Soil propagule banks of ectomycorrhizal fungi associated with Larix cajanderi above the treeline in the Siberian Arctic

Abstract

Microbial symbionts are essential for plant niche expansion into novel habitats. Dormant propagules of ectomycorrhizal (EM) fungi are thought to play an important role in seedling establishment in invasion fronts; however, propagule bank communities above the treeline are poorly understood in the Eurasian Arctic, where treelines are expected to advance under rapid climate change. To investigate the availability of EM fungal propagules, we collected 100 soil samples from Arctic tundra sites and applied bioassay experiments using Larix cajanderi as bait seedlings. We detected 11 EM fungal operational taxonomic units (OTUs) by obtaining entire ITS regions. Suillus clintonianus was the most frequently observed OTU, followed by Cenococcum geophilum and Sebacinales OTU1. Three Suillus and one Rhizopogon species were detected in the bioassay seedlings, indicating the availability of Larix-specific suilloid spores at least 30 km from the contemporary treeline. Spores of S. clintonianus and S. spectabilis remained infective after preservation for 14 mo and heat treatment at 60 °C, implying the durability of the spores. Long-distance dispersal capability and spore resistance to adverse conditions may represent ecological strategies employed by suilloid fungi to quickly associate with emerging seedlings of compatible hosts in treeless habitats.

1. Introduction

Microbial symbionts on plant roots positively influence host performance by improving growth and mediating environmental stresses (Smith & Read, 2008; Hayat, Ali, Amara, Khalid, & Ahmed, 2010). Association with compatible symbionts is thought to be essential for plant niche expansion into novel habitats (Parker, Malek, & Parker, 2006; Nuñez, Horton, & Simberloff, 2009; Pringle et al., 2009; Gerz, Bueno, Ozinga, Zobel, & Moora, 2018). Northern treelines are expected to advance with increasing temperatures, resulting in the transformation of tundra to forests in the Arctic (Kaplan et al., 2003; Harsch, Hulme, McGlone, & Duncan, 2009). At invasion fronts, seedling mortality may increase if they fail to form associations with symbiotic fungi in the early developmental stages (Collier & Bidartondo, 2009). Trees that form northern treelines are obligately associated with ectomycorrhizal (EM) fungi that improve water and nutrient absorption by the host plants. EM associations may provide plants with direct access to mineralized nutrients that are released from organic matter by fungal enzyme activity (Lindahl & Tunlid, 2015; Shah et al., 2016). Such functions are particularly important in northern ecosystems, where cold temperatures reduce decomposition rates and water accessibility (Bödeker et al., 2014; Deslippe, Hartmann, Grayston, Simard, & Mohn, 2016). Thus, the availability of compatible EM fungi is crucial for successful seedling establishment and subsequent tree range expansion into novel habitats (Germino, Smith, & Resor, 2002; Nuñez et al., 2009; Policelli, Bruns, Vilgalys, & Nunez, 2019).

Siberian Larix Mill. species form the longest continuous belt of Arctic treeline in Eurasia (> 5000 km) (Semerikov, Iroshnikov, & Lascoux, 2007). Given the extent of Larix distribution and the disproportionate rate of warming in the Arctic (Serreze & Barry, 2011; Overland, Walsh, & Kattsov, 2017), it is vital to clarify the migration potential of Larix trees. In primary habitats, pre-existing shrubs can provide EM fungal inocula to colonizer tree seedlings via common mycorrhizal networks (CMNs) (Nara & Hogetsu, 2004; Nara, 2006; Hewitt, Chapin, Hollingsworth, & Taylor, 2017). In the Arctic tundra, dwarf shrubs in the Betulaceae, Salicaceae, Rosaceae, and Ericaceae families form EM associations (Ryberg, Larsson, & Molau, 2009; Geml et al., 2012; Timling et al., 2012) and can act as reservoirs of compatible fungal symbionts above the treeline. However, a previous study found that EM fungal communities on Larix cajanderi Mayr [L. gmelinii (Rupr.) Rupr. sensu Farjon, 1990] were distinct from those on dwarf shrubs in the forest-tundra ecotone of eastern Siberia (Miyamoto, Maximov, Bryanin, Kononov, & Sugimoto, 2022). In that study, although dwarf shrubs at the ecotone harbored highly diverse EM fungal taxa, the EM fungal communities of L. cajanderi had low richness and were predominated by host specialist EM fungi. These observations imply that mycelial infection via existing tundra shrubs may be limited by low host-fungus compatibility. Fungal propagules (i.e., spores, sclerotia, and chlamydospores) are also important sources of EM formation in primary habitats. Population genetics studies have suggested that sexual reproduction and extensive gene flow (i.e., spore dispersal) are common among many EM fungal taxa in natural ecosystems because of the proliferation of microscopic spores per sporocarp (Vincenot & Selosse, 2017). Some EM fungi form ‘propagule banks’ in soil that can persist for extended periods, providing mycorrhizal inoculum for isolated seedlings in primary habitats (Ashkannejhad & Horton, 2006; Bruns et al., 2009; Nguyen, Hynson, & Bruns, 2012). To date, no information is available on propagule bank communities of EM fungi that can be compatible to Larix species above Arctic treelines.

Suilloid (specifically the genera Suillus Gray and Rhizopogon Fr.) species often dominate EM fungal communities of Larix roots in eastern Eurasia (Wang et al., 2021; Miyamoto et al., 2022). In particular, suilloid species have been reported to predominate on L. cajanderi populations growing at the Arctic treeline (Miyamoto et al., 2022), implying their key role in the establishment of Larix forests at the ecotone. Suilloid fungi exhibit strong specificity to the family Pinaceae (Molina & Trappe, 1994; Nguyen, Vellinga, Bruns, & Kennedy, 2016) and often promote growth and drought resistance in associated seedlings (Wen et al., 2018; Li et al., 2021; Wang et al., 2021). They produce abundant fruiting bodies containing proliferate spores that are dispersed long distances by wind and animals (Ashkannejhad & Horton, 2006; Peay, Schubert, Nguyen, & Bruns, 2012; Urcelay, Longo, Geml, Tecco, & Nouhra, 2017). Some suilloid species produce desiccation-resistant spores that can remain dormant for several years in soils (Bruns et al., 2009) and germinate in response to chemical cues produced by compatible hosts (Theodorou & Bowen, 1987; Kikuchi, Matsushita, Suzuki, & Hogetsu, 2007). Moreover, several studies have reported that spores are heat resistant and that germination may be stimulated by heat, suggesting an adaptation to fire disturbance (Izzo, Canright, & Bruns, 2006; Peay, Garbelotto, & Bruns, 2009; Murata, Nagata, & Nara, 2017b; Bruns, Hale, & Nguyen, 2019). These unique spore features are thought to promote quick establishment of EM associations with Pinaceae hosts at invasion fronts and in disturbed habitats (Baar, Horton, Kretzer, & Bruns, 1999; Ashkannejhad & Horton, 2006; Policelli et al., 2019). Thus, suilloid spores may play important roles in EM formation on Larix seedlings above contemporary treelines.

Bioassays are commonly used to examine soil propagule banks of EM fungi that are compatible with bait host plants (e.g., Bruns et al., 2009; Buscardo, Rodriguez-Echeverria, Martin, De Angelis, Pereira, Freitas, 2010; Kipfer, Moser, Egli, Wohlgemuth, & Ghazoul, 2011; Nguyen et al., 2012; Pickles, Gorzelak, Green, Egger, & Massicotte, 2015; Miyamoto & Nara, 2016). This method is effective for assessing the infectivity of dormant propagules and distinguishing EM formation via propagules and existing mycelia. The results often represent natural EM fungal communities on regenerating seedlings in primary habitats (Buscardo et al., 2010; Kipfer et al., 2011; Glassman, Levine, DiRocco, Battles, & Bruns, 2016). In this study, we conducted bioassay experiments to investigate propagule banks of EM fungi in the Arctic tundra of eastern Siberia. We predicted that the spores of suilloid fungi would be deposited in tundra soils where compatible hosts are absent. With special emphasis on suilloid species, we hypothesized that 1) propagules would remain infective with heating, 2) the richness and colonization of EM fungal species would be reduced at a distance from the treeline, and 3) EM fungal colonization would improve seedling growth.

2. Material and Methods

2.1. Study sites

We conducted a field survey in the Indigirka River lowlands near Chokurdakh, Sakha Republic, Russia. This region is a transition zone between forest and tundra underlain by continuous permafrost. The mean annual, Jul, and Jan temperatures are −13.3 °C, 10.6 °C, and −33.9 °C, respectively, and the mean annual and summer (Jun-Sep) precipitation amounts are 200.3 and 103 mm, respectively (CDIAC, 2012). Sampling was conducted at the Kytalyk (KTY) and Kodac (KOD) Stations of Sakha Flux Network Research, which are managed by the Institute for Biological Problems of the Cryolithozone, Siberian Branch of the Russian Academy of Sciences (Table 1). These two sites were approximately 30 km and 500 m from L. cajanderi stands, respectively. The vegetation was composed mainly of tundra dwarf shrubs including Betula nana L., Salix pulchra Cham., Salix glauca L., and ericaceous shrubs (Vaccinium vitis-idaea L., V. uliginosum L., Arctous alpina (L.) Nied., and Ledum palustre L.).

Table 1 Site descriptions.

Research Station (Site code)a	Kytalyk (KTY)b	Kodac (KOD)b
Latitude / Longitude	N70°49', E147°29'	N70°34', E148°19'
Elevation asl. (m)	15	85
Distance from treeline	30 km	500 m
Soil parameters (to 10 cm depth)
C (%)	4.08 ± 2.62 (1.17 - 11.80)	23.74 ± 10.63 (5.22 - 44.92)
N (%)	0.23 ± 0.13 (0.08 - 0.61)	1.00 ± 0.35 (0.25 - 2.18)
C/N	17.37 ± 2.34 (13.00 - 22.64)	23.07 ± 4.95 (15.29 - 36.63)
pH	5.15 ± 0.30 (4.61 - 5.84)	5.17 ± 0.31 (4.39 - 5.79)

^a asl., above sea level; C, total carbon concentration; N, total nitrogen concentration.

^b Values are means ± standard deviation. Minimum and maximum values are shown in parentheses.

In Aug 2018, a 50 m × 50 m plot was established at each site. Using a hand trowel, 50 soil cores (5 cm × 5 cm) were collected to a depth of 10 cm after removing surface litter to examine propagules in the surface soil layer. The cores were collected at intervals of ≥ 5 m between cores.

2.2. Bioassay of propagule bank communities

Soil samples were placed in paper bags to air dry at room temperature for 11 d in a clean laboratory. Subsequently, soil samples were placed in new plastic bags and stored in a container for 14 mo to prevent mycorrhizal formation from existing mycelia. To assess propagule heat resistance, each soil sample was divided in two, and one half was subjected to heat treatment at 60 °C for 1 h, followed by 50 °C for 1 h, with a 30-min transition interval of 50-60 °C, in a dry-heat oven (DX400, Yamato Inc., Japan). These settings were selected based on a fire-disturbance experiment in a forest of central Sakha (Takahashi, 2006) and a previous bioassay study that revealed increased frequencies of some EM fungal taxa at 60 °C (Murata, Kanetani, & Nara, 2017a; Murata et al., 2017b).

Bioassays were designed according to an established method (Miyamoto & Nara, 2016; Murata et al., 2017a, b). Each soil sample was passed through a 2-mm sieve to remove roots, organic debris, and small stones, and then placed in a 50-mL centrifuge tube with water drainage holes and a cotton ball in the bottom to prevent soil loss. We initially attempted to use L. cajanderi seeds collected at the treeline sites, but none germinated, possibly due to low seed quality. Therefore, we used commercially available seeds of the same species originating from China (Sheffield's Seed Co., Inc., USA) for the bioassay. The seeds were cold stratified for 3 mo and surface sterilized using 5% sodium hypochlorite before sowing. Seeds were placed on a moist paper towel to induce germination, and one germinated seed was placed in each bioassay tube. In total, 30 seedlings were grown in autoclaved soil (120 °C, 20 min) as controls. The seedlings were grown in a growth chamber with a 16-h/8-h day/night cycle at room temperature (24 °C) and watered with tap water every 4-5 d. After 6 mo of growth, fungal-associated roots, including those exhibiting discernible mantle structures and loosely surrounding hyphae, were morphologically differentiated into morphotypes according to the system described by Agerer (2001) using a dissecting microscope. We collected 1-5 root tips per morphotype per seedling for molecular analyses. The root tip samples were stored in cetyl trimethyl ammonium bromide (CTAB) solution at 4 °C until processing. After root tip collection was completed, the seedlings were separated into above- and belowground parts and dried at 60 °C for 24 h in a dry oven. Shoot and root biomass (above- and belowground dry weight, respectively) were measured to examine seedling growth performance.

2.3. Molecular analyses

The molecular methods and bioinformatics used for fungal identification in this study were identical to those described previously (Miyamoto, Danilov, & Bryanin, 2021). Briefly, total DNA was extracted using the CTAB method, and the entire internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of nuclear rDNA was amplified primarily using the ITS5 and ITS4 primers (White, Bruns, Lee, & Taylor, 1990). The amplicons were purified and sequenced using ITS1 (White et al., 1990) and/or ITS4 primers on an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). All sequencing chromatograms were visually checked and ITS sequences of sufficiently high quality (> 350 bp) were assembled into contigs at 100% similarity using the ATGC software (GENETYX Corp., Tokyo, Japan). One representative sequence from each contig was selected, and BLAST searches against the National Center for Biotechnology Information (NCBI) database were conducted for fungal identification. The EM status of the representative sequences was determined according to the literature and the UNITE database (Tedersoo, May, & Smith, 2010; Tedersoo & Smith, 2013; Koljalg et al., 2013). These representative sequences were subsequently assembled into operational taxonomic units (OTUs) at a 97% cutoff level using the ATGC software. Sequences were deposited to the DNA Data Bank of Japan (DDBJ) under accession nos. LC574750-LC574779.

2.4. Statistical analyses

All statistical analyses were conducted using the R v3.2.3 software (R Core Team, 2015), at a statistical significance of α < 0.05 unless otherwise stated. To examine the diversity of soil propagule banks, species accumulation curves and the Jackknife2 richness estimator were computed using EstimateS v9.1.0 software (Colwell, 2019) with 1000 randomizations without replacement. Fisher's exact test was used to examine the effects of site and heat treatment on the frequency of EM fungal colonization. The false discovery rate was used to adjust P values for multiple comparisons. The seedling growth parameters examined included total biomass and the shoot/root ratio. Seedlings colonized by non-EM fungal taxa were excluded from growth performance analyses. Simple linear regression analysis was used to examine associations between EM richness and growth parameters. One-way analysis of variance (ANOVA) was used to test differences in seedling growth parameters among frequently occurring EM fungal taxa and uncolonized seedlings. Seedlings that were colonized by multiple EM fungal OTUs were excluded from ANOVA.

3. Results

In total, 214 (185 field soil samples and 29 control) of 230 seedlings survived to the end of the experimental period. None of the control seedlings exhibited fungal colonization on the roots. Fungal colonization was detected on 66 seedlings. A total of 206 root tips were subjected to DNA analysis and ITS sequences were obtained for 186 tips. After removal of short, low-quality sequences, 171 DNA samples were clustered into 30 unique sequences (Supplementary Table S1). At a 97% similarity cutoff, 18 root-associated fungal OTUs were identified, of which 11 were classified as EM fungi (Table 2). We identified 6 and 9 EM fungal OTUs from 29 and 37 seedlings at KTY and KOD, respectively. The three most frequently encountered fungal OTUs were Suillus clintonianus (Peck) Kuntze (13.5%), Cenococcum geophilum Fr. (5.9%), and Sebacinales OTU1 (5.9%), all of which were detected at both sites. Sebacinales OTU1 belongs to the EM lineage of the /serendipita1 clade (Tedersoo & Smith, 2013). Closely related sequences (SH1577254.08FU, 93.9% match to the query sequence) were detected mainly on Pyrola asarifolia Michx. and occasionally on Bistorta vivipara (L.) Delarbre and other angiosperm trees (e.g., Betula L. and Populus L.) based on records in the UNITE database (accessed Nov 29, 2021). Four suilloid species were identified in tundra soils, where compatible host trees were absent. Suillus spectabilis (Peck) Kuntze, Suillus viscidus (L.) Roussel, and Rhizopogon laricinus Y. Miyamoto & T.C. Maximov had previously been confirmed on roots of existing L. cajanderi at treelines in the study region (Miyamoto et al., 2022; Table 2). By contrast, S. clintonianus was dominant in the propagule banks but was not detected on existing roots at the treeline.

Table 2 Identity and frequency of root-associated fungal operational taxonomic units (OTUs) detected from soil propagule banks. Frequency indicates the percentage of seedlings colonized by each fungal OTU among all seedlings examined per treatment.

Ectomycorrhizal fungal lineage	Fungal OTU	KTY		KOD			Existing root communities a
		N	H	N	H	Total
/suillus-rhizopogon	Suillus clintonianus	16.7	12.5	19.0	6.4	13.5	C2
	Suillus spectabilis	4.2	2.1	0	0	1.6	B3, C3, C4
	Suillus viscidus	2.1	0	0	0	0.5	B1, B2, B3
	Rhizopogon laricinus	0	0	2.4	0	0.5	B1, B2, B3, C1, C3, C4
/laccaria	Laccaria cf. laccata OTU1	0	0	2.4	0	0.5	KOD
	Laccaria cf. laccata OTU 2	0	0	2.4	2.1	1.1
/cenococcum	Cenococcum geophilum c	0 B	2.1 AB	16.7 A	6.4 AB	5.9	All sites
/meliniomyces	Hyaloscypha finlandica	2.1	0	4.8	0	1.6
	Meliniomyces bicolor	0	0	2.4	2.1	1.1	KOD, B2, C4
/serendipita1	Sebacinales OTU1	8.3	0	9.5	6.4	5.9
	Sebacinales OTU2	0	0	2.4	0	0.5
/non-ectomycorrhizal	Helotiales OTU1	0	0	0	2.1	0.5
	Archaeorhizomyces OTU1	2.1	0	0	0	0.5
	Phialocephala fortinii	10.4	2.1	2.4	4.3	4.9	KOD, B2, C1, C3, C4
	Pezoloma ericae OTU1	0	2.1	0	0	0.5
	Pezoloma ericae OTU2	2.1	2.1	0	0	1.1
	Sebacinales OTU3	2.1	0	0	0	0.5
unknown status	Pezizales OTU1	2.1	4.2	0	4.3	2.7
No. seedlings examined b		48 (15)	48 (9)	42 (22)	47 (11)	185 (53)
EMF observed richness		5	3	9	5	11
EMF Jackknife2 richness		7.78	6.25	17.42	8.39	16.88
Seedlings colonized by EMF (%) c		31.2 AB	18.8 B	52.4 A	23.4 AB	28.6

N, non-heat treatment; H, heat treatment; EMF, ectomycorrhizal fungi. DDBJ accession numbers are available in Supplementary Table S1.

^a Sites where identical fungal OTUs were detected in existing root communities in eastern Siberia (from Miyamoto et al. 2022). Site codes: B1-B3, treeline sites near Chokurdakh, C1-C2, forest sites near Yakutsk; C3, forest site near Ust' Maya; C4, forest site near Zeya.

^b Values in parentheses represent numbers of seedlings colonized by EMF.

^c Different uppercase letters indicate significant differences in colonization rate according to Fisher's exact test after false discovery rate P value corrections for multiple comparisons. Results are not shown for Suillus clintonianus and Sebacinales OTU1, which did not differ significantly among categories.

Observed EM fungal richness was highest in the non-heat treatment at KOD and lowest in the heat treatment at KTY, but the differences among the four categories were unclear because the confidence intervals of the rarefaction curves overlapped considerably (Fig. 1). Fisher's exact test revealed that the overall colonization rate was higher for the non-heat treatment at KOD than for the heat treatment at KTY (P = 0.0057; Table 2). Effects of site and heat treatment were examined for the three most frequently observed EM fungal OTUs. Cenococcum geophilum had higher frequencies in the non-heat treatment at KOD than in the heat treatment at KTY (P = 0.022; Table 2), whereas no frequency differences were detected among the four categories for S. clintonianus and Sebacinales OTU1 (P > 0.14).

Fig. 1 - Rarefaction curves showing observed richness. Dashed lines indicate 95% confidence intervals.

Because no effect of soil heating on fungal richness and colonization rates was detected, seedlings grown in the heat and non-heat treatments were pooled to increase sample size and the growth parameters were analyzed. Linear regression analysis indicated a tendency toward increased total seedling biomass with increasing EM fungal richness (F_203,1 = 3.12, P = 0.078, R² = 0.0103). However, seedling biomass showed large variations and no clear trend was detected when outliers (1.5-fold more extreme than the upper or lower quartile) were removed from the analysis (Supplementary Fig. S1; P = 0.99). Similarly, seedling biomass and EM fungal richness at each site exhibited no clear associations. Seedling growth parameters (i.e., total biomass and shoot/root ratio) did not change with colonization by any EM fungal taxa (ANOVA, P > 0.22; Supplementary Fig. S2).

4. Discussion

Propagule banks are generally less diverse and composed of different fungal taxa than communities of existing EM roots (Taylor & Bruns, 1999; Glassman et al., 2015; Miyamoto & Nara, 2016). In this study, the EM fungal richness of the propagule banks in the bioassays was 23.4% that of existing L. cajanderi roots at the treeline (Miyamoto et al., 2022). This reduction is comparable to those reported in previous studies that performed bioassay experiments in temperate and boreal forests (12-45%) (Glassman et al., 2015; Miyamoto & Nara, 2016; Murata et al., 2017a; Wen et al., 2018).

To our knowledge, this study is the first to investigate soil propagule bank communities of EM fungi in the tundra ecosystems of eastern Eurasia, where Larix trees form the extensive northern treeline. The detection of three Suillus species and R. laricinus from these tundra soils confirms that spores of Larix-specific EM fungi are deposited above the treeline, where compatible hosts are absent. All Suillus species were detected at KTY, indicating spore dispersal at least 30 km from L. cajanderi stands. We also found that S. clintonianus and S. spectabilis spores remained infective in soils after 14 mo of storage under dry conditions and heat treatment at 60 °C, indicating the durability of the spores (Peay et al., 2009; Nguyen et al., 2012). These results are consistent with the first hypothesis of spore heat resistance in the studied suilloid fungi. Moreover, Suillus species have been reported to mediate drought stress of infected seedlings by altering host physiology in water relations (Li et al., 2021; Wang et al., 2021). These features may support the rapid establishment of EM associations and increase seedling survival in arid regions.

Suillus clintonianus was the most frequently detected OTU in this bioassay; however, it was not detected from existing root communities of L. cajanderi trees at treelines in the same area (Table 2). In a previous study involving extensive sampling of L. cajanderi roots in eastern Siberia (252 samples), S. clintonianus was detected in only one sample in a forest composed of young L. cajanderi trees (Miyamoto et al., 2022). By contrast, S. viscidus and S. spectabilis were detected frequently on L. cajanderi roots at treeline sites but were less frequent in propagule banks in this tundra. These observations suggest species-specific strategies for forest occupation in terms of successional stage among Suillus species, but additional observations are needed to confirm this trend. Rhizopogon laricinus was the most frequently detected species on L. cajanderi roots at treelines but was almost negligible in propagule banks (Table 2). This pattern was unexpected because Rhizopogon species are often dominant components of propagule banks compared with existing root communities in mature forests (Taylor & Bruns, 1999; Murata et al., 2017a; Shemesh, Boaz, Millar, & Bruns, 2020). The ecology of R. laricinus remains unclear; further research will be required to clarify its dispersal strategies including fruiting habits (e.g., amount and seasonality) and spore characteristics (e.g., longevity and germination rates).

Cenococcum geophilum was detected at high frequencies in both propagule banks and existing root communities in tundra sites. Cenococcum geophilum is a globally distributed species complex that produces asexual sclerotia and often improves the drought resistance of associated hosts (Pigott, 1982; Douhan & Rizzo, 2005). Given the availability of sclerotia and mycelia in the tundra, C. geophilum may readily form EM associations with emerging seedlings whose growth and survival may be enhanced. The third most frequently occurring taxon, Sebacinales OTU1, had immature mantles characterized by grey hairy hyphae loosely surrounding root tips of L. cajanderi bioassay seedlings. Thus, Sebacinales OTU1 may be an endophyte or EM fungus primarily associated with tundra shrubs.

The second hypothesis, that the richness and colonization rates of EM fungal taxa would be reduced with increasing distance from the treeline, was not clearly supported in this study. The results suggest no strong dispersal limitation at this spatial scale, notably for S. clintonianus. The frequency of C. geophilum was reduced at KTY, perhaps due to soil conditions or other environmental factors rather than distance effects because this species is a common EM associate of tundra shrubs (Miyamoto et al., 2022). Although we did not detect distance effects between the two sites, the overall fungal colonization rates were lower in this tundra (28.6%) than in similar bioassay experiments conducted in mature forests (57-100%; Miyamoto & Nara, 2016; Murata et al., 2017a, b; Wen et al., 2018). Several bioassay studies have reported reduced EM colonization from soils collected beyond host range limits at local (1-3 km) and regional (~300 km) scales (Nuñez et al., 2009; Fujiyoshi et al., 2011; Reithmeier & Kernaghan, 2013; Pickles et al., 2015), primarily due to dispersal limitation of the fungal propagules (Peay et al., 2012). Spore dispersal tends to be highly spatiotemporally variable, resulting in patchy deposition in soils (Ashkannejhad & Horton, 2006; Peay & Bruns, 2014). Thus, emerging seedlings may have low opportunity to encounter propagules of compatible EM fungi in this tundra, similar to other primary habitats (Nuñez et al., 2009; Fujiyoshi et al., 2011; Reithmeier & Kernaghan, 2013).

Colonization by EM fungi often improves seedling performance (Murata et al., 2017b; Wen et al., 2018). Moreover, colonization by additional fungal taxa may improve seedling growth (Oliveira, Franco, & Castro, 2012; Wen et al., 2018) due to the complementary functions of various fungal taxa, driven by their enzyme activities (Courty, Pritsch, Schloter, Hartmann, & Garbaye, 2005). However, the third hypothesis of improved seedling growth with EM fungal colonization was not clearly supported in this study. This hypothesis could not be supported due to limited statistical power, which resulted from the underrepresentation of most fungal OTUs at the study sites. Fungal inoculation experiments would be an effective method for further clarifying the potential effects of individual fungal taxa and their interactions on seedling performance.

Some limitations of this study should be noted. We used bioassay experiments, which can detect only a subset of EM fungal taxa in propagule banks that persist in dried soils for an extended duration. Short-lived and seasonally dispersed spores (Peay & Bruns, 2014) can be an important source of EM inocula in natural settings. Moreover, indoor bioassay conditions may have resulted in low propagule germination rates. Soil storage duration may affect the detectability of EM fungal taxa, and spore germination may require additional stimuli specific to Arctic field conditions, such as freezing stratification or chemical cues produced by rhizosphere microorganisms. We also used commercially available seeds that originated from a southern L. cajanderi population, which may have disrupted plant-fungus genotype interactions in the glasshouse bioassay (Hoeksema, Hernandez, Rogers, Mendoza, & Thompson, 2012). Although we cannot detect all propagules in soil, the bioassay approach effectively demonstrated the infectivity of spores of Larix-specific EM fungi that were deposited above the treeline. Spores of specialist EM fungi likely play an important role in mycorrhiza formation on regenerating seedlings in novel habitats (Ashkannejhad & Horton, 2006). Seedling establishment is the first step in the range expansion of trees and subsequent forest development. The ecological role of specialized associations between suilloid fungi and Larix at the treeline may require further research to clarify tree establishment processes under climate change in the Eurasian Arctic.

Disclosure

The authors declare no conflicts of interest.

Appendix A. Supplementary Materials

Supplementary data related to this article is attached.

Acknowledgments

We thank Egor Starostin and Alexandra Alexeeva of the Institute for Biological Problems of Cryolithozone SB RAS, and Ruslan Shakmatov at the Graduate School of Environmental Sciences, Hokkaido University (EES-HU), for their help with field work and obtaining research permissions, and Dr. Kyoko Miwa in EES-HU and Dr. Yutaka Tamai at the School of Agriculture-HU for their assistance with bioassay experiments and molecular analyses. This work was partially funded by JSPS KAKENHI (Grant Number 17K15281; 20H03016) and the Grant for Joint Research Program of the Japan Arctic Research Network Center to Y.M., and JST Belmont Forum COPERA project partly supported by Hokkaido University to A.S.

References

• Agerer, R. (2001). Exploration types of ectomycorrhizae - A proposal to classify ectomycorrhizal mycelial systems according to their patterns of differentiation and putative ecological importance. Mycorrhiza, 11, 107-114. https://doi.org/10.1007/s005720100108
• Ashkannejhad, S., & Horton, T. R. (2006). Ectomycorrhizal ecology under primary succession on coastal sand dunes: interactions involving Pinus contorta, suilloid fungi and deer. New Phytologist, 169, 345-354. https://doi.org/10.1111/j.1469-8137.2005.01593.x
• Baar, J., Horton, T. R., Kretzer, A. M., & Bruns, T. D. (1999). Mycorrhizal colonization of Pinus muricata from resistant propagules after a stand-replacing wildfire. New Phytologist, 143, 409-418. https://doi.org/10.1046/j.1469-8137.1999.00452.x
• Bödeker, I. T. M., Clemmensen, K. E., de Boer, W., Martin, F., Olson, A., & Lindahl, B. D. (2014). Ectomycorrhizal Cortinarius species participate in enzymatic oxidation of humus in northern forest ecosystems. New Phytologist, 203, 245-256. https://doi.org/10.1111/nph.12791
• Bruns, T. D., Hale, M. L., & Nguyen, N. H. (2019). Rhizopogon olivaceotinctus increases its inoculum potential in heated soil independent of competitive release from other ectomycorrhizal fungi. Mycologia, 111, 936-941. https://doi.org/10.1080/00275514.2019.1657354
• Bruns, T. D., Peay, K. G., Boynton, P. J., Grubisha, L. C., Hynson, N. A., Nguyen, N. H., & Rosenstock, N. P. (2009). Inoculum potential of Rhizopogon spores increases with time over the first 4 yr of a 99-yr spore burial experiment. New Phytologist, 181, 463-470. https://doi.org/10.1111/j.1469-8137.2008.02652.x
• Buscardo, E., Rodriguez-Echeverria, S., Martin, M. P., De Angelis, P., Pereira, J. S., & Freitas, H. (2010). Impact of wildfire return interval on the ectomycorrhizal resistant propagules communities of a Mediterranean open forest. Fungal Biology, 114, 628-636. https://doi.org/10.1016/j.funbio.2010.05.004
• CDIAC (2012). Daily Temperature and Precipitation Data for 518 Russian Meteorological Stations. Bulygina ON, Razuvaev V N. Carbon Dioxide Information Analysis Center (CDIAC), Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee. https://doi.org/10.3334/CDIAC/cli.100
• Collier, F. A., & Bidartondo, M. I. (2009). Waiting for fungi: the ectomycorrhizal invasion of lowland heathlands. Journal of Ecology, 97, 950-963. https://doi.org/10.1111/j.1365-2745.2009.01544.x
• Colwell, R. K. (2019). EstimateS software version 9.1.0. http://viceroy.eeb.uconn.edu/estimates/
• Courty, P. E., Pritsch, K., Schloter, M., Hartmann, A., & Garbaye, J. (2005). Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests. New Phytologist, 167, 309-319. https://doi.org/10.1111/j.1469-8137.2005.01401.x
• Deslippe, J. R., Hartmann, M., Grayston, S. J., Simard, S. W., & Mohn, W. W. (2016). Stable isotope probing implicates a species of Cortinarius in carbon transfer through ectomycorrhizal fungal mycelial networks in Arctic tundra. New Phytologist, 210, 383-390. https://doi.org/10.1111/nph.13797
• Douhan, G. W., & Rizzo, D. M. (2005). Phylogenetic divergence in a local population of the ectomycorrhizal fungus Cenococcum geophilum. New Phytologist, 166, 263-271. https://doi.org/10.1111/j.1469-8137.2004.01305.x
• Fujiyoshi, M., Yoshitake, S., Watanabe, K., Murota, K., Tsuchiya, Y., Uchida, M., & Nakatsubo, T. (2011). Successional changes in ectomycorrhizal fungi associated with the polar willow Salix polaris in a deglaciated area in the High Arctic, Svalbard. Polar Biology, 34, 667-673. https://doi.org/10.1007/s00300-010-0922-9
• Geml, J., Timling, I., Robinson, C. H., Lennon, N., Nusbaum, H. C., Brochmann, C., Noordeloos, M. E., & Taylor, D. L. (2012). An arctic community of symbiotic fungi assembled by long-distance dispersers: phylogenetic diversity of ectomycorrhizal basidiomycetes in Svalbard based on soil and sporocarp DNA. Journal of Biogeography, 39, 74-88. https://doi.org/10.1111/j.1365-2699.2011.02588.x
• Germino, M. J., Smith, W. K., & Resor, A. C. (2002). Conifer seedling distribution and survival in an alpine-treeline ecotone. Plant Ecology, 162, 157-168. https://doi.org/10.1023/a:1020385320738
• Gerz, M., Bueno, C. G., Ozinga, W. A., Zobel, M., & Moora, M. (2018). Niche differentiation and expansion of plant species are associated with mycorrhizal symbiosis. Journal of Ecology, 106, 254-264. https://doi.org/10.1111/1365-2745.12873
• Glassman, S. I., Levine, C. R., DiRocco, A. M., Battles, J. J., & Bruns, T. D. (2016). Ectomycorrhizal fungal spore bank recovery after a severe forest fire: some like it hot. Isme Journal, 10, 1228-1239. https://doi.org/10.1038/ismej.2015.182
• Glassman, S. I., Peay, K. G., Talbot, J. M., Smith, D. P., Chung, J. A., Taylor, J. W., Vilgalys, R., & Bruns, T. D. (2015). A continental view of pine-associated ectomycorrhizal fungal spore banks: a quiescent functional guild with a strong biogeographic pattern. New Phytologist, 205, 1619-1631. https://doi.org/10.1111/nph.13240
• Harsch, M. A., Hulme, P. E., McGlone, M. S., & Duncan, R. P. (2009). Are treelines advancing? A global meta-analysis of treeline response to climate warming. Ecology Letters, 12, 1040-1049. https://doi.org/10.1111/j.1461-0248.2009.01355.x
• Hayat, R., Ali, S., Amara, U., Khalid, R., & Ahmed, I. (2010). Soil beneficial bacteria and their role in plant growth promotion: a review. Annals of Microbiology, 60, 579-598. https://doi.org/10.1007/s13213-010-0117-1
• Hewitt, R. E., Chapin, F. S., Hollingsworth, T. N., & Taylor, D. L. (2017). The potential for mycobiont sharing between shrubs and seedlings to facilitate tree establishment after wildfire at Alaska arctic treeline. Molecular Ecology, 26, 3826-3838. https://doi.org/10.1111/mec.14143
• Hoeksema, J. D., Hernandez, J. V., Rogers, D. L., Mendoza, L. L., & Thompson, J. N. (2012). Geographic divergence in a species-rich symbiosis: interactions between Monterey pines and ectomycorrhizal fungi. Ecology, 93, 2274-2285. https://doi.org/10.1890/11-1715.1
• Izzo, A., Canright, M., & Bruns, T. D. (2006). The effects of heat treatments on ectomycorrhizal resistant propagules and their ability to colonize bioassay seedlings. Mycological Research, 110, 196-202. https://doi.org/10.1016/j.mycres.2005.08.010
• Kaplan, J. O., Bigelow, N. H., Prentice, I. C., Harrison, S. P., Bartlein, P. J., Christensen, T. R., Cramer, W., Matveyeva, N. V., McGuire, A. D., Murray, D. F., Razzhivin, V. Y., Smith, B., Walker, D. A., Anderson, P. M., Andreev, A. A., Brubaker, L. B., Edwards, M. E., & Lozhkin, A. V. (2003). Climate change and Arctic ecosystems: 2. Modeling, paleodata-model comparisons, and future projections. Journal of Geophysical Research-Atmospheres, 108, Article 8171. https://doi.org/10.1029/2002jd002559
• Kikuchi, K., Matsushita, N., Suzuki, K., & Hogetsu, T. (2007). Flavonoids induce germination of basidiospores of the ectomycorrhizal fungus Suillus bovinus. Mycorrhiza, 17, 563-570. https://doi.org/10.1007/s00572-007-0131-8
• Kipfer, T., Moser, B., Egli, S., Wohlgemuth, T., & Ghazoul, J. (2011). Ectomycorrhiza succession patterns in Pinus sylvestris forests after stand-replacing fire in the Central Alps. Oecologia, 167, 219-228. https://doi.org/10.1007/s00442-011-1981-5
• Koljalg, U., Nilsson, R. H., Abarenkov, K., Tedersoo, L., Taylor, A. F. S., Bahram, M., Bates, S. T., Bruns, T. D., Bengtsson-Palme, J., Callaghan, T. M.,Douglas, B., Drenkhan, T., Eberhardt, U., Duenas, M., Grebenc, T., Griffith, G. W., Hartmann, M., Kirk, P. M., Kohout, P., Larsson, E., Lindahl, B. D., Luecking, R., Martin, M. P., Matheny, P. B., Nguyen, N. H., Niskanen, T., Oja, J., Peay, K. G., Peintner, U., Peterson, M., Poldmaa, K., Saag, L., Saar, I., Schuessler, A., Scott, J. A., Senes, C., Smith, M. E., Suija, A., Taylor, D. L., Telleria, M. T., Weiss, M., & Larsson, K. H. (2013). Towards a unified paradigm for sequence-based identification of fungi. Molecular Ecology, 22, 5271-5277. https://doi.org/10.1111/mec.12481
• Li, M., Wang, H., Zhao, X., Lu, Z., Sun, X., & Ding, G. (2021). Role of Suillus placidus in improving the drought tolerance of Masson pine (Pinus massoniana Lamb.) seedlings. Forests, 12, Article 332. https://doi.org/10.3390/f12030332
• Lindahl, B. D., & Tunlid, A. (2015). Ectomycorrhizal fungi - potential organic matter decomposers, yet not saprotrophs. New Phytologist, 205, 1443-1447. https://doi.org/10.1111/nph.13201
• Miyamoto, Y., Danilov, A. V., & Bryanin, S. V. (2021). The dominance of Suillus species in ectomycorrhizal fungal communities on Larix gmelinii in a post-fire forest in the Russian Far East. Mycorrhiza, 31, 55-66. https://doi.org/10.1007/s00572-020-00995-3
• Miyamoto, Y., Maximov, T. C., Bryanin, S. V., Kononov, A., & Sugimoto, A. (2022). Host phylogeny is the primary determinant of ectomycorrhizal fungal community composition in the permafrost ecosystem of eastern Siberia at a regional scale. Fungal Ecology, 55. https://doi.org/10.1016/j.funeco.2021.101117
• Miyamoto, Y., & Nara, K. (2016). Soil propagule banks of ectomycorrhizal fungi share many common species along an elevation gradient. Mycorrhiza, 26, 189-197. https://doi.org/10.1007/s00572-015-0658-z
• Molina, R., & Trappe, J. M. (1994). Biology of the ectomycorrhizal genus, Rhizopogon.1. Host associations, host-specificity and pure culture syntheses. New Phytologist, 126, 653-675. https://doi.org/10.1111/j.1469-8137.1994.tb02961.x
• Murata, M., Kanetani, S., & Nara, K. (2017a). Ectomycorrhizal fungal communities in endangered Pinus amamiana forests. Plos One, 12, e0189957. https://doi.org/10.1371/journal.pone.0189957
• Murata, M., Nagata, Y., & Nara, K. (2017b). Soil spore banks of ectomycorrhizal fungi in endangered Japanese Douglas-fir forests. Ecological Research, 32, 469-479. https://doi.org/10.1007/s11284-017-1456-1
• Nara, K. (2006). Pioneer dwarf willow may facilitate tree succession by providing late colonizers with compatible ectomycorrhizal fungi in a primary successional volcanic desert. New Phytologist, 171, 187-198. https://doi.org/10.1111/j.1469-8137.2006.01744.x
• Nara, K., & Hogetsu, T. (2004). Ectomycorrhizal fungi on established shrubs facilitate subsequent seedling establishment of successional plant species. Ecology, 85, 1700-1707. https://doi.org/10.1890/03-0373
• Nguyen, N. H., Hynson, N. A., & Bruns, T. D. (2012). Stayin' alive: survival of mycorrhizal fungal propagules from 6-yr-old forest soil. Fungal Ecology, 5, 741-746. https://doi.org/10.1016/j.funeco.2012.05.006
• Nguyen, N. H., Vellinga, E. C., Bruns, T. D., & Kennedy, P. G. (2016). Phylogenetic assessment of global Suillus ITS sequences supports morphologically defined species and reveals synonymous and undescribed taxa. Mycologia, 108, 1216-1228. https://doi.org/10.3852/16-106
• Nuñez, M. A., Horton, T. R., & Simberloff, D. (2009). Lack of belowground mutualisms hinders Pinaceae invasions. Ecology, 90, 2352-2359. https://doi.org/10.1890/08-2139.1
• Oliveira, R. S., Franco, A. R., & Castro, P. M. L. (2012). Combined use of Pinus pinaster plus and inoculation with selected ectomycorrhizal fungi as an ecotechnology to improve plant performance. Ecological Engineering, 43, 95-103. https://doi.org/10.1016/j.ecoleng.2012.01.021
• Overland, J., Walsh, J., & Kattsov, V. (2017). Chapter 2. Trends and feedbacks. In Arctic Monitoring and Assessment Programme (AMAP), Snow, Water, Ice and Permafrost in the Arctic (SWOPA) 2017 (pp. 9-24). Oslo, Norway
• Parker, M. A., Malek, W., & Parker, I. M. (2006). Growth of an invasive legume is symbiont limited in newly occupied habitats. Diversity and Distributions, 12, 563-571. https://doi.org/10.1111/j.1366-9516.2006.00255.x
• Peay, K. G., & Bruns, T. D. (2014). Spore dispersal of basidiomycete fungi at the landscape scale is driven by stochastic and deterministic processes and generates variability in plant-fungal interactions. New Phytologist, 204, 180-191. https://doi.org/10.1111/nph.12906
• Peay, K. G., Garbelotto, M., & Bruns, T. D. (2009). Spore heat resistance plays an important role in disturbance-mediated assemblage shift of ectomycorrhizal fungi colonizing Pinus muricata seedlings. Journal of Ecology, 97, 537-547. https://doi.org/10.1111/j.1365-2745.2009.01489.x
• Peay, K. G., Schubert, M. G., Nguyen, N. H., & Bruns, T. D. (2012). Measuring ectomycorrhizal fungal dispersal: macroecological patterns driven by microscopic propagules. Molecular Ecology, 21, 4122-4136. https://doi.org/10.1111/j.1365-294X.2012.05666.x
• Pickles, B. J., Gorzelak, M. A., Green, D. S., Egger, K. N., & Massicotte, H. B. (2015). Host and habitat filtering in seedling root-associated fungal communities: taxonomic and functional diversity are altered in ‘novel’ soils. Mycorrhiza, 25, 517-531. https://doi.org/10.1007/s00572-015-0630-y
• Pigott, C. D. (1982). Survival of mycorrhiza formed by Cenococcum geophilum Fr. in dry soils. New Phytologist, 92, 513-517. https://doi.org/10.1111/j.1469-8137.1982.tb03409.x
• Policelli, N., Bruns, T. D., Vilgalys, R., & Nunez, M. A. (2019). Suilloid fungi as global drivers of pine invasions. New Phytologist, 222, 714-725. https://doi.org/10.1111/nph.15660
• Pringle, A., Bever, J. D., Gardes, M., Parrent, J. L., Rillig, M. C., & Klironomos, J. N. (2009). Mycorrhizal symbioses and plant invasions. Annual Review of Ecology Evolution and Systematics, 40, 699-715. https://doi.org/10.1146/annurev.ecolsys.39.110707.173454
• Reithmeier, L., & Kernaghan, G. (2013). Availability of ectomycorrhizal fungi to black spruce above the present treeline in eastern Labrador. Plos One, 8, e77527, https://doi.org/10.1371/journal.pone.0077527
• Ryberg, M., Larsson, E., & Molau, U. (2009). Ectomycorrhizal diversity on Dryas octopetala and Salix reticulata in an alpine cliff ecosystem. Arctic Antarctic and Alpine Research, 41, 506-514. https://doi.org/10.1657/1938-4246-41.4.506
• Semerikov, V. L., Iroshnikov, A. I., & Lascoux, M. (2007). Mitochondrial DNA variation pattern and postglacial history of the Siberian larch (Larix sibirica ledeb.). Russian Journal of Ecology, 38, 147-154. https://doi.org/10.1134/s1067413607030010
• Serreze, M. C., & Barry, R. G. (2011). Processes and impacts of Arctic amplification: A research synthesis. Global and Planetary Change, 77, 85-96. https://doi.org/10.1016/j.gloplacha.2011.03.004
• Shah, F., Nicolas, C., Bentzer, J., Ellstroom, M., Smits, M., Rineau, F., Canback, B., Floudas, D., Carleer, R., Lackner, G., Braesel, J., Hoffmeister, D., Henrissat, B., Ahren, D., Johansson, T., Hibbett, D. S., Martin, F., Persson, P., & Tunlid, A. (2016). Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors. New Phytologist, 209, 1705-1719. https://doi.org/10.1111/nph.13722
• Shemesh, H., Boaz, B. E., Millar, C. I., & Bruns, T. D. (2020). Symbiotic interactions above treeline of long-lived pines: Mycorrhizal advantage of limber pine (Pinus flexilis) over Great Basin bristlecone pine (Pinus longaeva) at the seedling stage. Journal of Ecology, 108, 908-916. https://doi.org/10.1111/1365-2745.13312
• Smith, S. E., & Read, D. J. (2008). Mycorrhizal Symbiosis, 3rd Edition. Elsevier Academic Press Inc.
• Takahashi, K. (2006). Future perspective of forest management in a Siberian permafrost area. In Hatano, R., & Guggenberger, G. (Eds.), Symptom of Environmental Change in Siberian Permafrost Region. (pp. 163-170). Hokkaido Univ. Press.
• Taylor, D. L., & Bruns, T. D. (1999). Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities. Molecular Ecology, 8, 1837-1850. https://doi.org/10.1046/j.1365-294x.1999.00773.x
• R Core Team (2015). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.
• Tedersoo, L., May, T. W., & Smith, M. E. (2010). Ectomycorrhizal lifestyle in fungi: global diversity, distribution, and evolution of phylogenetic lineages. Mycorrhiza, 20, 217-263. https://doi.org/10.1007/s00572-009-0274-x
• Tedersoo, L., & Smith, M. E. (2013). Lineages of ectomycorrhizal fungi revisited: Foraging strategies and novel lineages revealed by sequences from belowground. Fungal Biology Reviews, 27, 83-99. https://doi.org/10.1016/j.fbr.2013.09.001
• Theodorou, C., & Bowen, G. D. (1987). Germination of basidiospores of mycorrhizal fungi in the rhizosphere of Pinus radiata D. Don. New Phytologist, 106, 217-223. https://doi.org/10.1111/j.1469-8137.1987.tb00137.x
• Timling, I., Dahlberg, A., Walker, D. A., Gardes, M., Charcosset, J. Y., Welker, J. M., & Taylor, D. L. (2012). Distribution and drivers of ectomycorrhizal fungal communities across the North American Arctic. Ecosphere, 3, 1-25, Article 111. https://doi.org/10.1890/es12-00217.1
• Urcelay, C., Longo, S., Geml, J., Tecco, P. A., & Nouhra, E. (2017). Co-invasive exotic pines and their ectomycorrhizal symbionts show capabilities for wide distance and altitudinal range expansion. Fungal Ecology, 25, 50-58. https://doi.org/10.1016/j.funeco.2016.11.002
• Vincenot, L., & Selosse, M. A. (2017). Population biology and ecology of ectomycorrhizal fungi. In L. Tedersoo (Ed.), Biogeography of Mycorrhizal Symbiosis (pp. 39-59). Springer. https://doi.org/10.1007/978-3-319-56363-3
• Wang, J., Zhang, H., Gao, J., Zhang, Y., Liu, Y., & Tang, M. (2021). Effects of ectomycorrhizal fungi (Suillus variegatus) on the growth, hydraulic function, and non-structural carbohydrates of Pinus tabulaeformis under drought stress. BMC Plant Biology, 21, Article 171. https://doi.org/10.1186/s12870-021-02945-3
• Wen, Z. G., Shi, L., Tang, Y. Z., Hong, L. Z., Xue, J. W., Xing, J. C., Chen, Y. H., & Nara, K. (2018). Soil spore bank communities of ectomycorrhizal fungi in endangered Chinese Douglas-fir forests. Mycorrhiza, 28, 49-58. https://doi.org/10.1007/s00572-017-0800-1
• White, T.J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In Innis, M. A., Gelfand, D. H., Sninsky, J. J., & White, T. J. (Eds.), PCR protocols: a guide to methods and applications (pp. 315-322). Academic Press, London, United Kingdom.