier A, Anzidei M, Alvarez-Buylla ER, Vendramin GG, Piñero D. 2007. Introgressive hybridization in Pinus montezumae Lamb and Pinus pseudostrobus Lindl. (Pinaceae): Morphological and molecular (cpSSR) evidence. International Journal of Plant Sciences 168: 861-875. DOI: [ Links ]

Duponnois R, Garbaye J. 1991. Mycorrhization helper bacteria associated with the Douglas fir- Laccaria laccata symbiosis: effect in aseptic and in glasshouse conditions. Annales des Sciences Forestières 48: 239-251. DOI: [ Links ]

Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792-1797. DOI: [ Links ]

Farfán-Heredia B, Casas A, Rangel-Landa S. 2018. Cultural, economic, and ecological factors influencing management of wild plants and mushrooms interchanged in Purépecha markets of Mexico. Journal of Ethnobiology and Ethnomedicine 14: 68. DOI: [ Links ]

Galindo-Flores G, Castillo-Guevara C, Campos-López A, Lara C. 2015. Caracterización de las ectomicorrizas formadas por Laccaria trichodermophora y Suillus tomentosus en Pinus montezumae. Botanical Sciences 93: 855-863. DOI: [ Links ]

Gardes M, Bruns D. 1993. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118. DOI: [ Links ]

Gardes M, Burns TD. 1996. Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above- and below-ground views. Canadian Journal of Botany 74: 1572-1583. DOI: [ Links ]

Gómez-Romero M, Villegas J, Sáenz-Romero C, Lindig-Cisneros R. 2013. Efecto de la micorrización en el establecimiento de Pinus pseudostrobus en cárcavas. Madera y Bosque 19: 51-63. DOI: [ Links ]

INEGI [Instituto Nacional de Estadística y Geografía]. 1997. Anuario estadístico del estado de Tlaxcala. Instituto Nacional de Estadistica y Geografía, Aguascalientes, México. [ Links ]

Ingleby K, Mason PA, Last FT, Fleming LV. 1990. Identification of ectomycorrhizae. London, UK: HMSO Publisher. ISBN: 0117014613 [ Links ]

Kalita K, Bezbaroa RN, Kumar R, Pandey S. 2016. Documentation of wild edible mushrooms from Meghalaya, Northeast India. Current Research in Environmental & Applied Mycology 6: 238-247. DOI: [ Links ]

Kang H, Chen X, Kemppainen M, Pardo AG, Veneault‐Fourrey C, Kohler A, Martin FM. 2020. The small secreted effector protein MiSSP7.6 of Laccaria bicolor is required for the establishment of ectomycorrhizal symbiosis. Environmental Microbiology 22: 1435-1446. DOI: [ Links ]

Kropp BR, Mueller GM. 1999. Laccaria. In: Cairney JWG, Chambers SM, eds. Ectomycorrhizal Fungi Key Genera in Profile. Berlin, Germany: Springer, pp. 65-88. DOI: [ Links ]

Li Q, Yang L, Xiang D, Wanm Y, Qi W, Huangm W, Zhao G. 2020. The complete mitochondrial genomes of two model ectomycorrhizal fungi (Laccaria): features, intron dynamics and phylogenetic implications. International Journal of Biological Macromolecules 145: 974-984. DOI: [ Links ]

Martin F, Aerts A, Ahren D, Brun A, Danchin EGJ, Duchaussoy F, Gibon J, Kohler A, Lindquist E, Pereda V, Salamov A, Shapiro HJ, Wuyts J, Blaudez D, Buée M, Brokstein P, Canbäck B, Cohen D, Courty PE, Coutinho PM, Delaruelle C, Detter JC, Deveau A, DiFazio S, Duplessis S, Fraissinet-Tachet L, Lucic E, Frey-Klett P, Fourrey C, Feussner I, Gay G, Grimwood J, Hoegger PJ, Jain P, Kilaru S, Labbé J, Lin YC, Legué V, Le Tacon F, Marmeisse R, Melayah D, Montanini B, Muratet M, Nehls U, Niculita-Hirzel H, Oudot-Le Secq MP, Peter M, Quesneville H, Rajashekar B, Reich M, Rouhier N, Schmutz J, Yin T, Chalot M, Henrissat B, Kües U, Lucas S, Van de Peer Y, Podila GK, Polle A, Pukkila PJ, Richardson PM, Rouzé P, Sanders IR, Stajich JE, Tunlid A, Tuskan G, Grigoriev IV. 2008. The genome of Laccaria bicolor provides insights into ectomycorrhizal symbiosis. Nature 452: 88-93. DOI: [ Links ]

McKnight KH, Peterson RT, McKnight VB. 1998. A field guide to mushrooms: North America New York: Houghton Mifflin Harcourt. ISBN: 0-395-42101-2 [ Links ]

Miller MA, Pfeiffer WT, Schwart ZT. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE10). New York: Association for Computing Machinery, pp. 1-8. DOI: [ Links ]

Mohan V, Natarajan K, Ingleby K. 1993. Anatomical studies on ectomycorrhizae. II. The ectomycorrhizae produced by Amanita muscaria, Laccaria laccata and Suillus brevipes on Pinus patula. Mycorrhiza 3: 43-49. DOI: [ Links ]

Molina R. 1980. Ectomycorrhizal inoculation of containerized western conifer seedlings. Oregon, Portland: United States Department of Agriculture Forest Service. [ Links ]

Molina R, Chamard J. 1983. Use of the ectomycorrhizal fungus Laccaria laccata in forestry. II. Effects of fertilizer forms and levels on ectomycorrhizal development and growth of container-grown Douglar-fir and ponderosa pine seedlings. Canadian Journal of Forest Research 13: 89-95. DOI: [ Links ]

Montoya L, Bandala VM, Baroni T, Horton TR. 2015. A new species of Laccaria in a montane cloud forest from Eastern Mexico. Mycoscience 56: 597-605. DOI: [ Links ]

Mueller GM. 1984. New North American species of Laccaria (Agaricales). Mycotaxon 20: 1010-1116. [ Links ]

Mueller GM. 1992. Systematics of Laccaria (Agaricales) in the continental United States and Canada, with discussions on extralimital taxa and descriptions of extant types. Fieldiana Botany 30: 1-158. DOI: [ Links ]

Nara K. 2015. The role of the ectomycorrhizal networks in seedlings establishment and primary succession. In: Horton TR, ed. Mycorrhizal networks Dordrecht: Springer, pp. 117-201. ISBN 978-94-017-7395-9 [ Links ]

Nara K, Nakaya H, Hogetsu T. 2003. Ectomycorrhizal sporocarps succession and production during the early primary succession on Mount Fuji. New Phytologist 158: 193-206. DOI: [ Links ]

Osmundson TW, Cripps CL, Mueller GM. 2005. Morphological and molecular systematics of Rocky Mountain alpine Laccaria. Mycologia 97: 949-972. DOI: [ Links ]

Pérez-Moreno J, Martínez-Reyes M, Yescas-Pérez A, Delgado-Alvarado A, Xoconostle-Cázares, B. 2008. Wild Mushroom Markets in Central Mexico and a Case Study at Ozumba. Economic Botany 62: 425-436. DOI: [ Links ]

Pérez-Moreno J, Martínez-Reyes M, Hernández-Santiago F, Ortiz-López I. 2020. Climate change, biotechnology, and Mexican Neotropical edible ectomycorrhizal mushrooms. In: Pérez-Moreno J, Guerin-Laguette A, Flores-Arzú R, Yu FQ, eds. Mushrooms, humans and nature in a changing world. Switzerland: Springer Nature, pp. 61-100. DOI: [ Links ]

Perry JP. 1991. The pines of Mexico and Central America. Oregon, US: Timber Press. ISBN: 978-1604691108 [ Links ]

Popa F, Castillo-Jiménez SYC, Weisenborn J, Donges K, Rexer KH, Piepenbring M. 2016. A new Laccaria species from cloud forest of Fortuna, Panama. Mycological Progress 15: 1-8. DOI: [ Links ]

Popa F, Rexer K H, Donges K, Yang ZL, Kost G. 2014. Three new Laccaria species from Southwest China (Yunnan). Mycological Progress 13: 1105-1117. DOI: [ Links ]

Ramos A, Bandala VM, Montoya L. 2017. A new species and a new record of Laccaria (Fungi, Basidiomycota) found in a relict forest of the endangered Fagus grandifolia var. mexicana. MycoKeys 27: 77-94. DOI: [ Links ]

Reverchon F, Ortega-Larrocea MP, Bonilla-Rosso G, Pérez-Moreno J. 2012. Structure and species composition of ectomycorrhizal fungal communities colonizing seedlings and adult trees of Pinus montezumae in Mexican neotropical forests. FEMS Microbiology Ecology 80: 479-487. DOI: [ Links ]

Rodríguez-Gutiérrez I, Ramírez-Martínez D, Garibay-Orijel R, Jacob-Cervantes V, Pérez-Moreno J, Ortega-Larrocea MP, Arellano-Torres E. 2019. Sympatric species develop more efficient ectomycorrhizae in the Pinus-Laccaria symbiosis. Revista Mexicana de Biodiversidad 90: e902868. DOI: [ Links ]

Santiago-Martínez G, Estrada-Torres A, Varela L, Herrera T. 2003. Crecimiento en siete medios nutritivos y síntesis in vitro de una cepa de Laccaria bicolor. Agrociencia 37: 575-584. [ Links ]

Sheedy E, Van de Wouw AP, Howlett BJ, May TW. 2013. Multigene sequence data reveal morphologically cryptic phylogenetic species within the genus Laccaria in southern Australia. Mycologia 105: 547-563. DOI: [ Links ]

Smith SE, Read DJ. 2008. Mycorrhizal Symbiosis. UK, London: Academic Press. ISBN: 978-0-12-370526-6 [ Links ]

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6. Molecular Biology and Evolution 30: 2725-2729. DOI: [ Links ]

Tedersoo L, Jairus T, Horton BM, Abarenkov K, Suvi T, Saar I, Kõljalg U. 2008. Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers. New Phytologist 180: 479-490. DOI: [ Links ]

Trappe JM. 1962. Fungus associates of ectotrophic mycorrhizae. Botanical Review 28: 538-606. DOI: [ Links ]

Valencia AS, Flores-Franco G. 2006. Catálogo de Autoridad Taxonómica del género Quercus, Fagaceae en México. Base de Datos SNIB-CONABIO proyecto CS008. México: Facultad de Ciencias, Universidad Nacional Autónoma de México. [ Links ]

Vincenot L, Nara K, Sthultz C, Labbé J, Dubois MP, Tedersoo L, Martin F, Selosse MA. 2012. Extensive gene flow over Europe and possible speciation over Eurasia in the ectomyc orrhizal basidiomycete Laccaria amethystina complex. Molecular Ecology 21: 281-299. DOI: [ Links ]

Werner A, Zadworny M. 2003. In vitro evidence of mycoparasitism of the ectomycorrhizal fungus Laccaria laccata against Mucor hiemalis in the rhizosphere of Pinus sylvestris. Mycorrhiza 13: 41-47. DOI: [ Links ]

White TJ, Bruns TD, Lee SB, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JN, White TJ, eds. PCR protocols: a guide to methods and applications. New York: Academic Press, pp. 315-322. DOI: [ Links ]

Wilson AW, Hosaka K, Perry BA, Mueller GM. 2013. Laccaria (Agaricomycetes, Basidiomycota) from Tibet (Xizang Autonomous Region, China). Mycoscience 54: 406-419. DOI: [ Links ]

Wilson AW, Hosaka K, Mueller GM. 2017. Evolution of ectomycorrhizae as a driver of diversification and biogeographic patterns in the model mycorrhizal mushroom genus Laccaria. New Phytologist 213: 1862-1873. [ Links ]

Wu F, Zhou LW, Yang ZL, Bau T, Li TH, Dai YC. 2019. Resource diversity of Chinese marofungi: edible, medicinal, poisonous species. Fungal Diversity 98: 1-7. DOI: [ Links ]

Received: December 29, 2020; Accepted: July 09, 2021; Published: December 16, 2021

*Corresponding author:

Associate editor: Miguel Olvera Vargas

Author contributions: MH and JPM, study conception, design and manuscript writing. FQY, MMR and FHS, material preparation, data collection and analysis. DRR, enhancement of ectomycorrhiza descriptions and images. CC manuscript writing. All authors read and approved the final manuscript.

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