Dual-mycorrhizal colonization is determined by plant age and host identity in two species of Populus

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Cregger, Christopher Schadt, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6531386/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jun, 2025 Read the published version in Mycorrhiza → Version 1 posted 9 You are reading this latest preprint version Abstract Plants have evolved symbioses with mycorrhizal and endophytic fungi that are essential for their growth and survival. While most plants associate with a single guild of mycorrhizal fungi, a select group termed “dual-mycorrhizal plants” associate with both arbuscular mycorrhizal and ectomycorrhizal fungi. Although a shift from arbuscular mycorrhizal to ectomycorrhizal colonization with plant development has been demonstrated on other dual-mycorrhizal hosts, it is not known how mycorrhizal colonization shifts with plant age in Populus species. We performed an in planta mycorrhization experiment to test for the occurrence of mycorrhizal switching in response to plant age and for host-specific patterns of fungal colonization in two species of Populus ( P. tremuloides and P. trichocarpa ). We found that only P. trichocarpa displayed dual-mycorrhizal colonization, while P. tremuloides associated with ectomycorrhizal fungi, but not arbuscular mycorrhizal fungi. On P. trichocarpa , both guilds of mycorrhizal fungi increased in abundance with plant age, while root endophytic fungal colonization decreased. Many of the early-colonizing endophytic fungi that we documented have strong saprotrophic capabilities, which may be an important trait for fast colonization. Dark septate endophytes were more abundant than either guild of mycorrhizal fungi, and are likely functionally important members of the Populus root fungal community. Our findings represent a novel pattern in the development of dual-mycorrhizal colonization and illustrate that Populus species vary in their association with arbuscular mycorrhizal fungi. Our results also stress the importance of a “third guild” of root fungal symbionts – the dark septate endophytes – on dual-mycorrhizal plants. Dual-mycorrhizal dark septate endophytes Populus succession ectomycorrhizae arbuscular mycorrhizae Figures Figure 1 Figure 2 Figure 3 Introduction Plants have evolved symbioses with diverse mycorrhizal fungal symbionts that serve perform diverse functions in the plant root environment, including nutrient transfer and protection from abiotic and biotic stress (Behie & Bidochka, 2014 ; Branco et al., 2022 ). The two most common guilds of mycorrhizal fungal symbionts are arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi, which colonize 72% and 2% of vascular plants respectively (Brundrett & Tedersoo, 2018 ). Although associations with both of these groups of fungi have evolved in multiple plant lineages, the mycorrhizal type of plants is generally conserved within the same genus, family, or order, suggesting some degree of phylogenetic conservation (Brundrett & Tedersoo, 2018 ). While vascular plants associate with a single guild of mycorrhizal fungi, there are plants termed “dual-mycorrhizal” that can associate with both AM and EM fungi. Although this group of dual-mycorrhizal plants is relatively small, estimated at between 1500 to 7300 plant species or < 3% of flowering plants (Brundrett & Tedersoo, 2019 ; Teste et al., 2020 ), it includes dominant trees on multiple continents, such as Populus spp. and Eucalyptus spp. While dual-mycorrhizal associations are often conserved within the same plant genus or family, there have been relatively few studies testing for differences in the extent of AM and EM colonization within individual dual-mycorrhizal plant lineages. Some dual-mycorrhizal plants can engage in mycorrhizal switching in which they shift towards AM or EM colonization in response to environmental or plant cues (Teste et al., 2020 ). Mycorrhizal switching has been documented in response to topographic position, seasonal drought, soil age, habitat type, and geographic range (Watson et al., 1990 ; Moyersoen & Fitter, 1999 ; Querejeta et al., 2009 ; Albornoz et al., 2016 ; Van Nuland et al., 2023 ). Plant development has also been implicated as a trigger for mycorrhizal switching on some dual-mycorrhizal hosts, with a frequently observed transition from high AM colonization in young plants, towards increasing EM colonization as plants age (Chilvers et al., 1987 ; Chen et al., 2000 ; Dickie et al., 2001 ; Santos et al., 2001). Teste et al. ( 2020 ) proposed the classification of such plants as “temporally dependent dual-mycorrhizal hosts”. Preferential association with AM fungi at the seedling stage is hypothesized to facilitate seedling establishment (Teste et al., 2020 ). While some temporally dependent dual-mycorrhizal hosts retain low levels of AM colonization throughout development, in extreme cases hosts can completely lose AM colonization and become entirely EM after approximately one year (de Mendonça Bellei et al., 1992 ). Although studies of dual-mycorrhizal plants have hinted at variability in the degree of temporal-dependence, there have been few systematic studies comparing successional trajectories on closely related hosts. In this study, we focus on the dual-mycorrhizal tree genus Populus to test the effects of plant host species identity and plant age on AM and EM colonization. Trees in the genus Populus have been widely used in plant-microbe interactions research and have been proposed as model dual-mycorrhizal systems (Cregger et al., 2021 ; Karst et al., 2021 ). Although there have been studies documenting variation in AM and EM colonization in Populus in response to tree genotype, temperature, soil moisture, soil depth, seasonality, heavy metal contamination, and leaf litter extract application (Lodge, 1989 ; Neville et al., 2002 ; Gehring et al., 2006 ; Piotrowski et al., 2008 ; Karliński et al., 2010 ; Van Nuland et al., 2023 ; Nash et al., 2025 ), there has been very little work examining AM and EM colonization throughout plant development and it is unclear if Populus species should be classified as temporally dependent dual-mycorrhizal hosts (Dominik, 1958 ; Fracchia et al., 2021 ). Although the composition of the fungal and bacterial microbiome of Populus is known to change with plant age (Fracchia et al., 2021 ; Xie et al., 2023 ; Argiroff et al., 2024 ), very few studies have tracked changes in AM and EM colonization through plant development on Populus (Dominik, 1958 ; Dove et al., 2021 ; Fracchia et al., 2024 ). Thus, it is not known whether Populus species undergo a shift from AM colonization to EM colonization as plants age as has been demonstrated on other temporally dependent dual-mycorrhizal species (Teste et al., 2020 ). The Populus genus contains around 30 species, distributed across six sections, which are all presumably dual-mycorrhizal (Eckenwalder, 1996 ; Wang et al., 2020 ). These species occupy unique biogeographic distributions and occupy a range of habitats, including boreal forest, semi-arid shrublands, mixed hardwood forests, and riparian zones. A DNA metabarcoding and microscopy-based field survey of five species of Populus in North America found a gradient of EM and AM colonization levels with P. angustifolia and P. tremuloides having the highest EM colonization and P. deltoides having the highest AM colonization (though P. tremuloides had the highest AM fungal richness). Our study makes use of a pot culture common garden approach with a field sampled soil inoculum to isolate the effect of plant species identity on mycorrhizal guild abundance during the first year of growth on the species P. tremuloides and P. trichocarpa . We used metabarcoding and root staining/microscopy to profile fungal colonization on both Populus spp. at 5 and 12 months of plant growth. We used RNA metabarcoding over DNA based methods, as we have previously found it to recover AM sequences at much higher levels (Nash et al., 2025 ). We hypothesized that 1) both Populus hosts will be temporally dependent dual-mycorrhizal species, 2) P. tremuloides will display a preference for EM over AM colonization compared to P. trichocarpa which will have greater AM colonization, and 3) in addition to guild-wide patterns, there will be consistently early and late colonizing species. Methods Overview of methods We inoculated 20 P. tremuloides and 20 P. trichocarpa seedlings with field soils sampled from P. tremuloides stands in southern Utah, along with four uninoculated controls of each species and grew them in pots for 12 months in a controlled growth chamber at Duke University’s Phytotron facility. Fine roots were collected from each plant at 5 and 12 months and used for ink-vinegar staining and LSU RNA metabarcoding to measure colonization levels of AM, EM, and endophytic fungi as well as sequence abundance of individual taxa. Inoculum collection and plant growth conditions In September 2021, we collected 20 soil samples from the top 15 cm of soil in four P. tremuloides stands in Fishlake National Forest, Utah, United States. Soils were shipped back to Duke University and kept at 4°C for four months until inoculation. One month prior to inoculation, P. tremuloides and P. trichocarpa seeds were sewn into nursery trays with autoclaved vermiculite and grown covered under fluorescent lights until inoculation. A sterile potting mix was prepared by mixing vermiculite, perlite, peat, and sand at a 2:2:1:1 ratio and autoclaving twice at 121°C for two hours with two days in between each autoclave cycle. We inoculated one seedling of each plant host with each of the 20 soil inocula at a ratio of 1 part soil inoculum to 2 parts sterile potting mix to maintain biological replication and capture the natural heterogeneity of soil inocula. For some soil samples, we had to decrease this ratio because of a low quantity of inoculum, though inoculum levels were always the same between the two hosts to avoid systemic bias and we found no impact of inoculum levels on alpha or beta diversity metrics. Seedlings were planted into 2.7” diameter by 10” deep conical tree pots until 6 months of age at which point they were transplanted into 5” by 5” by 12” square tree pots filled with the sterile potting mix to prevent root binding. Plants were grown in a controlled growth chamber with a 16 h day/ 8 h night light cycle with a light intensity of approximately 400 umol m − 2 s − 1 , 60% humidity, 23° C daytime temperature, and 18° C night temperature. Plants were fertilized throughout the experiment by watering pots with half strength Hoagland’s solution until saturation once per week. Plant height was collected at the time of sampling (see following section) at both 5 and 12 months by measuring the distance from the soil line to the apical bud. Sample collection and molecular methods At 5 and 12 months of plant growth, seedlings were gently extricated from pots and fine root samples were clipped from plants non-destructively with dissecting scissors and a scalpel to allow the same plants to be resampled. Fine roots with tightly adhering soil were flash frozen in liquid nitrogen immediately on collection and stored at -80°C until RNA extraction. A separate subsample of fine roots was rinsed of all adhering soil and stored in 10% KOH for staining and microscopic colonization scoring. For RNA extraction, approximately 1 gram of root tissue was cryogenically ground with a mortar and pestle and liquid nitrogen. RNA was extracted from ground roots using the Qiagen RNeasy PowerSoil Total RNA Kit (Qiagen, Carlsbad, CA, USA), followed by DNase treatment with TURBO DNase (Thermo Fisher Scientific, Waltham, USA), and then by a column cleanup with the Qiagen RNeasy PowerClean Pro Kit. RNA samples were tested for DNA contamination by amplification of the LSU gene with the primers LR0R and LR3 primers (Vilgalys & Hester, 1990 ) and those that had DNA contamination were subjected to additional DNase treatments until they failed to amplify. RNA samples were reverse transcribed into cDNA using the reverse primer LR5 (Vilgalys & Hester, 1990 ) and the Qiagen Omniscript RT Kit. cDNA was amplified using a two-step PCR barcoding approach with the primers LR0R and LR5 and the Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, USA). Amplicons were submitted to the Duke Sequencing Center for library preparation, pooling, and sequencing on a PacBio Sequel. Root staining and colonization scoring The fine root samples that were collected and preserved in 10% KOH were used to quantify the degree of root colonization by EM, AM, and endophytic fungi using the ink-vinegar staining method (Vierheilig et al., 1998 ) followed by scoring of colonization using the magnified intersections method (McGonigle et al., 1990 ). For staining, we extended the KOH clearing step to 1 to 2 hours (judged by visually checking pigmentation of roots) because of the high levels of pigmentation in the roots. After destaining, roots were cut into 1 cm segments and mounted in vinegar on a microscope slide. The roots were scored for a minimum of 100 root intersections. Each root intersection was scored using the following categories: ectomycorrhizal root tip, AM vesicles, AM hyphal coils, arbuscules, AM hyphae, microsclerotia, endophytic hypha, or uncolonized. Total AM colonization was calculated by summing together vesicle, hyphal coil, arbuscule, and AM hyphal colonization. Total endophytic colonization was calculated by summing together microsclerotia and endophytic hyphal colonization. Sequence Analysis and Statistics PacBio LSU sequencing data were analyzed in QIIME2 version 2024.2.0 (Bolyen et al., 2019 ). CCS sequences were denoised, trimmed of primers, and chimera-checked with Dada2 (Callahan et al., 2016 ). Taxonomy was assigned to ASV (Amplicon sequence variant) sequences using the classify-consensus-blast command in QIIME2 (Bokulich et al., 2018 ) against a database of NCBI eukaryotic LSU sequences. Taxonomic assignments were reviewed for the 200 most abundant ASVs and other seemingly erroneous taxonomic assignments (notably ASVs assigned as Terfezia and Alnicola , which were erroneous) by manually examining BLAST results and adjusted taxonomic assignments when necessary (see Supplemental File S1 for taxonomy edits). Sequence data were rarefied to 816 sequences per sample and samples with fewer sequences than this were excluded from further analysis. The diversity of most samples had saturated at this rarefaction depth. Fungal ASVs were assigned to guilds based on the FUNGuild database (Nguyen et al., 2016 ) along with manual annotation of known endophytes (see Supplemental File S1). We also conducted Sanger sequencing of some visibly colonized EM root tips by first extracting in a solution of Tris, EDTA, and KCl ph 9.5, followed by amplification with the primers 5.8SR and LR3, and submission for Sanger sequencing by Eurofins USA. We tested for differences in guild colonization using both microscopic colonization scoring data and sequencing data using ANOVAs with square root transformations of response variables to meet assumptions of normality. We identified individual fungal genera that varied between conditions ( Populus species or plant age) by 1) first identifying genera were entirely absent from one condition and present in at least samples in the other conditions and 2) running ANCOMBC-2 tests with false discovery rate p-value corrections to identify differences in sequence abundance of remaining genera – genera were considered differentially abundance if they either met the presence/absence filter or had a significant ANCOMBC-2 result. Data were visualized using the ggplot2 package (Wickham & Sievert, 2009 ) in R (R Core Team, 2013 ). Results Overview of fungal communities We detected 742 ASVs (Amplicon sequence variants) that were distributed across 73 fungal genera with LSU RNA metabarcoding. The root-associating fungal communities had generally low diversity and showed a decrease in diversity through time from 29.6 ± 10.4 ASVs at 5 months of growth down to 14.9 ± 4.7 ASVs at 12 months of growth ( P < 0.0001). Fungal richness was slightly higher on P. trichocarpa than on P. tremuloides at both timepoints ( P = 0.04). Many of the most abundant ASVs were from known Populus endophytes including Hyaloscypha spp. and Ilyonectria spp (Bonito et al., 2016 ). The most abundant EM ASVs were Hebeloma spp., Tuber spp., Sphaerosporella spp., and Cenococcum geophilum . We also observed high abundance of the biotrophic genera Ceratobasidium , Serendipita , Flagelloscypha , and Tulasnella , though it is unclear what type of symbiotic interaction these taxa form with Populus . Distinct EM morphotypes were observed on plant roots that could be identified in some cases by Sanger sequencing (see Fig. 1 a, 1 b). We also documented fungal endophytic structures such as microsclerotia, although we are unable to link specific endophytic structures with taxonomic information (see Fig. 1 c). We documented AM colonization based on the presence of AM hyphae, arbuscules, vesicles, and hyphal coils (see Fig. 1 d-f). Patterns in guild abundance EM and AM colonization on both Populus hosts increased with plant age (confirmed by microscopy and metabarcoding), while endophytic colonization decreased with age (confirmed by microscopy but not metabarcoding; see Fig. 2 ). Based on microscopy, the rate of EM fungal recruitment seemed to be faster for P. tremuloides than P. trichocarpa , with greater EM colonization on P. tremuloides than P. trichocarpa at 5 months (Wilcoxon test, P = 0.045), but equal EM colonization between hosts by the 12 month timepoint (although this pattern was not confirmed by LSU metabarcoding). The two hosts had similar endophytic colonization at 5 months, but at 12 months P. tremuloides had higher endophytic colonization than P. trichocarpa based on microscopy, although this was not reflected in the sequencing dataset (see Fig. 2 ). The vast majority (> 90%) of the endophytic sequence abundance was attributable to dark septate endophytic fungi in the order Helotiales. Among taxa in the Helotiales, Hyaloscypha was the most abundant. P. trichocarpa had substantial AM colonization based on microscopy (24% ± 29%; see Fig. 2 a), but modest sequence abundances (3.6% ± 5.3%; see Fig. 2 b). P. tremuloides was entirely devoid of AM colonization, which was confirmed by both sequencing and microscopy. Most of the AM colonization on P. trichocarpa was attributable to hyphal colonization (13.2% ± 15.6% root length colonized), while vesicles (8% ± 15.3%), arbuscules (2.4% ± 3.3%), and hyphal coils (0.49% ± 0.81%) were observed at lower rates. On P. trichocarpa , 41% of plants were dual-mycorrhizal, 35% of plants were entirely AM, 21% were entirely EM, and 3% were non-mycorrhizal based on microscopic colonization scoring. Dual-mycorrhizal P. trichocarpa plants had a weak tendency to be taller than entirely AM plants at 12 months plant age, but the effect was not significant ( P = 0.097). Early and late colonizing species The genera Mycena , Diaporthe , Polyphilus , Solicoccozyma , Trichocladium , Cenococcum , Aquilomyces , Exophiala , and Tulasnella were frequent colonizers on 5 month old plants, but then declined in abundance or disappeared entirely on the 12 month old plants (see Fig. 3 ). In the case of Cenococcum , this shift was readily observable during microscopic examination of the roots due to their easily identifiable black ectomycorrhizae (personal observation, see Fig. 1 b). The only genus that increased in abundance with plant age was Sphaerosporella , but this may have been an airborne contaminant because it is a common environmental contaminant and was observed fruiting in one pot before quickly spreading to other pots (confirmed by Sanger sequencing of fruiting bodies and root tips) shortly before the 12 month sampling timepoint. The AM fungal genera Glomus and Rhizophagus occurred on P. trichocarpa but not on P. tremuloides and were the only fungal genera that displayed plant host preference. Both Glomus and Rhizophagus seemed to increase in abundance with plant age on P. trichocarpa (see Fig. 3 ), but this pattern was not significant. Discussion We hypothesized that both Populus spp. would behave as temporally dependent dual-mycorrhizal species and exhibit a transition from AM to EM colonization during the first year of growth. We found little evidence for the hypothesized pattern of mycorrhizal switching in either of the Populus spp. examined. Both species of Populus demonstrated an increase in EM colonization and a decrease in endophytic colonization with plant age. Only P. trichocarpa was colonized by AM fungi, which increased in abundance with plant age. These observations suggest that both EM and AM colonzation may continue increasing beyond the first year of growth in the Populus spp. examined and it is not known if or when a decline in AM colonization would occur. Studies on other plant hosts have found that mycorrhizal switching can occur on timescales ranging from 3 to 16 months (Chen et al., 2000 ; Dickie et al., 2001 ; Santos et al., 2001), while an early study of European Populus spp. suggests that AM colonization can be maintained during the first two years of growth (Dominik, 1958 ). A time-series study of the first 50 days of growth in Populus tremula x alba cuttings in pot culture found a steady increase in both EM and endophytic colonization (Fracchia et al., 2021 ), while another study in a Populus plantation found a an increase in EM colonization during the first two years of growth (Argiroff et al., 2024 ). Collectively, these results show that colonization by AM, EM, and endophytic fungi are dynamic through time and these different guilds may peak in abundance at different periods of plant development. In addition to temporal patterns in guild abundance, we also identified specific early-colonizing fungal genera that declined in abundance or disappeared entirely from the root system by 12 months. This list included the genera Mycena , Diaporthe , Polyphilus , Solicoccozyma , Trichocladium , Cenococcum , Aquilomyces , Exophiala , and Tulasnella . The EM fungus Cenococcum has been previously documented as an early fungal colonizer on Populus (Dominik, 1958 ), although others have found extensive colonization by Cenococcum in mature stands (Vélez et al., 2021 ; Nash et al., 2025 ). This group of early-colonizing fungi includes taxa that are notable for having high saprotrophic capabilities, despite their lifestyles as plant biotrophs. Tulasnella is a genus of orchid mycorrhizal fungi and endophytes that has strong plant cell degradative capabilities (Adamo et al., 2020 ). Mycena spp. have been classically considered to be a free-living saprotrophs, but recently have been documented as potential endophytes in healthy living roots and some species may transfer nutrients to plants (Thoen et al., 2020 ; Harder et al., 2023 ). Trichocladium , Exophiala , Polyphilus and Aquilomyces are all dark septate endophytic genera (Knapp et al., 2015 ; Ashrafi et al., 2018 ) which have been linked to high saprotrophic capabilities (Caldwell et al., 2000 ). Our findings suggest that this saprotrophic capability may be a key trait enabling fungal symbionts to quickly colonize plants, but may not be associated with long-term persistence of fungal symbionts as plants age. Fracchia et al. ( 2021 ) found similar results in a time-series study of Populus on a much shorter timescale, with saprotrophic fungi being very abundant on roots in the first few days of growth. Dark septate endophytes are understudied, but some experimental work shows that they can transfer nutrients to plants (Almario et al., 2017 ; Xu et al., 2020 ) and thus may represent an important nutrient acquisition symbiosis during early plant growth. In this study, the dark septate endophytic genus Hyaloscypha was found to be one of the most prolific colonizers on both Populus spp. throughout the growth period. Past studies have identified both Hyaloscypha and Exophiala as being core members of the Populus root fungal community (Shakya et al., 2013 ; Nash et al., 2025 ). A past culture-based study of Populus root fungal symbionts found that endophytes were among the most common culturable members of root communities (Bonito et al., 2016 ), with frequent isolations of Ilyonectria , Hyaloscypha , and Exophiala which were abundant in our dataset. Despite the growing body of work showing that dark septate endophytes are prolific colonizers on dual-mycorrhizal hosts (including Populus ), many mycorrhizal studies still overlook the role of this guild in studies of plant nutrition and stress tolerance. The temporal dynamics of fungal endophytic colonization on root systems that are co-colonized by AM and EM fungi remains an underexplored area of research. Fungal alpha diversity decreased by half from 5 months to 12 months of plant growth. The pot culture method that we employed did not allow for recruitment of new fungal symbionts by dispersal and is thus somewhat unrealistic compared to natural conditions where continuous dispersal allows for recruitment of new symbionts over the lifespan of a tree. However, this time-dependent decrease in alpha diversity suggests that in the absence of dispersal, root associated fungal communities experience loss of species diversity as plants age. Mycorrhizal fungal diversity is thought to promote plant growth and higher rates of nutrient transfer to plants (Nash et al., 2020 ; Anthony, 2025 ) and it is unknown how the observed decrease in fungal diversity with plant development affected the quality of mycorrhizal benefits provided to the plant hosts. Our study utilized a common garden approach to isolate the role of Populus host identity on structuring root-associated fungal communities without the confounding influence of environmental variation. The two Populus spp. examined exhibited distinct preferences for AM and EM colonization, suggesting that they exist along a spectrum of dual-mycorrhizal colonization. P. trichocarpa displayed moderate levels of AM colonization, while P. tremuloides showed very little evidence for AM colonization but had higher EM colonization rates as determined by microscopy (though similar levels based on LSU sequencing). The findings regarding AM colonization of P. tremuloides are consistent with other studies showing that the species is a poor AM host (Kaldorf et al., 2002 ; Karst et al., 2021 ). However, a microscopy-based study on mature P. tremuloides found an average AM colonization rate of 6% (Neville et al., 2002 ) and another molecular-based study found modest AM association with P. tremuloides (Nash et al., 2025 ). Interestingly, P. tremuloides x P. tremula hybrids have been shown to exhibit greatly decreased AM colonization in response to nitrogen fertilization, while AM colonization of P. trichocarpa was unaffected and so the usage of an inorganic fertilizer in our experiment may have prevented AM colonization of P. tremuloides (Baum & Makeschin, 2000 ). Thus, P. tremuloides should still be assumed to be dual-mycorrhizal despite the lack of evidence for AM colonization in this study. The higher rate of EM colonization on P. tremuloides than P. trichocarpa that we found is consistent with a continental-scale comparison of five Populus species (Van Nuland et al., 2023 ). These findings suggest that previously observed differences in mycorrhizal colonization among Populus spp. (Van Nuland et al., 2023 ) are not solely the result of environmental variation and fungal biogeography, but are also due differences in host preference for AM and EM colonization. Although we found stark host-dependent differences in mycorrhizal colonization between the two Populus spp., we found few individual fungal taxa that differed in abundance between the two hosts. Additional controlled common garden experiments are needed that use a wider phylogenetic diversity of Populus species planted with diverse soil inocula to advance our knowledge of fungal host-specificity in the Populus genus. Our experiment made use of a simplified growth chamber environment to isolate the effects of hosts species identity and plant age on root fungal colonization, but there are still many unresolved questions about the impact of environmental context on determining dual-mycorrhizal colonization during early plant development and the mechanisms that underlie mycorrhizal switching. The density and proximity of adult mycorrhizal hosts may influence colonization dynamics of seedlings due to hyphal spread (Dickie et al., 2001 ). Soil moisture levels can shift the balance between AM and EM fungi and may alter the rate of microbiome development (Lodge, 1989 ; Xu et al., 2018 ). The long-term trajectories of dual-mycorrhizal colonization is also likely influenced by the ability of mycorrhizal symbionts to persist during winter when plant carbon availability is low and fungal symbionts may experience freezing. EM fungi are more resistant to soil freezing than AM fungi and successive freeze-thaw cycles may result in a decline in AM colonization over time on dual-mycorrhizal hosts (Kilpeläinen et al., 2016 ). Additionally, Lodge and Wentworth ( 1990 ) have documented potential antagonism between EM and AM fungi in the same root system, which could result in transitions between these guilds if one of them gains a competitive advantage. In our study, AM and EM colonization increased in tandem as plants aged while endophytic colonization decreased, hinting at a possible unexplored role of competition with endophytic fungi in structuring Populus root-associated fungal communities. Changes in host metabolism through plant development are likely also strong controls on fungal community composition (Xie et al., 2023 ; Fracchia et al., 2024 ). To conclude, our study demonstrates that plant age-dependent changes in root fungal community composition in Populus are distinct from patterns observed in other dual-mycorrhizal hosts and demonstrates that there is variation in association with AM fungi among different Populus spp. We show that saprotrophic capabilities may be an important trait of early colonizing fungal endophytes. Our findings indicate the potential for diverse developmental trajectories in dual-mycorrhizal hosts and stress the importance of examining endophytic fungal communities in addition to mycorrhizal fungi. Developmentally induced shifts in root-associated fungal communities likely have functional implications for rates of nutrient transfer and ecosystem processes that provide an exciting avenue for future research. Declarations The authors have no relevant financial or non-financial interests to disclose Author Contribution J.N. designed the experiment, conducted the experiment, analyzed the data, and wrote the paper. B.L. designed the experiment and conducted the experiment. M.A.C. designed the experiment. C.W.S. designed the experiment. R.V. Designed the experiment. All authors reviewed the manuscript. Acknowledgement We greatly appreciate the assistance of the Duke University Phytotron Facility in conducting this research. We received tremendous intellectual support and advice from the scientists in the Plant Microbe Interfaces Science Focus Area at Oak Ridge National Laboratory. We thank the managers of the Fishlake National Forest for allowing us to sample soil from their lands. This research was funded by the U.S. Department of Energy Office of Biological and Environmental Research, Genomic Science Program as part of the Plant Microbe Interfaces Science Focus (http://pmi.ornl.gov). Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. Data Availability Code underlying the analyses presented in this paper is available at https://github.com/jakenash12/PopulusSuccession. All data underlying the analyses presented in this paper are included in Supplemental File S1. Representative sequences for OTUs sequenced for this project will also be made available on GenBank upon acceptance of this paper for permanent data deposition. 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Mycorrhiza 8:247–253. https://doi.org/10.1007/s005720050241 Nash J, Laushman R, Schadt C (2020) Ectomycorrhizal fungal diversity interacts with soil nutrients to predict plant growth despite weak plant-soil feedbacks. Plant Soil 453:445–458. https://doi.org/10.1007/s11104-020-04616-y Nash J, Tremble K, Schadt C, Cregger M, Bryan C, Vilgalys R (2025) Time-series RNA metabarcoding of the active Populus tremuloides root microbiome reveals hidden temporal dynamics and dormant core members. bioRxiv:2025.2002. 2019.639079. https://doi.org/10.1101/2025.02.19.639079 Neville J, Tessier J, Morrison I, Scarratt J, Canning B, Klironomos J (2002) Soil depth distribution of ecto-and arbuscular mycorrhizal fungi associated with Populus tremuloides within a 3-year-old boreal forest clear-cut. Appl Soil Ecol 19:209–216. https://doi.org/10.1016/S0929-1393(01)00193-7 Nguyen NH, Song Z, Bates ST, Branco S, Tedersoo L, Menke J, Schilling JS, Kennedy PG (2016) FUNGuild: an open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecol 20:241–248. https://doi.org/10.1016/j.funeco.2015.06.006 Piotrowski J, Morford S, Rillig M (2008) Inhibition of colonization by a native arbuscular mycorrhizal fungal community via Populus trichocarpa litter, litter extract, and soluble phenolic compounds. Soil Biol Biochem 40:709–717. https://doi.org/10.1016/j.soilbio.2007.10.005 Querejeta J, Egerton-Warburton LM, Allen MF (2009) Topographic position modulates the mycorrhizal response of oak trees to interannual rainfall variability. Ecology 90:649–662. https://doi.org/10.1890/07-1696.1 Core Team R (2013) R. R: A language and environment for statistical computing Santos VLd, Muchovej RM, Borges AC, Neves JCL, Kasuya MCM (2001) Vesicular-arbuscular-/ecto-mycorrhiza succession in seedlings of Eucalyptus spp. Braz J Microbiol 32:81–86. https://doi.org/10.1590/S1517-83822001000200002 Shakya M, Gottel N, Castro H, Yang ZK, Gunter L, Labbé J, Muchero W, Bonito G, Vilgalys R, Tuskan G (2013) A multifactor analysis of fungal and bacterial community structure in the root microbiome of mature Populus deltoides trees. PLoS ONE 8:e76382. https://doi.org/10.1371/journal.pone.0076382 Teste FP, Jones MD, Dickie IA (2020) Dual-mycorrhizal plants: their ecology and relevance. New Phytol 225:1835–1851. https://doi.org/10.1111/nph.16190 Thoen E, Harder CB, Kauserud H, Botnen SS, Vik U, Taylor AF, Menkis A, Skrede I (2020) In vitro evidence of root colonization suggests ecological versatility in the genus Mycena . New Phytol 227:601–612. https://doi.org/10.1111/nph.16545 Van Nuland ME, Daws SC, Bailey JK, Schweitzer JA, Busby PE, Peay KG (2023) Above-and belowground fungal biodiversity of Populus trees on a continental scale. Nat Microbiol 8:2406–2419. https://doi.org/10.1038/s41564-023-01514-8 Vélez JM, Morris RM, Vilgalys R, Labbé J, Schadt CW (2021) Phylogenetic diversity of 200 + isolates of the ectomycorrhizal fungus Cenococcum geophilum associated with Populus trichocarpa soils in the Pacific Northwest, USA and comparison to globally distributed representatives. PLoS ONE 16:e0231367. https://doi.org/10.1371/journal.pone.0231367 Vierheilig H, Coughlan AP, Wyss U, Piché Y (1998) Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl Environ Microbiol 64:5004–5007. https://doi.org/10.1128/AEM.64.12.5004-5007.1998 Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 Wang M, Zhang L, Zhang Z, Li M, Wang D, Zhang X, Xi Z, Keefover-Ring K, Smart LB, DiFazio SP (2020) Phylogenomics of the genus Populus reveals extensive interspecific gene flow and balancing selection. New Phytol 225:1370–1382. https://doi.org/10.1111/nph.16215 Watson GW, von der Heide-Spravka KG, Howe VK (1990) Ecological significance of endo-/ectomycorrhizae in the oak sub-genus Erythrobalanus . Arboricultural J 14:107–116. https://doi.org/10.1080/03071375.1990.9746833 Wickham H, Sievert C (2009) ggplot2: elegant graphics for data analysis. springer New York Xie J, Ma Y, Li X, Wu J, Martin F, Zhang D (2023) Multifeature analysis of age-related microbiome structures reveals defense mechanisms of Populus tomentosa trees. New Phytol 238:1636–1650. https://doi.org/10.1111/nph.18847 Xu L, Naylor D, Dong Z, Simmons T, Pierroz G, Hixson KK, Kim Y-M, Zink EM, Engbrecht KM, Wang Y (2018) Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria. Proc Natl Acad Sci 115:E4284–E4293. https://doi.org/10.1073/pnas.1717308115 Xu R, Li T, Shen M, Yang ZL, Zhao Z-W (2020) Evidence for a dark septate endophyte ( Exophiala pisciphila , H93) enhancing phosphorus absorption by maize seedlings. Plant Soil 452:249–266. https://doi.org/10.1007/s11104-020-04538-9 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6531386","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":449181990,"identity":"55015cb9-91a5-42ca-9c3b-aa31c1723083","order_by":0,"name":"Jake Nash","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACxgYG9g8fKpgZJEA8HiK1sDHOOEOKFiBgY+ZtI0ULc/vZYw9nzrNOnNnewPjgbRsxDuvJSzf4uC09cTbPAWbDuURpacgxkJy57XDiPIkENmleorT0vzGQ5p0D1CL/gP03cVpm5JhJ8zYcTpwtAQ4HorS8MTaccSzdeGZPYrPknHNEaDHszzF88KHGWnbG8cMHP7wpI0ZLA8LCBpyqUIA8ccpGwSgYBaNgRAMAJIk35Zni8JEAAAAASUVORK5CYII=","orcid":"","institution":"Duke University","correspondingAuthor":true,"prefix":"","firstName":"Jake","middleName":"","lastName":"Nash","suffix":""},{"id":449181995,"identity":"2250798d-3f2f-4306-acf1-9c7eeeaa8332","order_by":1,"name":"Brian Looney","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"prefix":"","firstName":"Brian","middleName":"","lastName":"Looney","suffix":""},{"id":449182001,"identity":"7a90990c-fd74-4aa2-beb0-3ec8bb6eb1e2","order_by":2,"name":"Melissa A. Cregger","email":"","orcid":"","institution":"Oak Ridge National Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Melissa","middleName":"A.","lastName":"Cregger","suffix":""},{"id":449182003,"identity":"263289c6-2568-4e63-a837-2103535c0f80","order_by":3,"name":"Christopher Schadt","email":"","orcid":"","institution":"Oak Ridge National Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Schadt","suffix":""},{"id":449182005,"identity":"8386e3c5-bf39-48f9-b444-45fdcb98d3f7","order_by":4,"name":"Rytas Vilgalys","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"prefix":"","firstName":"Rytas","middleName":"","lastName":"Vilgalys","suffix":""}],"badges":[],"createdAt":"2025-04-25 20:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6531386/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6531386/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00572-025-01215-6","type":"published","date":"2025-06-09T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81630054,"identity":"bf96382a-0803-4416-99eb-ddc037c282b3","added_by":"auto","created_at":"2025-04-29 11:17:30","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":549468,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of fungal colonization observed throughout the experiment. (a) an ectomycorrhiza of \u003cem\u003eTuber anniae\u003c/em\u003e, (b) ectomycorrhizae of \u003cem\u003eCenococcum geophilum \u003c/em\u003eobserved on 5 month old plants, (c) endophytic microsclerotia, (d) arbuscular mycorrhizal arbuscules, (e) arbuscular mycorrhizal vesicles, (f) arbuscular mycorrhizal hyphal coils\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6531386/v1/13b7f7187c5b80aec7d55d28.jpeg"},{"id":81629837,"identity":"71cdcb97-aa6d-4a6d-a7d9-d7d1128f8fc4","added_by":"auto","created_at":"2025-04-29 11:09:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":109223,"visible":true,"origin":"","legend":"\u003cp\u003eStacked bar plots indicating the (a) relative root colonization and (b) relative sequence abundance of arbuscular mycorrhizal fungi (AM), ectomycorrhizal fungi (EM), and endophytic fungi (Endo). For the microscopic colonization scoring data (a), the uncolonized category refers to the proportion of root length that was uncolonized. For the sequencing dataset (b), fungi that were not annotated as plant symbiotic taxa were grouped into the “other” category. (c) Significance of differences in guild abundance by plant age and species identity is displayed. Asterisks in the top left corner of each box indicate significance determined by microscopy and those in the lower right corner indicate significance determined by LSU RNA metabarcoding\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6531386/v1/477b2839f9600e7d829ce865.png"},{"id":81629838,"identity":"55ef0615-e7bc-41c7-b8a1-e6cce47a48b7","added_by":"auto","created_at":"2025-04-29 11:09:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50846,"visible":true,"origin":"","legend":"\u003cp\u003eA heatmap showing the abundance of fungal genera that differed either by plant age or plant host species. Columns represent individual samples and locations in white represent the absence of that genus in a sample. Sequence abundance of each genus was scaled between 0 and 1 to allow for convenient visualization\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6531386/v1/e2051c9ddb290c2957770207.png"},{"id":84726442,"identity":"91bc0fc5-76c1-4562-a8cc-781ee373ff1c","added_by":"auto","created_at":"2025-06-16 16:03:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1337023,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6531386/v1/0ba3d312-a053-4f82-9495-65e56ca45868.pdf"},{"id":81629831,"identity":"5267b3c9-d3a4-4b8e-ae3d-6ccdf8159a97","added_by":"auto","created_at":"2025-04-29 11:09:30","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":361062,"visible":true,"origin":"","legend":"","description":"","filename":"PAMB1SupplementalS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6531386/v1/d8e21b23a93671bdc06632c3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual-mycorrhizal colonization is determined by plant age and host identity in two species of Populus","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants have evolved symbioses with diverse mycorrhizal fungal symbionts that serve perform diverse functions in the plant root environment, including nutrient transfer and protection from abiotic and biotic stress (Behie \u0026amp; Bidochka, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Branco et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The two most common guilds of mycorrhizal fungal symbionts are arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi, which colonize 72% and 2% of vascular plants respectively (Brundrett \u0026amp; Tedersoo, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Although associations with both of these groups of fungi have evolved in multiple plant lineages, the mycorrhizal type of plants is generally conserved within the same genus, family, or order, suggesting some degree of phylogenetic conservation (Brundrett \u0026amp; Tedersoo, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While vascular plants associate with a single guild of mycorrhizal fungi, there are plants termed \u0026ldquo;dual-mycorrhizal\u0026rdquo; that can associate with both AM and EM fungi. Although this group of dual-mycorrhizal plants is relatively small, estimated at between 1500 to 7300 plant species or \u0026lt;\u0026thinsp;3% of flowering plants (Brundrett \u0026amp; Tedersoo, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Teste et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), it includes dominant trees on multiple continents, such as \u003cem\u003ePopulus\u003c/em\u003e spp. and \u003cem\u003eEucalyptus\u003c/em\u003e spp. While dual-mycorrhizal associations are often conserved within the same plant genus or family, there have been relatively few studies testing for differences in the extent of AM and EM colonization within individual dual-mycorrhizal plant lineages.\u003c/p\u003e \u003cp\u003eSome dual-mycorrhizal plants can engage in mycorrhizal switching in which they shift towards AM or EM colonization in response to environmental or plant cues (Teste et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Mycorrhizal switching has been documented in response to topographic position, seasonal drought, soil age, habitat type, and geographic range (Watson et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Moyersoen \u0026amp; Fitter, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Querejeta et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Albornoz et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Van Nuland et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Plant development has also been implicated as a trigger for mycorrhizal switching on some dual-mycorrhizal hosts, with a frequently observed transition from high AM colonization in young plants, towards increasing EM colonization as plants age (Chilvers et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Dickie et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Santos et al., 2001). Teste et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) proposed the classification of such plants as \u0026ldquo;temporally dependent dual-mycorrhizal hosts\u0026rdquo;. Preferential association with AM fungi at the seedling stage is hypothesized to facilitate seedling establishment (Teste et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While some temporally dependent dual-mycorrhizal hosts retain low levels of AM colonization throughout development, in extreme cases hosts can completely lose AM colonization and become entirely EM after approximately one year (de Mendon\u0026ccedil;a Bellei et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Although studies of dual-mycorrhizal plants have hinted at variability in the degree of temporal-dependence, there have been few systematic studies comparing successional trajectories on closely related hosts.\u003c/p\u003e \u003cp\u003eIn this study, we focus on the dual-mycorrhizal tree genus \u003cem\u003ePopulus\u003c/em\u003e to test the effects of plant host species identity and plant age on AM and EM colonization. Trees in the genus \u003cem\u003ePopulus\u003c/em\u003e have been widely used in plant-microbe interactions research and have been proposed as model dual-mycorrhizal systems (Cregger et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Karst et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although there have been studies documenting variation in AM and EM colonization in \u003cem\u003ePopulus\u003c/em\u003e in response to tree genotype, temperature, soil moisture, soil depth, seasonality, heavy metal contamination, and leaf litter extract application (Lodge, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Neville et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Gehring et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Piotrowski et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Karliński et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Van Nuland et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Nash et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), there has been very little work examining AM and EM colonization throughout plant development and it is unclear if \u003cem\u003ePopulus\u003c/em\u003e species should be classified as temporally dependent dual-mycorrhizal hosts (Dominik, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1958\u003c/span\u003e; Fracchia et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although the composition of the fungal and bacterial microbiome of \u003cem\u003ePopulus\u003c/em\u003e is known to change with plant age (Fracchia et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Argiroff et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), very few studies have tracked changes in AM and EM colonization through plant development on \u003cem\u003ePopulus\u003c/em\u003e (Dominik, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1958\u003c/span\u003e; Dove et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fracchia et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, it is not known whether \u003cem\u003ePopulus\u003c/em\u003e species undergo a shift from AM colonization to EM colonization as plants age as has been demonstrated on other temporally dependent dual-mycorrhizal species (Teste et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The \u003cem\u003ePopulus\u003c/em\u003e genus contains around 30 species, distributed across six sections, which are all presumably dual-mycorrhizal (Eckenwalder, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These species occupy unique biogeographic distributions and occupy a range of habitats, including boreal forest, semi-arid shrublands, mixed hardwood forests, and riparian zones. A DNA metabarcoding and microscopy-based field survey of five species of \u003cem\u003ePopulus\u003c/em\u003e in North America found a gradient of EM and AM colonization levels with \u003cem\u003eP. angustifolia\u003c/em\u003e and \u003cem\u003eP. tremuloides\u003c/em\u003e having the highest EM colonization and \u003cem\u003eP. deltoides\u003c/em\u003e having the highest AM colonization (though \u003cem\u003eP. tremuloides\u003c/em\u003e had the highest AM fungal richness). Our study makes use of a pot culture common garden approach with a field sampled soil inoculum to isolate the effect of plant species identity on mycorrhizal guild abundance during the first year of growth on the species \u003cem\u003eP. tremuloides\u003c/em\u003e and \u003cem\u003eP. trichocarpa\u003c/em\u003e. We used metabarcoding and root staining/microscopy to profile fungal colonization on both \u003cem\u003ePopulus\u003c/em\u003e spp. at 5 and 12 months of plant growth. We used RNA metabarcoding over DNA based methods, as we have previously found it to recover AM sequences at much higher levels (Nash et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). We hypothesized that 1) both \u003cem\u003ePopulus\u003c/em\u003e hosts will be temporally dependent dual-mycorrhizal species, 2) \u003cem\u003eP. tremuloides\u003c/em\u003e will display a preference for EM over AM colonization compared to \u003cem\u003eP. trichocarpa\u003c/em\u003e which will have greater AM colonization, and 3) in addition to guild-wide patterns, there will be consistently early and late colonizing species.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOverview of methods\u003c/h2\u003e \u003cp\u003eWe inoculated 20 \u003cem\u003eP. tremuloides\u003c/em\u003e and 20 \u003cem\u003eP. trichocarpa\u003c/em\u003e seedlings with field soils sampled from \u003cem\u003eP. tremuloides\u003c/em\u003e stands in southern Utah, along with four uninoculated controls of each species and grew them in pots for 12 months in a controlled growth chamber at Duke University\u0026rsquo;s Phytotron facility. Fine roots were collected from each plant at 5 and 12 months and used for ink-vinegar staining and LSU RNA metabarcoding to measure colonization levels of AM, EM, and endophytic fungi as well as sequence abundance of individual taxa.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInoculum collection and plant growth conditions\u003c/h3\u003e\n\u003cp\u003eIn September 2021, we collected 20 soil samples from the top 15 cm of soil in four \u003cem\u003eP. tremuloides\u003c/em\u003e stands in Fishlake National Forest, Utah, United States. Soils were shipped back to Duke University and kept at 4\u0026deg;C for four months until inoculation. One month prior to inoculation, \u003cem\u003eP. tremuloides\u003c/em\u003e and \u003cem\u003eP. trichocarpa\u003c/em\u003e seeds were sewn into nursery trays with autoclaved vermiculite and grown covered under fluorescent lights until inoculation. A sterile potting mix was prepared by mixing vermiculite, perlite, peat, and sand at a 2:2:1:1 ratio and autoclaving twice at 121\u0026deg;C for two hours with two days in between each autoclave cycle. We inoculated one seedling of each plant host with each of the 20 soil inocula at a ratio of 1 part soil inoculum to 2 parts sterile potting mix to maintain biological replication and capture the natural heterogeneity of soil inocula. For some soil samples, we had to decrease this ratio because of a low quantity of inoculum, though inoculum levels were always the same between the two hosts to avoid systemic bias and we found no impact of inoculum levels on alpha or beta diversity metrics. Seedlings were planted into 2.7\u0026rdquo; diameter by 10\u0026rdquo; deep conical tree pots until 6 months of age at which point they were transplanted into 5\u0026rdquo; by 5\u0026rdquo; by 12\u0026rdquo; square tree pots filled with the sterile potting mix to prevent root binding. Plants were grown in a controlled growth chamber with a 16 h day/ 8 h night light cycle with a light intensity of approximately 400 umol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 60% humidity, 23\u0026deg; C daytime temperature, and 18\u0026deg; C night temperature. Plants were fertilized throughout the experiment by watering pots with half strength Hoagland\u0026rsquo;s solution until saturation once per week. Plant height was collected at the time of sampling (see following section) at both 5 and 12 months by measuring the distance from the soil line to the apical bud.\u003c/p\u003e\n\u003ch3\u003eSample collection and molecular methods\u003c/h3\u003e\n\u003cp\u003eAt 5 and 12 months of plant growth, seedlings were gently extricated from pots and fine root samples were clipped from plants non-destructively with dissecting scissors and a scalpel to allow the same plants to be resampled. Fine roots with tightly adhering soil were flash frozen in liquid nitrogen immediately on collection and stored at -80\u0026deg;C until RNA extraction. A separate subsample of fine roots was rinsed of all adhering soil and stored in 10% KOH for staining and microscopic colonization scoring. For RNA extraction, approximately 1 gram of root tissue was cryogenically ground with a mortar and pestle and liquid nitrogen. RNA was extracted from ground roots using the Qiagen RNeasy PowerSoil Total RNA Kit (Qiagen, Carlsbad, CA, USA), followed by DNase treatment with TURBO DNase (Thermo Fisher Scientific, Waltham, USA), and then by a column cleanup with the Qiagen RNeasy PowerClean Pro Kit. RNA samples were tested for DNA contamination by amplification of the LSU gene with the primers LR0R and LR3 primers (Vilgalys \u0026amp; Hester, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) and those that had DNA contamination were subjected to additional DNase treatments until they failed to amplify. RNA samples were reverse transcribed into cDNA using the reverse primer LR5 (Vilgalys \u0026amp; Hester, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) and the Qiagen Omniscript RT Kit. cDNA was amplified using a two-step PCR barcoding approach with the primers LR0R and LR5 and the Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, USA). Amplicons were submitted to the Duke Sequencing Center for library preparation, pooling, and sequencing on a PacBio Sequel.\u003c/p\u003e\n\u003ch3\u003eRoot staining and colonization scoring\u003c/h3\u003e\n\u003cp\u003eThe fine root samples that were collected and preserved in 10% KOH were used to quantify the degree of root colonization by EM, AM, and endophytic fungi using the ink-vinegar staining method (Vierheilig et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) followed by scoring of colonization using the magnified intersections method (McGonigle et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). For staining, we extended the KOH clearing step to 1 to 2 hours (judged by visually checking pigmentation of roots) because of the high levels of pigmentation in the roots. After destaining, roots were cut into 1 cm segments and mounted in vinegar on a microscope slide. The roots were scored for a minimum of 100 root intersections. Each root intersection was scored using the following categories: ectomycorrhizal root tip, AM vesicles, AM hyphal coils, arbuscules, AM hyphae, microsclerotia, endophytic hypha, or uncolonized. Total AM colonization was calculated by summing together vesicle, hyphal coil, arbuscule, and AM hyphal colonization. Total endophytic colonization was calculated by summing together microsclerotia and endophytic hyphal colonization.\u003c/p\u003e\n\u003ch3\u003eSequence Analysis and Statistics\u003c/h3\u003e\n\u003cp\u003ePacBio LSU sequencing data were analyzed in QIIME2 version 2024.2.0 (Bolyen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). CCS sequences were denoised, trimmed of primers, and chimera-checked with Dada2 (Callahan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Taxonomy was assigned to ASV (Amplicon sequence variant) sequences using the classify-consensus-blast command in QIIME2 (Bokulich et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) against a database of NCBI eukaryotic LSU sequences. Taxonomic assignments were reviewed for the 200 most abundant ASVs and other seemingly erroneous taxonomic assignments (notably ASVs assigned as \u003cem\u003eTerfezia\u003c/em\u003e and \u003cem\u003eAlnicola\u003c/em\u003e, which were erroneous) by manually examining BLAST results and adjusted taxonomic assignments when necessary (see Supplemental File S1 for taxonomy edits). Sequence data were rarefied to 816 sequences per sample and samples with fewer sequences than this were excluded from further analysis. The diversity of most samples had saturated at this rarefaction depth. Fungal ASVs were assigned to guilds based on the FUNGuild database (Nguyen et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) along with manual annotation of known endophytes (see Supplemental File S1). We also conducted Sanger sequencing of some visibly colonized EM root tips by first extracting in a solution of Tris, EDTA, and KCl ph 9.5, followed by amplification with the primers 5.8SR and LR3, and submission for Sanger sequencing by Eurofins USA.\u003c/p\u003e \u003cp\u003eWe tested for differences in guild colonization using both microscopic colonization scoring data and sequencing data using ANOVAs with square root transformations of response variables to meet assumptions of normality. We identified individual fungal genera that varied between conditions (\u003cem\u003ePopulus\u003c/em\u003e species or plant age) by 1) first identifying genera were entirely absent from one condition and present in at least samples in the other conditions and 2) running ANCOMBC-2 tests with false discovery rate p-value corrections to identify differences in sequence abundance of remaining genera \u0026ndash; genera were considered differentially abundance if they either met the presence/absence filter or had a significant ANCOMBC-2 result. Data were visualized using the \u003cem\u003eggplot2\u003c/em\u003e package (Wickham \u0026amp; Sievert, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) in R (R Core Team, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eOverview of fungal communities\u003c/h2\u003e \u003cp\u003eWe detected 742 ASVs (Amplicon sequence variants) that were distributed across 73 fungal genera with LSU RNA metabarcoding. The root-associating fungal communities had generally low diversity and showed a decrease in diversity through time from 29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4 ASVs at 5 months of growth down to 14.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 ASVs at 12 months of growth (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Fungal richness was slightly higher on \u003cem\u003eP. trichocarpa\u003c/em\u003e than on \u003cem\u003eP. tremuloides\u003c/em\u003e at both timepoints (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). Many of the most abundant ASVs were from known \u003cem\u003ePopulus\u003c/em\u003e endophytes including \u003cem\u003eHyaloscypha\u003c/em\u003e spp. and \u003cem\u003eIlyonectria\u003c/em\u003e spp (Bonito et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The most abundant EM ASVs were \u003cem\u003eHebeloma\u003c/em\u003e spp., \u003cem\u003eTuber\u003c/em\u003e spp., \u003cem\u003eSphaerosporella\u003c/em\u003e spp., and \u003cem\u003eCenococcum geophilum\u003c/em\u003e. We also observed high abundance of the biotrophic genera \u003cem\u003eCeratobasidium\u003c/em\u003e, \u003cem\u003eSerendipita\u003c/em\u003e, \u003cem\u003eFlagelloscypha\u003c/em\u003e, and \u003cem\u003eTulasnella\u003c/em\u003e, though it is unclear what type of symbiotic interaction these taxa form with \u003cem\u003ePopulus\u003c/em\u003e. Distinct EM morphotypes were observed on plant roots that could be identified in some cases by Sanger sequencing (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We also documented fungal endophytic structures such as microsclerotia, although we are unable to link specific endophytic structures with taxonomic information (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We documented AM colonization based on the presence of AM hyphae, arbuscules, vesicles, and hyphal coils (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePatterns in guild abundance\u003c/h3\u003e\n\u003cp\u003eEM and AM colonization on both \u003cem\u003ePopulus\u003c/em\u003e hosts increased with plant age (confirmed by microscopy and metabarcoding), while endophytic colonization decreased with age (confirmed by microscopy but not metabarcoding; see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on microscopy, the rate of EM fungal recruitment seemed to be faster for \u003cem\u003eP. tremuloides\u003c/em\u003e than \u003cem\u003eP. trichocarpa\u003c/em\u003e, with greater EM colonization on \u003cem\u003eP. tremuloides\u003c/em\u003e than \u003cem\u003eP. trichocarpa\u003c/em\u003e at 5 months (Wilcoxon test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045), but equal EM colonization between hosts by the 12 month timepoint (although this pattern was not confirmed by LSU metabarcoding). The two hosts had similar endophytic colonization at 5 months, but at 12 months \u003cem\u003eP. tremuloides\u003c/em\u003e had higher endophytic colonization than \u003cem\u003eP. trichocarpa\u003c/em\u003e based on microscopy, although this was not reflected in the sequencing dataset (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The vast majority (\u0026gt;\u0026thinsp;90%) of the endophytic sequence abundance was attributable to dark septate endophytic fungi in the order Helotiales. Among taxa in the Helotiales, \u003cem\u003eHyaloscypha\u003c/em\u003e was the most abundant. \u003cem\u003eP. trichocarpa\u003c/em\u003e had substantial AM colonization based on microscopy (24% \u0026plusmn; 29%; see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), but modest sequence abundances (3.6% \u0026plusmn; 5.3%; see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). \u003cem\u003eP. tremuloides\u003c/em\u003e was entirely devoid of AM colonization, which was confirmed by both sequencing and microscopy. Most of the AM colonization on \u003cem\u003eP. trichocarpa\u003c/em\u003e was attributable to hyphal colonization (13.2% \u0026plusmn; 15.6% root length colonized), while vesicles (8% \u0026plusmn; 15.3%), arbuscules (2.4% \u0026plusmn; 3.3%), and hyphal coils (0.49% \u0026plusmn; 0.81%) were observed at lower rates. On \u003cem\u003eP. trichocarpa\u003c/em\u003e, 41% of plants were dual-mycorrhizal, 35% of plants were entirely AM, 21% were entirely EM, and 3% were non-mycorrhizal based on microscopic colonization scoring. Dual-mycorrhizal \u003cem\u003eP. trichocarpa\u003c/em\u003e plants had a weak tendency to be taller than entirely AM plants at 12 months plant age, but the effect was not significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.097).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEarly and late colonizing species\u003c/h2\u003e \u003cp\u003eThe genera \u003cem\u003eMycena\u003c/em\u003e, \u003cem\u003eDiaporthe\u003c/em\u003e, \u003cem\u003ePolyphilus\u003c/em\u003e, \u003cem\u003eSolicoccozyma\u003c/em\u003e, \u003cem\u003eTrichocladium\u003c/em\u003e, \u003cem\u003eCenococcum\u003c/em\u003e, \u003cem\u003eAquilomyces\u003c/em\u003e, \u003cem\u003eExophiala\u003c/em\u003e, and \u003cem\u003eTulasnella\u003c/em\u003e were frequent colonizers on 5 month old plants, but then declined in abundance or disappeared entirely on the 12 month old plants (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the case of \u003cem\u003eCenococcum\u003c/em\u003e, this shift was readily observable during microscopic examination of the roots due to their easily identifiable black ectomycorrhizae (personal observation, see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The only genus that increased in abundance with plant age was \u003cem\u003eSphaerosporella\u003c/em\u003e, but this may have been an airborne contaminant because it is a common environmental contaminant and was observed fruiting in one pot before quickly spreading to other pots (confirmed by Sanger sequencing of fruiting bodies and root tips) shortly before the 12 month sampling timepoint. The AM fungal genera \u003cem\u003eGlomus\u003c/em\u003e and \u003cem\u003eRhizophagus\u003c/em\u003e occurred on \u003cem\u003eP. trichocarpa\u003c/em\u003e but not on \u003cem\u003eP. tremuloides\u003c/em\u003e and were the only fungal genera that displayed plant host preference. Both \u003cem\u003eGlomus\u003c/em\u003e and \u003cem\u003eRhizophagus\u003c/em\u003e seemed to increase in abundance with plant age on \u003cem\u003eP. trichocarpa\u003c/em\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e), but this pattern was not significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe hypothesized that both \u003cem\u003ePopulus\u003c/em\u003e spp. would behave as temporally dependent dual-mycorrhizal species and exhibit a transition from AM to EM colonization during the first year of growth. We found little evidence for the hypothesized pattern of mycorrhizal switching in either of the \u003cem\u003ePopulus\u003c/em\u003e spp. examined. Both species of \u003cem\u003ePopulus\u003c/em\u003e demonstrated an increase in EM colonization and a decrease in endophytic colonization with plant age. Only \u003cem\u003eP. trichocarpa\u003c/em\u003e was colonized by AM fungi, which increased in abundance with plant age. These observations suggest that both EM and AM colonzation may continue increasing beyond the first year of growth in the \u003cem\u003ePopulus\u003c/em\u003e spp. examined and it is not known if or when a decline in AM colonization would occur. Studies on other plant hosts have found that mycorrhizal switching can occur on timescales ranging from 3 to 16 months (Chen et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Dickie et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Santos et al., 2001), while an early study of European \u003cem\u003ePopulus\u003c/em\u003e spp. suggests that AM colonization can be maintained during the first two years of growth (Dominik, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1958\u003c/span\u003e). A time-series study of the first 50 days of growth in \u003cem\u003ePopulus tremula\u003c/em\u003e x \u003cem\u003ealba\u003c/em\u003e cuttings in pot culture found a steady increase in both EM and endophytic colonization (Fracchia et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), while another study in a \u003cem\u003ePopulus\u003c/em\u003e plantation found a an increase in EM colonization during the first two years of growth (Argiroff et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Collectively, these results show that colonization by AM, EM, and endophytic fungi are dynamic through time and these different guilds may peak in abundance at different periods of plant development.\u003c/p\u003e \u003cp\u003eIn addition to temporal patterns in guild abundance, we also identified specific early-colonizing fungal genera that declined in abundance or disappeared entirely from the root system by 12 months. This list included the genera \u003cem\u003eMycena\u003c/em\u003e, \u003cem\u003eDiaporthe\u003c/em\u003e, \u003cem\u003ePolyphilus\u003c/em\u003e, \u003cem\u003eSolicoccozyma\u003c/em\u003e, \u003cem\u003eTrichocladium\u003c/em\u003e, \u003cem\u003eCenococcum\u003c/em\u003e, \u003cem\u003eAquilomyces\u003c/em\u003e, \u003cem\u003eExophiala\u003c/em\u003e, and \u003cem\u003eTulasnella\u003c/em\u003e. The EM fungus \u003cem\u003eCenococcum\u003c/em\u003e has been previously documented as an early fungal colonizer on \u003cem\u003ePopulus\u003c/em\u003e (Dominik, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1958\u003c/span\u003e), although others have found extensive colonization by \u003cem\u003eCenococcum\u003c/em\u003e in mature stands (V\u0026eacute;lez et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nash et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This group of early-colonizing fungi includes taxa that are notable for having high saprotrophic capabilities, despite their lifestyles as plant biotrophs. \u003cem\u003eTulasnella\u003c/em\u003e is a genus of orchid mycorrhizal fungi and endophytes that has strong plant cell degradative capabilities (Adamo et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eMycena\u003c/em\u003e spp. have been classically considered to be a free-living saprotrophs, but recently have been documented as potential endophytes in healthy living roots and some species may transfer nutrients to plants (Thoen et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Harder et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eTrichocladium\u003c/em\u003e, \u003cem\u003eExophiala\u003c/em\u003e, \u003cem\u003ePolyphilus\u003c/em\u003e and \u003cem\u003eAquilomyces\u003c/em\u003e are all dark septate endophytic genera (Knapp et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ashrafi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) which have been linked to high saprotrophic capabilities (Caldwell et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Our findings suggest that this saprotrophic capability may be a key trait enabling fungal symbionts to quickly colonize plants, but may not be associated with long-term persistence of fungal symbionts as plants age. Fracchia et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found similar results in a time-series study of \u003cem\u003ePopulus\u003c/em\u003e on a much shorter timescale, with saprotrophic fungi being very abundant on roots in the first few days of growth. Dark septate endophytes are understudied, but some experimental work shows that they can transfer nutrients to plants (Almario et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and thus may represent an important nutrient acquisition symbiosis during early plant growth. In this study, the dark septate endophytic genus \u003cem\u003eHyaloscypha\u003c/em\u003e was found to be one of the most prolific colonizers on both \u003cem\u003ePopulus\u003c/em\u003e spp. throughout the growth period. Past studies have identified both \u003cem\u003eHyaloscypha\u003c/em\u003e and \u003cem\u003eExophiala\u003c/em\u003e as being core members of the \u003cem\u003ePopulus\u003c/em\u003e root fungal community (Shakya et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nash et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). A past culture-based study of \u003cem\u003ePopulus\u003c/em\u003e root fungal symbionts found that endophytes were among the most common culturable members of root communities (Bonito et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with frequent isolations of \u003cem\u003eIlyonectria\u003c/em\u003e, \u003cem\u003eHyaloscypha\u003c/em\u003e, and \u003cem\u003eExophiala\u003c/em\u003e which were abundant in our dataset. Despite the growing body of work showing that dark septate endophytes are prolific colonizers on dual-mycorrhizal hosts (including \u003cem\u003ePopulus\u003c/em\u003e), many mycorrhizal studies still overlook the role of this guild in studies of plant nutrition and stress tolerance. The temporal dynamics of fungal endophytic colonization on root systems that are co-colonized by AM and EM fungi remains an underexplored area of research.\u003c/p\u003e \u003cp\u003eFungal alpha diversity decreased by half from 5 months to 12 months of plant growth. The pot culture method that we employed did not allow for recruitment of new fungal symbionts by dispersal and is thus somewhat unrealistic compared to natural conditions where continuous dispersal allows for recruitment of new symbionts over the lifespan of a tree. However, this time-dependent decrease in alpha diversity suggests that in the absence of dispersal, root associated fungal communities experience loss of species diversity as plants age. Mycorrhizal fungal diversity is thought to promote plant growth and higher rates of nutrient transfer to plants (Nash et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Anthony, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and it is unknown how the observed decrease in fungal diversity with plant development affected the quality of mycorrhizal benefits provided to the plant hosts.\u003c/p\u003e \u003cp\u003eOur study utilized a common garden approach to isolate the role of \u003cem\u003ePopulus\u003c/em\u003e host identity on structuring root-associated fungal communities without the confounding influence of environmental variation. The two \u003cem\u003ePopulus\u003c/em\u003e spp. examined exhibited distinct preferences for AM and EM colonization, suggesting that they exist along a spectrum of dual-mycorrhizal colonization. \u003cem\u003eP. trichocarpa\u003c/em\u003e displayed moderate levels of AM colonization, while \u003cem\u003eP. tremuloides\u003c/em\u003e showed very little evidence for AM colonization but had higher EM colonization rates as determined by microscopy (though similar levels based on LSU sequencing). The findings regarding AM colonization of \u003cem\u003eP. tremuloides\u003c/em\u003e are consistent with other studies showing that the species is a poor AM host (Kaldorf et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Karst et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, a microscopy-based study on mature \u003cem\u003eP. tremuloides\u003c/em\u003e found an average AM colonization rate of 6% (Neville et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and another molecular-based study found modest AM association with \u003cem\u003eP. tremuloides\u003c/em\u003e (Nash et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Interestingly, \u003cem\u003eP. tremuloides\u003c/em\u003e x \u003cem\u003eP. tremula\u003c/em\u003e hybrids have been shown to exhibit greatly decreased AM colonization in response to nitrogen fertilization, while AM colonization of \u003cem\u003eP. trichocarpa\u003c/em\u003e was unaffected and so the usage of an inorganic fertilizer in our experiment may have prevented AM colonization of \u003cem\u003eP. tremuloides\u003c/em\u003e (Baum \u0026amp; Makeschin, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Thus, \u003cem\u003eP. tremuloides\u003c/em\u003e should still be assumed to be dual-mycorrhizal despite the lack of evidence for AM colonization in this study. The higher rate of EM colonization on \u003cem\u003eP. tremuloides\u003c/em\u003e than \u003cem\u003eP. trichocarpa\u003c/em\u003e that we found is consistent with a continental-scale comparison of five \u003cem\u003ePopulus\u003c/em\u003e species (Van Nuland et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings suggest that previously observed differences in mycorrhizal colonization among \u003cem\u003ePopulus\u003c/em\u003e spp. (Van Nuland et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) are not solely the result of environmental variation and fungal biogeography, but are also due differences in host preference for AM and EM colonization. Although we found stark host-dependent differences in mycorrhizal colonization between the two \u003cem\u003ePopulus\u003c/em\u003e spp., we found few individual fungal taxa that differed in abundance between the two hosts. Additional controlled common garden experiments are needed that use a wider phylogenetic diversity of \u003cem\u003ePopulus\u003c/em\u003e species planted with diverse soil inocula to advance our knowledge of fungal host-specificity in the \u003cem\u003ePopulus\u003c/em\u003e genus.\u003c/p\u003e \u003cp\u003eOur experiment made use of a simplified growth chamber environment to isolate the effects of hosts species identity and plant age on root fungal colonization, but there are still many unresolved questions about the impact of environmental context on determining dual-mycorrhizal colonization during early plant development and the mechanisms that underlie mycorrhizal switching. The density and proximity of adult mycorrhizal hosts may influence colonization dynamics of seedlings due to hyphal spread (Dickie et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Soil moisture levels can shift the balance between AM and EM fungi and may alter the rate of microbiome development (Lodge, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The long-term trajectories of dual-mycorrhizal colonization is also likely influenced by the ability of mycorrhizal symbionts to persist during winter when plant carbon availability is low and fungal symbionts may experience freezing. EM fungi are more resistant to soil freezing than AM fungi and successive freeze-thaw cycles may result in a decline in AM colonization over time on dual-mycorrhizal hosts (Kilpel\u0026auml;inen et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, Lodge and Wentworth (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) have documented potential antagonism between EM and AM fungi in the same root system, which could result in transitions between these guilds if one of them gains a competitive advantage. In our study, AM and EM colonization increased in tandem as plants aged while endophytic colonization decreased, hinting at a possible unexplored role of competition with endophytic fungi in structuring \u003cem\u003ePopulus\u003c/em\u003e root-associated fungal communities. Changes in host metabolism through plant development are likely also strong controls on fungal community composition (Xie et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fracchia et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo conclude, our study demonstrates that plant age-dependent changes in root fungal community composition in \u003cem\u003ePopulus\u003c/em\u003e are distinct from patterns observed in other dual-mycorrhizal hosts and demonstrates that there is variation in association with AM fungi among different \u003cem\u003ePopulus\u003c/em\u003e spp. We show that saprotrophic capabilities may be an important trait of early colonizing fungal endophytes. Our findings indicate the potential for diverse developmental trajectories in dual-mycorrhizal hosts and stress the importance of examining endophytic fungal communities in addition to mycorrhizal fungi. Developmentally induced shifts in root-associated fungal communities likely have functional implications for rates of nutrient transfer and ecosystem processes that provide an exciting avenue for future research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.N. designed the experiment, conducted the experiment, analyzed the data, and wrote the paper. B.L. designed the experiment and conducted the experiment. M.A.C. designed the experiment. C.W.S. designed the experiment. R.V. Designed the experiment. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe greatly appreciate the assistance of the Duke University Phytotron Facility in conducting this research. We received tremendous intellectual support and advice from the scientists in the Plant Microbe Interfaces Science Focus Area at Oak Ridge National Laboratory. We thank the managers of the Fishlake National Forest for allowing us to sample soil from their lands. This research was funded by the U.S. Department of Energy Office of Biological and Environmental Research, Genomic Science Program as part of the Plant Microbe Interfaces Science Focus (http://pmi.ornl.gov). Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eCode underlying the analyses presented in this paper is available at https://github.com/jakenash12/PopulusSuccession. All data underlying the analyses presented in this paper are included in Supplemental File S1. 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Plant Soil 452:249\u0026ndash;266. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11104-020-04538-9\u003c/span\u003e\u003cspan address=\"10.1007/s11104-020-04538-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycorrhiza","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcor","sideBox":"Learn more about [Mycorrhiza](http://link.springer.com/journal/572)","snPcode":"572","submissionUrl":"https://submission.nature.com/new-submission/572/3","title":"Mycorrhiza","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Dual-mycorrhizal, dark septate endophytes, Populus, succession, ectomycorrhizae, arbuscular mycorrhizae","lastPublishedDoi":"10.21203/rs.3.rs-6531386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6531386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlants have evolved symbioses with mycorrhizal and endophytic fungi that are essential for their growth and survival. While most plants associate with a single guild of mycorrhizal fungi, a select group termed \u0026ldquo;dual-mycorrhizal plants\u0026rdquo; associate with both arbuscular mycorrhizal and ectomycorrhizal fungi. Although a shift from arbuscular mycorrhizal to ectomycorrhizal colonization with plant development has been demonstrated on other dual-mycorrhizal hosts, it is not known how mycorrhizal colonization shifts with plant age in \u003cem\u003ePopulus\u003c/em\u003e species. We performed an \u003cem\u003ein planta\u003c/em\u003e mycorrhization experiment to test for the occurrence of mycorrhizal switching in response to plant age and for host-specific patterns of fungal colonization in two species of \u003cem\u003ePopulus\u003c/em\u003e (\u003cem\u003eP. tremuloides\u003c/em\u003e and \u003cem\u003eP. trichocarpa\u003c/em\u003e). We found that only \u003cem\u003eP. trichocarpa\u003c/em\u003e displayed dual-mycorrhizal colonization, while \u003cem\u003eP. tremuloides\u003c/em\u003e associated with ectomycorrhizal fungi, but not arbuscular mycorrhizal fungi. On \u003cem\u003eP. trichocarpa\u003c/em\u003e, both guilds of mycorrhizal fungi increased in abundance with plant age, while root endophytic fungal colonization decreased. Many of the early-colonizing endophytic fungi that we documented have strong saprotrophic capabilities, which may be an important trait for fast colonization. Dark septate endophytes were more abundant than either guild of mycorrhizal fungi, and are likely functionally important members of the \u003cem\u003ePopulus\u003c/em\u003e root fungal community. Our findings represent a novel pattern in the development of dual-mycorrhizal colonization and illustrate that \u003cem\u003ePopulus\u003c/em\u003e species vary in their association with arbuscular mycorrhizal fungi. Our results also stress the importance of a \u0026ldquo;third guild\u0026rdquo; of root fungal symbionts \u0026ndash; the dark septate endophytes \u0026ndash; on dual-mycorrhizal plants.\u003c/p\u003e","manuscriptTitle":"Dual-mycorrhizal colonization is determined by plant age and host identity in two species of Populus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 11:09:25","doi":"10.21203/rs.3.rs-6531386/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-24T08:05:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T16:24:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-07T21:14:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20221138025016305741622894175812329411","date":"2025-04-29T13:26:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333667121627643171576590353567091113989","date":"2025-04-28T14:36:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T14:17:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T13:19:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-28T08:18:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycorrhiza","date":"2025-04-25T19:52:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycorrhiza","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcor","sideBox":"Learn more about [Mycorrhiza](http://link.springer.com/journal/572)","snPcode":"572","submissionUrl":"https://submission.nature.com/new-submission/572/3","title":"Mycorrhiza","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"506379ce-8170-44c7-9a8e-f0122a48f9f0","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T15:58:30+00:00","versionOfRecord":{"articleIdentity":"rs-6531386","link":"https://doi.org/10.1007/s00572-025-01215-6","journal":{"identity":"mycorrhiza","isVorOnly":false,"title":"Mycorrhiza"},"publishedOn":"2025-06-09 15:56:55","publishedOnDateReadable":"June 9th, 2025"},"versionCreatedAt":"2025-04-29 11:09:25","video":"","vorDoi":"10.1007/s00572-025-01215-6","vorDoiUrl":"https://doi.org/10.1007/s00572-025-01215-6","workflowStages":[]},"version":"v1","identity":"rs-6531386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6531386","identity":"rs-6531386","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}