Symbiosis (2018) 74:67–78 DOI 10.1007/s13199-017-0496-1

Persistence of ecto- and ectendomycorrhizal fungi associated with Pinus montezumae in experimental microcosms

Edith Garay–Serrano1,2 & Ma. del Pilar Ortega–Larrocea1 & Frédérique Reverchon3 & Iris Suárez–Quijada1

Received: 9 August 2016 /Accepted: 13 June 2017 /Published online: 30 June 2017 # Springer Science+Business Media B.V. 2017

Abstract Ectomycorrhizal (ECM) and ectendomycorrhizal Keywords ITS . Extramatrical mycelium . Morphotyping . fungal species associated with Pinus montezumae were re- Species co-existence corded in 8 year-old trees established in microcosms and com- pared with those associated with 2 year-old trees, in order to determine their persistence over the long-term. Mycorrhizal 1 Introduction root tips were morphologically and anatomically character- ized and sequenced. The extension of extramatrical mycelium Tree roots in coniferous forests are colonized by a variety of of ECM fungi with long exploration strategies was evaluated. ectomycorrhizal (ECM) and, to a lesser extent, by In total, 11 mycorrhizal species were registered. Seven mycor- ectendomycorrhizal fungi, which improve plant water uptake rhizal species were detected on both 2 and 8 year-old pines: and nutrient availability, increase the tolerance of roots to high Atheliaceae sp., Rhizopogon aff. fallax, R. aff. occidentalis, temperatures or soil acidity, and protect roots against patho- Suillus pseudobrevipes, Tuber separans, Wilcoxina mikolae gens (Horton and Van der Heijden 2008; Futai et al. 2008). and Wilcoxina rehmii.Onespecies,Thelephora terrestris, The fungal components involved in the ECM symbiotic asso- was exclusively associated with two year–old seedlings, while ciation are the intraradical mycelium (or Hartig net), the Cenococcum geophilum, Pezizaceae sp. and Pyrenomataceae extramatrical mycelium (including rhizomorphs) and resis- sp. were exclusively found on 8 year-old trees. Atheliaceae sp. tance structures as sclerotia (Futai et al. 2008). External my- was the ECM fungal species that presented the most abundant celium plays an important ecological role in seedling estab- mycelium. Finally, we report one new fungal species of lishment (Reverchon et al. 2015), as an extension of roots. It Pezizaceae occurring as a symbiont of P. montezumae. also constitutes a reservoir of carbon and an efficient way of exploring the soil, taking up and transporting water and nutri- ents as phosphorus, nitrogen and other minerals (Ekblad et al. Electronic supplementary material The online version of this article 2013; Hendricks et al. 2016). External mycelium constitutes (doi:10.1007/s13199-017-0496-1) contains supplementary material, which is available to authorized users. up to 80% of the total biomass of the ECM fungi (Wallander et al. 2001), and is thereby considered as the main organ for * Edith Garay–Serrano nutrient fluxes (Futai et al. 2008). Differences in the develop- [email protected] ment of external mycelium have been recorded between ECM fungal (ECMF) species (Agerer 2001), but the different pat- terns existing in mycelial growth have seldom been registered, 1 Instituto de Geología, Departamento de Edafología, Universidad Nacional Autónoma de México (UNAM), 04510 Ciudad de mainly due to the difficulty to study extramatrical mycelium in Mexico, Mexico field conditions (Anderson and Cairney 2007). 2 Posgrado de Ciencias de la Tierra, Universidad Nacional Autónoma The establishment of microcosms as experimental units has de México (UNAM), Ciudad de Mexico, Mexico allowed to gain insights into several ecological questions. The 3 Red de Estudios Moleculares Avanzados, Instituto de Ecología, A.C, development and persistence of extramatrical mycelium, the Carretera antigua a Coatepec, El Haya, Xalapa, strategy of substrate occupation by ECM fungi, the role of 91070 Veracruz, Mexico mycorrhizal associations in seedling growth and nutrient 68 Garay–Serrano E. et al. exchange, and the weathering activities of ECM fungi have all microcosm experimental units were maintained under green- been explored using microcosms (Donnelly et al. 2004; house conditions, at 25 °C with 45–50% relative humidity, Heinonsalo et al. 2001, 2010; Koele and Hildebrand 2011; and watered weekly with tap water; since trees were 7 years Read et al. 2004; Rosenstock 2009; Saccone et al. 2012). old, modified Melin & Norkrans solution (MMN) was applied More recently, microcosms have been used to disentangle every fortnight to the microcosms. The MMN solution was the relationship between biodiversity and ecosystem function- prepared as follows: CaCl2, 0.05 g; NaCl, 0.025 g; KH2PO4, . ing by testing the effect of intra- and interspecific identity and 0.5 g; (NH4)2HPO4, 0.125 g; MgSO4 7H2O, 0.15 g; richness of ECM fungi on the regulation of plant productivity, FeNaEDTA, 0.027 g, adjusted to 1000 ml with distilled water. soil CO2 efflux and soil nutrient retention (Hazard et al. 2017). During one year (2014), the presence of mycorrhizal Mexico is the second centre of diversity of Pinus species morphotypes was registered monthly on the roots of pines with 49 of the 120 species reported worldwide, of which 22 growing in the 19 experimental units. Mycorrhizal tips were are endemic (Gernandt and Pérez-de la Rosa 2014). As pines collected by removing one of the plates of the microcosms. As are obligate ECM symbionts (Pérez-Moreno and Read 2004), mycorrhizas may differ in color depending on their age, the it is important to gain knowledge about the composition of identity of ECM morphotypes was confirmed through molec- their fungal symbionts and about the development of ular identification. associated extramatrical mycelium under different conditions. Reverchon et al. (2010, 2012) studied the ECMF 2.2 Description of mycorrhizal morphotypes communities established in neotropical forests dominated by Pinus montezumae, in Corredor Biológico Chichinautzin The sampled ECM root tips were described using criteria such (CBC), central México. The authors also established bioassays as branching morphology, color, presence of cystidia, emanat- with P. montezumae growing in microcosms filled with soil ing hyphae, rhizomorphs, and characteristics of mantle sur- from CBC, in order to identify the ECMF propagules able to face, as described by Agerer (1987–2002). Descriptions were colonize the roots of this pine species (Reverchon et al. 2015). recorded in microphotographic images taken under a stereo- Those pines have been maintained in microcosms for 8 years scopic microscope. Longitudinal sections of ectotrophic my- and this study aimed at assessing the persistence and coexis- corrhizas were made by hand cutting and mounted with poly- tence of ECMF species associated with P. montezumae grow- vinyl alcohol – lactic acid – glycerol (PVLG), either stained ing in microcosms, and at evaluating the extramatrical myce- with 0.01% acid fuchsin in lactic acid or stain free. lium development of those ECMF species presenting long- Microscopic characteristics (mantle type with presence or ab- distance exploration type of external mycelia. Furthermore, sence of gelatinous matrix, latex and anatomical features of we aimed at describing in detail the morphological and ana- external elements and Hartig net type) were recorded and tomical characteristics of ECMF morphotypes associated with photographed under optical Leica DME and/or petrographic P. montezumae, since such morphology features may differ Olympus BX-51 microscopes. depending on the host and soil type (Ma et al. 2010). 2.3 Molecular identification of ECM morphotypes and sequence analysis 2 Materials and methods Total DNAwas extracted from 143 collected root tips with the 2.1 Pinus maintenance in microcosms and sampling RedExtract–N–Amp Plant PCR kit (Sigma-Aldrich, Mexico) of ECM root tips as described in Reverchon et al. (2015). Internal transcribed spacer 1 (ITS1) and 2 (ITS2), and 5.8S rDNA were amplified Two year-old trees of P. montezumae were established in mi- by PCR. The 25–μl PCR mixture contained 3 μl of DNA crocosms in 2008, with non-sterilized soil as a substrate, as (diluted in distilled water 1/10), 2.5 μlof10Xbuffer,2.5μl reported by Reverchon et al. (2015). The soil used to set up the of 25 mM MgCl2,2.5μl of 2 mM dNTPs, 0.25 μlof50μM microcosms was sampled in the CBC, under mature neotrop- ITS1F primer; 0.25 μlof50μM ITS4 primer, 0.35 μlof5U ical forests dominated by P. montezumae, at an altitude of Taq polymerase (Axygen, California) and 13.65 μlofsterile 3100 m. The characteristics of this volcanic soil are described deionized ultrapure water. PCR cycling parameters were as in Reverchon et al. (2015). Briefly, soil pH ranged from 4.8 to follows: 1 cycle at 95 °C for 2 min, followed by 35 cycles 5.7, soil total carbon content from 7.7 to 17.8 kg m−2,soiltotal with a denaturation step at 95 °C for 1 min, an annealing step nitrogen content from 0.42 to 1.04 kg m−2, and soil available at 55 °C for 1 min, and an extension step at 72 °C for 1 min, phosphorus from 1.38 to 2.39 g m−2. Microcosms were then finally 1 cycle at 72 °C for 8 min for final extension. scaled up and trees were transferred to bigger units consisting Amplified PCR products were sent to High Throughput of acrylic plates of 50 × 50 cm, filled with additional sterilized Genomics Center in Washington University for purification soil from the CBC, with one tree per microcosm. Nineteen and sequencing reactions. Persistence of ecto- and ectendomycorrhizal fungi 69

The obtained fungal ITS sequences were manually edited ecto- and ectendomycorrhizal fungi were identified from the and assembled using the Sequencher software (V4.1, Gene obtained sequences (Supplementary Table T1). Five ECMF Codes, Ann Arbor, MI, USA). The consensus sequences were species were identified to the species level: Cenococcum contrasted with sequences of the ITS region in NCBI and geophilum, Rhizopogon aff. fallax, R. aff. occidentalis, UNITE gene databases and species names were assigned Suillus pseudobrevipes and Tuber separans;furthermore, when the 97% sequence similarity criterion, recommended two of the identified fungal species were previously reported by Tedersoo et al. (2003), was reached. as ectendomycorrhizas: Wilcoxina mikolae and W. rehmii (Mikola 1988); finally, three ECMF morphotypes could only 2.4 Persistence of species through time be identified at the family level: Atheliaceae sp., Pezizaceae sp. and Pyrenomataceae sp. Sequences obtained from the present study (8 year-old trees) All the obtained ecto- and ectendomycorrhizal sequences and from ECM fungi associated with 2 year-old trees were belonged to the Ascomycota and Basidiomycota phyla. The integrated in a single dataset, in order to verify the taxonomic former group includes: Cenococcum geophilum, Pezizaceae assignment of sequences obtained by Reverchon et al. (2015). sp., Pyrenomataceae sp., Tuber separans, Wilcoxina mikolae The dataset was divided into three matrices: Ascomycetes, and W. rehmii. The Basidiomycota group includes Atheliaceae Atheliales and Boletales (data not shown in paper, available sp., Rhizopogon aff. fallax, R. aff. occidentalis and Suillus upon request to the corresponding author). Sequences from pseudobrevipes. matrices were aligned with a multiple sequence alignment program (MAFFT). Phylogenetic trees for each dataset were 3.2 Morphological and anatomical description of ecto generated using the Maximum Likelihood (ML) algorithm and ectendomycorrhizas with 1000 replicates of bootstrapping using Mega6 software (Tamura et al. 2013). The newly obtained sequences of ECMF The ECM morphotypes described in this study varied in color, species were uploaded to GenBank and their accession num- length of mycorrhizal systems, presence of emanating hyphae, bers are indicated in Table 1. rhizomorphs and sclerotia. Moreover, differences were ob- Additionally, we used an analysis of similarity (ANOSIM) served in the anatomy of mantle, Hartig net, ornamentation in order to compare the species composition of ECMF asso- of emanating hyphae wall, and rhizomorphs. A shared feature ciated with 2 year-old and 8 year-old trees. The ANOSIM was among the analyzed ECM root tips was the dichotomous ram- based on a presence/absence matrix of OTUs and was com- ification of mycorrhizal systems. The morphological and an- puted in PAST (Hammer et al. 2001)with9999permutations, atomical characteristics of ECM fungi found in this study are using the Raup-Crick similarity index as a probabilistic mea- described in Table 2. Details and photos of the morphology sure based on occurrence data (Raup and Crick 1979). and anatomy of mycorrhizas are provided as Supplementary material (Suppl. S1 and Suppl. Fig. 1–5). 2.5 Development of extramatrical mycelium of ectomycorrhizas 3.3 Persistence of species through time Once the identity of ECM root tips was confirmed by the ITS sequences, the development pattern of the The phylogenetic analysis of the data reported by Reverchon extramatrical mycelium was recorded in order to describe et al. (2015) allowed us to retrieve 8 mycorrhizal species oc- exploration types, according to Agerer (2001). During curring in 2 year-old microcosms. In the present study, we 2014, the area covered by mycelium of those ECMF spe- recorded 10 fungal species associated with 8 year-old trees, cies with long-distance exploration type was registered and obtained a total of 11 mycorrhizal fungal species for both monthly in the corresponding microcosms. Some mea- periods. Only one species, Thelephora terrestris, was exclu- surements were taken weekly when fast mycelial growth sively identified on roots of 2 year–old pines (Table 3); shared occurred. The area covered by extramatrical mycelia was species in both periods were Atheliaceae sp., Rhizopogon aff. estimated with the program Adobe®Photoshop®CS4. fallax, R. aff. occidentalis, Suillus pseudobrevipes, Tuber separans, Wilcoxina mikolae and W. rehmii.Fungalspecies that were exclusively present in 8 year–old pines were 3Results Cenococcum geophilum, Pezizaceae sp. and Pyrenomataceae sp. The relative similarity in species composition of ECM 3.1 Mycorrhizal fungal species present in microcosms fungi associated with 2 year-old and 8 year-old pines was further confirmed by the ANOSIM, which showed little (al- Ninety sequences were retrieved from the 143 processed root though significant) differences in species occurrence tips (63% success in the amplification step), and 10 species of (R = 0.09595, p =0.045). 70 Garay–Serrano E. et al.

Table 1 Sequences obtained for each ECMF taxon in ecto- and ectendomycorrhizas associated with 8 year–old Pinus montezumae trees in microcosms. Access numbers of sequences as uploaded in Genbank are shown

ECMF species Number of Genbank access number of newly obtained sequences sequences obtained

Atheliaceae sp. 14 KU245964, KU245965, KU245966, KU245968, KU245969 Cenococcum geophilum 14 KU245933, KU245934, KU245935, KU245936, KU245937, KU245938 Pezizaceae sp. 6 KU245950, KU245951, KU245952 Pyrenomataceae sp. 5 KU245947, KU245948, KU245949 Rhizopogon aff. fallax 14 KU245958, KU245959, KU245960, KU245961, KU245962, KU245963 Rhizopogon aff. occidentalis 2 KU245955, KU245956 Suillus pseudobrevipes 1 KU245957 Tuber separans 6 KU245953, KU245954 Wilcoxina mikolae 18 KU245939, KU245940, KU245941, KU245942, KU245943, KU245944 Wilcoxina rehmii 10 KU245944, KU245945, KU245946, KU245970

3.4 Development of extramatrical mycelium of ecto- by Atheliaceae sp. The external mycelium of R. aff. fallax was and ectendomycorrhizas formed by white rhizomorphs growing radially, integrating colonization circles that could extend up to 6 cm in diameter The identified ECMF species presented distinct explorations in the microcosm (Fig. 2a). Rhizomorphs formed a loose net strategies: Pezizaceae sp. developed a mycelium of contact (Fig. 2 d-f) with fan shaped hyphae at the distal growing point. exploration type. Short-distance exploration type mycelium The mycelium remained present from three to four months. were recorded in C. geophilum, Pyrenomataceae sp., Tuber External mycelium and rhizomorphs spread in the microcosm separans, and ectendomycorrhizal W. mikolae and and covered an area of 1.3 cm2 in March 2014 (Fig. 2a). In W. rehmii. Long distance exploration type mycelium were April 2014, mycelium and rhizomorphs covered 13.2 cm2, produced by Atheliaceae sp., Rhizopogon aff. fallax, R.aff. and in May 2014 mycelium development reached 19.5 cm2, occidentalis and Suillus pseudobrevipes. which corresponds to 0.8% of the microcosm surface, after The recorded development of external mycelium produced which mycelium started to become less dense. The maximum by ECMF species with long exploration patterns is presented surface of microcosm covered by R. aff. fallax mycelium was in Table 4. The extramatrical mycelium of Atheliaceae sp. was less than 1%. The presence of R. aff. fallax ECM was regis- abundant and its development was remarkable through time tered from March to November 2014. Morphological varia- (Fig. 1 a-f). Some isolated hyphae started to grow in January tions in ECM root tips and rhizomorphs produced by R. aff. 2014 (not visible in Fig. 1a). Subsequently, white and cottony fallax are shown in Fig. 2 (b-g). mycelium started spreading, becoming abundant and invading The mycelium of R. aff. occidentalis grew less than that of almost all the microcosm soil surface (Fig. 1b-f). In R.aff.fallax, generally covering an area ranging from 3.5 to March 2014, visible mycelium covered 65% of microcosm 3.8 cm2, although the external mycelium of several root tips surface (Fig. 1b), and extended to cover up to 93% of the could reach up to 16.4 cm2 (0.65% of the microcosm surface). microcosm surface in August of the same year (Fig. 1d). As Its development in microcosms was registered in co- it became limited by space, mycelium visibly continued grow- occurrence with other ECMF species. The rhizomorphs ing in biomass density; in December 2014 and January 2015, formed a compact net in the soil. ECM root tip development the mycelium produced by Atheliaceae sp. covered about 94% occurred in a lapse of 4–5 months. Some tips extended for a of the total microcosm surface (Fig. 1f). The extended myce- few centimeters (1.2 cm2) before becoming senescent. lial growth was observed in all microcosms where Atheliaceae Suillus pseudobrevipes presented an external mycelium sp. was present, although the starting time of active mycelium growing radially onto the soil surface, and formed growth varied depending on the microcosm under study. rhizomorphs (Suppl. Fig. 4a) that anastomosed with external Mycelium turned pale olive-green and non-cottony when it hyphae to form an extended mycelial network, covering up to aged, and remained confined to the edges of microcosms just 40 cm2 (1.6% of the microcosm surface). This ECMF was before the new active mycelium started growing. recorded in only one microcosm, and the presence of root tips The extramatrical mycelium produced by Rhizopogon aff. was registered in a period ranging from July to October 2014. fallax, although belonging to the long exploration type, was Suillus pseudobrevipes grew on the posterior side of the mi- not as widespread in our microcosm units as the one produced crocosm unit, with less exposure to air. essec fet-adetnoyoria fungi ectendomycorrhizal and ecto- of Persistence

Table 2 Anatomical and morphological characteristics of morphotypes from ectomycorrhizas and ectendomycorrhizas associated with Pinus montezumae in microcosms

Mycorrhizal Colour Mycorrhizal system Outer mantle Inner mantle Hartig net Emanating hyphae Cystidia Rhizomorphs Sclerotia species

Ectomycorrhizas: Atheliaceae sp. Yellowish to pale Dichotomous, ramified Thin mantle, Thin mantle, Palmetti type, Septate, clamped, Absent Absent Absent brown 3–4 times plectenchymatous plectenchymatous periepidermal hyaline to pale brown, with fine ornamented walls Cenococcum Black Single systems or once Plectenchymatous, Plectenchymatous, Palmetti type, Straight, dark brown Absent Absent Cylindrical, geophilum carbonaceous ramified brown hyphae with without a specific paraepidermal black dichotomously star –like pattern pattern, hyphae brown Pezizaceae sp. Yellowish brown Dichotomously Plectenchymatous, Transitional Palmetti type, Absent Absent Absent Absent ramified, up to two hyaline hyphae, periepidermal. times loosely woven Pyrenomataceae Brown with orange Dichotomously Plectenchymatous, Plectenchymatous with Palmetti type, Hyalinetopalebrown, Absent Absent Absent sp. tinges, tips beige ramified, coralloid hyalinetopalebrown yellowish brown to cylindrical with short and in a start-like pattern, pale-ochre hyphae among cortical dispersed crystals in some gelatin cells. the wall Rhizopogon aff. Silvery Dichotomously Plectenchymatous, Plectenchymatous with Palmetti type, Hyaline, present in Absent White, roundish, Absent fallax ramified, coralloid hyaline hyphae most hyphae periepidermal bunch, scattered, smooth, not disposed parallel to disposed differentiated the root perpendicular, forming a compact tissue Rhizopogon aff. Whitish with Dichotomously Plectenchymatous, Plectenchymatous, Palmetti type, Hyaline hyphae, scarse Absent White, roundish, Absent occidentalis brown–red color ramified, coralloid hyaline hyphae, hyphae in several paraepidermal and smooth, especially in old loosely woven. directions, repeatedly parts Hyphae embedded in compacted, ramified, a gelatinous matrix embedded in a undifferentiated gelatinous matrix Suillus White, in some Dichotomously Plectenchymatous with Plectenchymatous, Palmetti type, Septate, hyaline, with Absent White, roundish, Absent pseudobrevipes areas pale brown ramified, coralloid hyphae hyaline composed by paraepidermal small warts on the undifferentiated, with orange yellowish brown surface of hyphae. with a gelatinous tinges hyphae matrix. Tuber separans Brown Dichotomously ramified Pseudoparenchymatous Transitional, with Palmetti type. Absent Awl-shaped, Absent Absent up to 3 times with angular cells, septate hyphae, Paraepidermal hyaline, with slightly gelatinized, hyaline to yellowish a septum near hyaline to yellowish to the base. Ectendomycorrizas: Wilcoxina Brown, young parts Dichotomously Plectenchymatous, thin, Plectenchymatous, thin, Lobulated hyphae, Brown– ochre, with Absent Absent Absent mikolae ochre, distal ramified, up to five hyphae embedded in with hyphae paraepidermal. warty walls, some ends whitish or times. Few a rigid and thick embedded in a Cells beaded hyphae ramified translucent monopodial gelatinized matrix gelatinous matrix around cortical dichotomously cells Wilcoxina rehmii Dark brown, apex Dichotomously Thin mantle, Absent or composed just Palmetti type, Brown, smooth wall Absent Absent Absent orange to pale ramified, until 5 plectenchymatous, by one layer, paraepidermal surface but in distal yellow or times just two layers of plectenchymatous. parts of hyphae the whitish. cells discernible surface become finely warty 71 72 Garay–Serrano E. et al.

Table 3 Ecto- and ectendomycorrhizal fungi on ECM taxa Seedlings in field Adults in field 2year-oldtrees 8 year-old trees a a b roots of Pinus montezumae conditions conditions in microcosms in microcosms growing in field conditions or in microcosms. Information Atheliaceae sp. 1 + + + + regarding the presence on field Atheliaceae sp. 2* + + + seedlings and adults, as well as in Cantharellales sp + 2year–old seedlings grown in microcosms was obtained and re- Cenococcum geophilum ++ analyzed from Reverchon et al. Clavulina sp. + + (2015) Cortinarius sp. + Inocybe geophylla (ex sp. 2) + Inocybe praetervisa ++ Inocybe sp. 3 + Inocybe sp. 4 + + Pezizaceae sp. + Pyrenomataceae sp. + Piloderma sp. + Pseudotomentella tristis (ex sp.) + Rhizopogon fallax/ salebrosus ++ Rhizopogon occidentalis ++ Rhizopogon subcaerulescens + Russula abietina ++ Russula sp. 1 + + Russula sp.3 + Sebacinaceae sp. 1 + + Sebacinaceae sp. 2 + Sebacinaceae sp. 3 + + Suillus sp. + + Thelephora terrestris + Thelephoraceae sp. 1 + + Thelephoraceae sp. 2 + + Thelephoraceae sp. 4 + Tomentella bryophila + Tomentella sp. 1 + + Tomentellopsis sp. + Tuber separans ++ Wilcoxina mikolae ++ Wilcoxina rehmii ++

a Information in Reverchon et al. (2012)/b reported in Reverchon et al. (2015) *Analysis with Maximum likelihood used in this paper/ established Atheliaceae sp. 1 and sp. 2 in the same clade/ as similar species

3.5 Co-occurrence of ectomycorrhizas present in 37% of experimental units. In contrast, Suillus and and ectendomycorrhizas Tuber species were only recorded in one microcosm. Fungal richness varied from 1 to 4 species per microcosm, with dif- There was a predominance of Ascomycota taxa in 8 year-old ferent assemblages of ECMF species (Table 5). All micro- pine trees and the most frequently recorded ecto– and cosms contained different fungal species composition ectendomycorrhizal species were Atheliaceae sp., (Fig. 3), except two units which only contained Atheliaceae Cenococcum geophilum, Rhizopogon aff. fallax and species sp. Most microcosms presented two ECMF species (32%), belonging to the Wilcoxina genus. Wilcoxina mikolae was the while 26% of microcosms only contained one ECMF species most abundant fungal species and was recorded in more than and 21% of experimental units presented 3 or 4 co-occurring 60% of microcosms. Atheliaceae sp. and C. geophilum were ECMF species (Fig. 3 a-g). Persistence of ecto- and ectendomycorrhizal fungi 73

Table 4 Characteristics of external mycelium development of ectomycorrhizal species with long exploration strategy registered in microcosms

Mycorrhizal species Exploration structures Area covered by external mycelia Duration of growth period Registered months in MCC

Atheliaceae sp. Emanating hyphae 2342 cm2 All year All year Rhizopogon aff. fallax Rhizomorphs 20 cm2 3–4 months March to November Rhizopogon aff. occidentalis Rhizomorphs 3.5 cm2,upto16cm2 4–5 months June–October Suillus pseudobrevipes Rhizomorphs 40 cm2 4 months July–October

4Discussion detected by Palfner et al. (2005) in ECMF associated with Picea sitchensis. Both Ma’sandPalfner’s experiments were The ECMF species richness was higher for 8 year-old trees carried out under field conditions. As microcosms constitute a (10 OTUs) than for 2 year-old trees (8 OTUs). The increase of closed system, we expected the occurrence of new ECMF ECMF species richness with the age of host plant has been species to be restricted. We observed that most ECMF species reported by Ma et al. (2010), who found that morphotype detected on roots of 2 year-old pine trees persisted over the diversity increased in P. densiflora seedlings evaluated from years, with the exception of T. terrestris. Moreover, we detect- 1 to 5 years for ECMF colonization. The same pattern was ed the occurrence of new ECM species belonging to the

Fig. 1 a-f Development of extramatrical mycelium of ECM Atheliaceae sp. associated with P. montezumae tree growing in 50 × 50 cm microcosm during a year; g, h Mycelium and ECM root tip of the same fungal species; white ball in g indicating mycelium and tips. Bar a-f = 5 cm, Bar g, h = 4 mm 74 Garay–Serrano E. et al.

Fig. 2 a Development of extramatrical mycelium of ECM Rhizopogon aff. fallax (arrows) associated with P. montezumae tree growing in microcosm during January–August 2014; b-f Variation of extramatrical mycelium and ECM tips of R. aff. fallax in microcosms. g ECM root tip and mycelium of individual senescent tip. Bar a = 5 cm. Bar b- g=2mm

Ascomycota phylum in 8 year-old trees. Ascomycota mem- may offer insights into the ECMF communities of Pinus bers are commonly associated with P. montezumae,asrecent- montezumae occurring in the CBC. Pyrenomataceae sp. has ly described by Garibay-Orijel et al. (2013), who reported 16 already been reported to be associated with P. montezumae in Ascomycota species on P. montezumae roots collected from Nevado de Toluca, Malinche e Iztaccihuatl volcanoes in several regions of Mexico. In our study, one species belonging Mexico (reported as a Pezizaceae JN704828 in Garibay- to the Ascomycota phylum constituted a new record for Orijel et al. 2013). Most of the taxa reported in the present P. montezumae symbionts: Pezizaceae sp., which was differ- study have been described as early-stage fungi, predominantly ent from the existing records in Genbank and UNITE data- forming mycorrhizal associations with young trees, which bases. It has been shown that some ECMF species of Pezizales could confer them a competitive advantage in early succes- could obtain their carbon resources from the degradation of sional stages (Nguyen et al. 2012; Palfner et al. 2005; Peterson plant cell walls, as pathogens would do, although less effi- 2012; Visser 1995). Thelephora terrestris was exclusively ciently (Nagendran et al. 2009; Kernaghan 2013). This found to be associated with 2 year–old pines, which is consis- pathogen-like behavior may be the cause of their late root tent with findings of Nguyen et al. (2012) and Klavina et al. colonization, when the tree is older and stronger to bear the (2013) who report the presence of that species in early stages cost of the symbiosis. Franco et al. (2014) also detected of root colonization in conifer forests. Cenococcum Pezizales in 5 year-old trees of P. pinaster, but not on younger geophilum was detected as frequent fungal symbiont on roots individuals. The appearance of new species in microcosms of 8 year-old pines in microcosms. It is likely that it was not could also be due to greenhouse contamination. Although detected on pine roots at 2 year-old due to a low frequency, as contamination could not be completely discounted, other stud- this species has been reported on P. montezumae seedlings in ies reported those newly occurring species as ECMF symbi- field conditions (Reverchon et al. 2012). Cenococcum onts in field conditions (Garibay-Orijel et al. 2013; Reverchon geophilum has also been detected as a frequent and abundant et al. 2012; Tedersoo et al. 2006). As such, the present study species on seedling roots of seedlings of Pinus densiflora in essec fet-adetnoyoria fungi ectendomycorrhizal and ecto- of Persistence

Table 5 Presence of ecto- and ectendomycorrhizal fungi on roots of Pinus montezumae in microcosms. In some microcosms (MCC), co-existence of species occurs

ID MCC Atheliaceae sp. C. geophilum Pezizaceae sp. Pyrenomataceae sp. R. aff. fallax R. aff. occidentalis Suillus pseudobrevipes Tuber separans W. mikolae W. rehmii Species by MCC

1P X 1 MCC-C1 X 1 MCC-C2 X 1 MCC-P10 X X X X 4 MCC-P1 X X X X4 MCC-P2 X X X3 4P X1 11P XX2 MCC-P7 X X X3 MCC-P8 X X2 3P X X XX4 MCC-P9 X X X X 4 MCC-P4 XX2 6Ch XX 2 10Ch X1 5P X X2 9P X X X3 2P X X 2 12P–Ch X X X 3 75 76 Garay–Serrano E. et al.

Fig. 3 Interaction of fungal species in different microcosms a General view of Pinus montezumae sapling with mycorrhizal root tips and external mycelium of ECMF species; b Close-up of Atheliaceae sp. mycelium, old mycelium with green colours; new, white mycelium growing on the old one; c Fan mycelium of Atheliaceae sp. in interaction with C. geophilum mycorrhizas; d-g Several of ectendo and ECMF assemblages present in different microcosm; Abbreviations: Atheliaceae sp. (Ath), C. geophilum (Cg), sclerotia of C. geophilum (Sc), Pyrenomataceae sp. (Pyr), Rhizopogon aff. fallax (Rf), R. aff. occidentalis (Ro); Wilcoxina mikolae (Wm); and W. rehmii (Wr). Bar a = 5 cm; bar b-g = 1 cm

Northeastern China forests (Ma et al. 2010). Cenococcum exploration type mycelium, an acknowledged strategy to in- geophilum is known to be resistant to drought (Nedelin vest fewer resources in mycelium growth (Shahin et al. 2013), 2014) and to associate with a broad host range (Fernandez which could be an advantage in soils where C availability is et al. 2013). The ability of establishing symbiosis with a broad reduced (Ekblad et al. 2013). Mycelium of Atheliaceae, range of host trees is a feature of early stage fungi and consti- Rhizopogon and Suillus belonged to the long-distance explo- tutes a strategy that could favor their long permanence (Bigg ration type, and those taxa have been reported to invest in the 2000). A broad host range has been reported for species of the formation of differentiated rhizomorphs or abundant genus Suillus (Şesan et al. 2010), and for W. mikolae and W. extramatrical mycelium (Kennedy et al. 2011;Peayetal. rehmii (Egger 2006;Mikola1988;Nedelin2014; Trevor et al. 2007) that could provide the host with a higher resistance to 2001;Peterson2012). drought (Bakker et al. 2006). Furthermore, an extensive my- Different strategies of mycelial growth and exploration celial network can promote carbon and nutrient fluxes be- were recorded in this study: Pezizaceae sp. presented a contact tween the tree and its symbionts (Anderson and Cairney exploration type; C. geophilum, Pyrenomataceeae sp., Tuber 2007) and provide the fungi with the ability to persist and separans, W. mikolae and W. rehmii presented short spread over the soil (Taylor and Bruns 1999). Atheliaceae Persistence of ecto- and ectendomycorrhizal fungi 77 differs from Rhizopogon and Suillus species in being a more ectomycorrhizas and its implications for forest carbon and nutrient – aggressive colonizer, as its mycelium extends and is main- cycles. Soil Biol Biochem 65:141 143 Franco AR, Sousa NR, Ramos MA, Oliveira RS, Castro PM (2014) tained for larger periods of time (annual pattern). This family Diversity and persistence of ectomycorrhizal fungi and their effect has been reported to have a high foraging capacity, being able on nursery-inoculated Pinus pinaster in a post-fire plantation in to establish extensive mycelial networks that could connect a northern Portugal. Microb Ecol 68:761–772 large amount of roots, which allows them to function as car- Futai K, Taniguchi T, Kataoka R (2008) Ectomycorrhizae and their im- bon reservoirs (Kennedy et al. 2009). Atheliaceae sp. was also portance in forest ecosystems. In: Mycorrhizae: sustainable agricul- ture and forestry. Springer, Netherlands, pp 241–285 found by Reverchon et al. (2012)tobedominantinseedling Garibay-Orijel R, Morales–Marañon E, Domínguez–Gutiérrez M, and adult pine trees in CBC. Flores–García A (2013) Caracterización morfológica y genética de The present work allowed us to gain insights into the las ectomicorrizas formadas entre Pinus montezumae y los hongos ECMF colonizing P. montezumae in long-term microcosms. presentes en los bancos de esporas en la Faja Volcánica Transmexicana. Rev Mex Biodivers 84:153–169 Moreover, the anatomical descriptions of the ectomycorrhizal Gernandt DS, Pérez-de la Rosa JA (2014) Biodiversidad de Pinophyta and ectendomycorrhizal species provided in this study are the (coníferas) en México. Rev Mex Biodivers 85:126–133 first descriptions considering Pinus montezumae as host. This Hammer Ř, Harper DAT, Ryan PD (2001) PAST: Paleontological statis- is also the first report of the presence of frequent rhizomorphs tics software package for education and data analysis. Palaeontol formed by S. pseudobrevipes, contrasting with findings from Electron 4(1):9. Available at: http://palaeo-electronica.org/2001_1/ past/issue1_01.htm Garibay-Orijel et al. (2013) who report the absence of Hazard C, Kruitbos L, Davidson H, Taylor AF, Johnson D (2017) rhizomorphs in natural conditions. Contrasting effects of intra- and interspecific identity and richness of ectomycorrhizal fungi on host plants, nutrient retention and – Acknowledgements This work was supported by PAPIME-DGAPA, multifunctionality. New Phytol 213:852 863 UNAM (project PE108915). 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