Disturbance tolerance of arbuscular mycorrhizal fungi: trait-based characterization along a disturbance gradient in a coastal dune ecosystem

Disturbance tolerance of arbuscular mycorrhizal fungi: trait-based characterization along a disturbance gradient in a coastal dune ecosystem | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Disturbance tolerance of arbuscular mycorrhizal fungi: trait-based characterization along a disturbance gradient in a coastal dune ecosystem Anjar Cahyaningtyas, Tatsuhiro Ezawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2758973/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Oct, 2023 Read the published version in Plant and Soil → Version 1 posted 4 You are reading this latest preprint version Abstract Purpose Arbuscular mycorrhizal fungi that regenerate rapidly after disturbance play a significant role in resilience of vegetation after disturbance. We experimentally characterized disturbance-tolerance traits of the fungi that inhabit a coastal dune ecosystem. Methods Root-zone soils, including root fragments, were collected from the seaward and landward slopes that differ in disturbance severity. The native grass Miscanthus sinensis (donor plants) were grown in the soils for two months, and from a half of the plants, disturbance-tolerant fungi that regenerate from spores and extraradical hyphae (soil propagule-mediated regenerators, SP) and those that regenerate from the colonized roots (root-direct regenerators, RD) were trapped separately with new seedlings (assessment plants). The other half of the donor plants were further grown for four months together with assessment plants, during which the fungi trapped by the assessment plants were categorized as disturbance-sensitive slow regenerators (SL). DNA was extracted from the assessment plants, and fungal taxa were identified based on LSU rDNA sequences. Results All fungi occurred in the seaward soil samples showed the SP and/or RD traits, whereas those occurred in the landward samples showed not only SP and/or RD traits but also SL traits. The seaward fungal communities were nested within the landward communities, implying that they were selected from the landward communities. Conclusion These observations suggest that rapid regeneration from soil-borne propagules and colonized roots are key traits of the fungi for survival in dune ecosystems, providing a new insight into the life-history strategies of AM fungi in frequently and severely disturbed ecosystems. arbuscular mycorrhizal fungi coastal dune community nestedness disturbance-tolerance trait soil disturbance Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Soil disturbance occurs through natural processes, e.g., volcanic ash fall (Crisafulli et al. 2015 ; del Moral 2010 ), landslide in steep slopes (Hungr et al. 2014 ; Van Eynde et al. 2017 ), wildfire (Hosseini et al. 2016 ; Neary 2019 ), and sand movement by wind and tidal waves in coastal dunes (Delgado-Fernandez and Davidson-Arnott 2011 ), which cause environmental issues, such as erosion (Kurothe et al. 2014 ; Rey 2003 ; Suescún et al. 2017 ) and eutrophication of ground and marine water ecosystems (Smith 2003 ). Arbuscular mycorrhizal (AM) fungi form symbiotic association with most land plants and deliver mineral nutrients to the host (Smith and Read 2008 ). In the association the fungi construct hyphal networks in the soil, that is, extraradical mycelia, from which water and nutrients are taken up and translocated to arbuscules in the root cortex where the fungal symbionts release nutrients to the host (Ezawa and Saito 2018 ). In exchange for the nutrients, the fungi receive lipid and sugar as carbon source (Lanfranco et al. 2018 ). Soil disturbance has a serious impact on mycorrhizal association via destruction of extraradical hyphal networks, which inhibits nutrient exchange between the symbionts and reduces new colonization mediated by the networks. It has been demonstrated that soil disturbance acts as selection pressure for the fungi that inhabit agroecosystems (Jansa et al. 2002 ), semi-natural grassland (Schnoor et al. 2011 ), coastal dunes (Corkidi and Rincón 1997 ; Kawahara and Ezawa 2013 ), and volcanic slopes (Atunnisa and Ezawa 2019 ; Fujiyoshi et al. 2006 ), indicating that sensitivity to soil disturbance is different among AM fungal species. Rapid regeneration of hyphal networks after disturbance would be an important trait for disturbance-tolerant fungi; they would have short life cycles (Declerck et al. 2001 ), high-hyphal growth rates (Avio et al. 2006 ; Hart and Reader 2005 ), and efficient hyphal-healing mechanisms (de la Providencia et al. 2007 ). Chagnon et al. ( 2013 ) proposed a concept of AM fungal life-history strategy according to the C-S-R (competitor, stress-tolerator, and ruderal) framework, which was originally raised by Grime ( 1977 ) to classify plant life-history strategies. Competitor fungi (C) construct a high-density of hyphal networks in the soil and produce spores at later growth stages. Stress-tolerator fungi (S) can tolerate biotic stresses, such as high-soil acidity and low temperature, have long-lived mycelia, and grow slowly. Ruderal fungi (R) grow rapidly, show high rates of hyphal turnover, produce abundant spores at early growth stages or constitutively, and allocate more biomass in the roots than in the soil. In this context, the ruderal traits are likely involved in disturbance tolerance, whereas competitor fungi would be susceptible to soil disturbance. Disturbance-tolerant/sensitive traits of the fungi, however, have not yet been experimentally characterized. In this study, disturbance-tolerance traits of AM fungi were characterized by employing Ishikari coastal dunes as a model ecosystem in which vegetation is zonally distributed along soil-disturbance gradients (Kawahara and Ezawa 2013 ). We addressed two hypotheses that i) disturbance-tolerant AM fungi that are capable of establishing new colony rapidly from soil-borne propagules (rapid regenerators) are enriched in a more severely disturbed habitat and ii) disturbance-sensitive fungi that require longer time to establish colony after disturbance (slow regenerators) are enriched in a less severely disturbed habitat. To test these hypotheses, a nylon mesh-separated compartment culture system was applied for assessing inoculum potential of extraradical and intraradical mycelia of the fungi separately. Material And Methods Sampling sites A sampling area of 100 × 1,000 m was defined along Ishikari coastal dune, Hokkaido, Japan (Figs. 1 a and b). This ecosystem belongs to the subarctic zone with the annual mean temperature and rainfall are 8.3˚C and 651.0 mm, respectively. The primary dunes are 2–6 m in height and located about 50–100 m inland from the coastal line. Vegetation in the dune has been described in Kawahara and Ezawa ( 2013 ). Briefly, the landward slopes of the primary dune are 80–150 m in width and largely dominated by the C4 perennial grass Miscanthus sinensis Andersson (Fig. 1 c). In addition, Rosa rugosa Thunb. and Lathyrus japonicus Willd. are also patchily distributed. The seaward slopes are 40–70 m in width and dominated by Leymus mollis (Trin. ex Spreng.) Pilg. (Fig. 1 d). Calystegia soldanella (L.) Riom. et Schult, Arabis stelleri var. japonica (A. Gray) Fr. Schm., Glehnia littoralis F. Schmidt ex Miq, Carex kobomugi Ohwi., Linaria japonica Miq., and Ixeris repens (L.) A. Gray are also patchily distributed in the slopes. The habitats of M. sinensis and L. mollis are clearly segregated between the slopes, and this typical zonal distribution is observed more than 5 km along the coastal line. The topsoil layer of the seaward slopes is constantly disturbed by wind thus unstable. Whereas the thick root system of M. sinensis stabilized the landward slopes, and thus an organic layer (3–5 cm) originated from M. sinensis litter has been developed. The soil chemical properties in the two slopes are described in Kawahara and Ezawa ( 2013 ); it is noteworthy that sodium (Na) concentration in the root-zone soils were not significantly different between the seaward and landward slopes (140 and 128 mg-Na kg − 1 , respectively, on average). Soil sampling Within the area, five sampling plots were defined at 200 m intervals along the coastal line in each of the seaward and landward slopes (Fig. 1 b). Four individuals of M. sinensis and L. mollis were selected in each sampling plot (5 × 5 m), and root-zone soil samples, including root fragments, were collected from the four plants on June 17, 2020 by using a stainless-steel core sampler [5 × 5 cm (diam/height), 100 mL in vol] for M. sinensis and with a small shovel for L. mollis (ca. 100–200 g soil plant − 1 ). The core sampler was not used for the latter because the root-zone soil (sand) was too fragile to collect by the sampler due to the low density of L. mollis roots. Twenty kilograms of sand was also collected from the seaward slope where no vegetation was present and autoclaved for 2 h as a base medium for soil trap culture. Trap culture The soil samples collected by the core samplers (landward slopes) and those collected by the shovel (seaward slopes) were transferred to cylinder-shaped 37-µm nylon mesh bags [5 × 5 cm (diam/height)] (non-destructively for the landward samples) in 350-mL plastic pots in which the base medium was filled around the mesh bag (four-replicated samples per plot and five plots in each of the two slopes in a total of 40 pots). M. sinensis seeds (Snow Brand Seed Co., Ltd., Sapporo) were sown onto all soil samples in the nylon mesh bag, covered with a thin layer of sterilized sand, and grown in a temperature/light/humidity-controlled greenhouse (day/night temperature 26/20˚C; 14-h day length; 60% relative humidity). The seedlings were grown with tap water for the first month and with liquid fertilizer made of Peters Professional 25-5-20 (ICL, St. Louis, MO) at 50 µM phosphate once a week thereafter and thinned to three plants per pot one month after sowing. After two months, the seedlings were removed together with the mesh bag from two out of the four-replicated pots. These seedlings were used as donor plants of "root-direct regenerator" fungi, whereas spore/mycelia formed in the medium outside the mesh bag (remained in the pots) were used as inocula of "soil propagule-mediated regenerator" fungi as follows. The hole formed after the removal of the mesh bag was filled with the base medium to which five three-week-old new seedlings of M. sinensis grown in the base medium were transplanted and grown for two months as assessment plants for soil propagule-mediated regenerators (two pots per plot) (Fig. S1 ). Roots of the donor plants (i.e., the seedlings from the mesh bags) were washed on a stainless mesh with gently pressured tap water to remove adhering spores and extraradical mycelia, and the plants were transplanted outside the mesh bag of a new pot filled with the base medium. Then five three-week-old M. sinensis seedlings were transplanted inside the mesh bag and grown for two months as assessment plants for root-direct regenerators (two pots per sampling plot). To raise "slow regenerator" fungi, five three-week-old M. sinensis seedlings were transplanted outside the mesh bag in the remaining two pots and grown for further four months without removing the donor plants in the mesh bag. In all treatments the two pots from each sampling plot were treated as technical replication, and thus the data from the two were combined prior to analysis as described in the later section. After harvesting the assessment plants, the roots were washed with pressured tap water, cut into 1-cm segments, randomized in water, blotted on a paper towel, immersed in RNA later (Thermo Fisher Scientific, Tokyo) for more than 48 h at room temperature to fix nucleic acid, blotted on a paper towel to remove excess RNA later , transferred to a 3-mL tube with an O-ring sealed cap (Yasui Kikai, Osaka), and stored at -80°C for DNA extraction. Molecular identification The frozen roots in the 3-mL tube were ground with a metal cone in the presence of liquid nitrogen at 2,500 rpm for 2 × 5 s using Multi-Beads Shocker (Yasui Kikai, Osaka), and DNA was extracted and purified from approx. 100 mg of ground sample by Maxwell RSC Instrument (Promega, Madison, WI) using Maxwell RSC PureFood GMO and Authentication Kit (Promega) according to the manufacturer's instructions, stored at -30˚C, and used as template for PCR amplification. The divergent domain 2 of large-subunit ribosomal RNA gene (LSU rDNA) was amplified in a 25-µL reaction mixture of Expand High-Fidelity PCR System (Roche Diagnostics, Tokyo), 0.5 nmol µL − 1 each of FLd3 (forward) and FLR2 (reverse) primers that were linked to TruSeq-type forward- and reverse-adapter sequences (Illumina, Tokyo), respectively, at the 5'-end (Niwa et al. 2018 ), and 0.2–2 µL template DNA using C1000 TouchTM Thermal Cycler (BIO-RAD, Tokyo) with the following program: initial denaturation at 94˚C for 2 min, followed by 30 cycles of denaturation at 94˚C for 15 s, annealing at 48˚C for 40 s, polymerization at 72˚C for 1 min, and final elongation at 72˚C for 10 min. The PCR products were sequenced on the Illumina MiSeq platform (2 × 300 bp), and high-quality paired end reads (read 1 and read 2) were merged with a minimum overlap length of 10 nt using FLASH ( http://ccb.jhu.edu/software/FLASH/ ) at Bioengineering Lab (Sagamihara, Kanagawa, Japan). The merged reads were subjected to BLASTn searches against the fungal LSU rDNA database consisted of 82,494 operational taxonomic units (OTUs) of fungi, including 524 OTUs of AM fungi, constructed by Niwa et al. ( 2018 ) and assigned to the OTUs at ≥ 95% similarities over 330-bp alignment with an E-value cut-off of 1e-30. Data processing and trait definition The read numbers of the OTUs were subjected to pairwise correlation analysis within the same genera/families of AM fungi, and pairs that showed a correlation coefficient more than 0.9 were merged into single OTUs. The OTUs occurred uniquely in the soil propagule-mediated regenerator (SP) and root-direct regenerator (RD) treatments across all samples, irrespective of the occurrence in the slow regenerator (SL) treatment, were categorized as SP and RD, respectively, and those that occurred in both treatments were defined as SP/RD. Only the OTUs occurred uniquely in the SL treatment were categorized as SL. Total read numbers in each sample were standardized to 5,000 reads, whereas zero-data were assigned to all OTUs in the samples from which no PCR product was obtained (i.e., total read number was zero per sample). In this step, OTUs with less than one read were excluded from the datasets. Then total read number from the same sampling plots [two technical replications × three (SP, RD, and SL) treatments] were standardized to 30,000 reads per plot, irrespective of whether zero-data samples were present or not. The read number data were transformed to logarithmic values (log 2 ) or binary data (presence/absence) prior to analysis. Statistical analysis F -test for equality of variance and Welch's t -test were conducted in the R 3.6.3 platform (R Core Team 2020), in which Bonferroni correction was applied for multiple comparisons. Non-metric multidimensional scaling (NMDS) were performed with the vegan package (Oksanen et al. 2020 ) in the R platform using Bray-Curtis dissimilarity index as a distance metric. Bray-Curtis index was also employed for b-diversity analysis and applied only to the trait categories in which fungal OTUs were detected from three or more plots. Community nestedness was assessed with a maximally stacked matrix constructed with the presence-absence dataset, in which the order of the columns (plots) and rows (OTUs) were sorted by OTU richness and the frequency of OTU, respectively, on the web interface Nestedness for Dummies (Strona et al. 2014 ) ( http://purl.oclc.org/ned ), employing nested overlap and decreasing fill (NODF) as a metric (Almeida-Neto et al. 2008 ) with the proportional row and column totals null model (Ulrich and Gotelli 2007 ). The metric NODF ranges from 0 (non-nested) to 100 (fully nested), and a positive Z -score indicate nestedness, and a negative Z -score indicates anti-nestedness. Result OTU definition and trait assignment PCR products were obtained from all samples from the landward slopes, except for one of the RD samples, and from five SP, five RD, and 10 SL samples from the seaward slopes. The amplicon sequencing generated 50,617 paired reads per sample on average, which were quality-filtered and generated 41,460 merged reads per sample on average. About 86% of the merged reads were assigned to the AM fungal OTUs, and among them, two Rhizophagus OTUs, 003_Rhz and 011_Rhz, and two Uncultured Glomeraceae OTUs, 107_UnG and 157_UnG, were merged, respectively, into single OTUs (Supplemental material Table S2 ). In the landward slopes 35, 15, and 53 OTUs occurred in the SP, RD, and SL treatments, respectively (60 OTUs in total), whereas 8, 5, and 5 OTUs occurred in the SP, RD, and SL treatments, respectively (10 OTUs in total), in the seaward slopes. Based on their occurrence patterns, 15, 23, 2, and 22 OTUs were categorized as SP/RD, SP, RD, and SL types, respectively (Supplemental material Table S3). Trait-based characterization of the landward and seaward communities NMDS species (OTU)-habitat biplots at the genus level provided an overview of taxon-trait distributions in the two slope habitats (Fig. 2 a – d). In these biplots, OTUs that are located closer to the center of habitat (sample) plots occur more specifically/abundantly in the habitat, while those that are located between the sample plots are broadly distributed in both habitats. Overall, diverse OTUs with various traits occurred in the landward slopes, whereas the seaward slopes were dominated by several SP/RD- and SP-type OTUs of limited taxa. Many of Rhizophagus OTUs were the SP/RD type, and two of them were broadly distributed across the habitats (Supplemental material Table S3). The Glomus OTUs consisted of the SP/RD, SP, and SL types and distributed only in the landward slopes. The OTUs assigned to uncultured Glomeraceae contained all types, and those of the SP/RD type were distributed in both slopes. A number of Scutellospora OTUs occurred in the landward slopes, in which all, except for one with the SL trait, showed the SP trait. The three Acaulospora OTUs showed the SP traits, and one of the OTUs occurred specifically in the seaward slopes. All three Diversispora OTUs showed the SL trait and occurred only in the landward slopes. The two Paraglomus OTUs with the SP/RD traits occurred both in the seaward slopes, but the other two with SP and SL traits were distributed only in the landward slopes. All five Claroideoglomus OTUs occurred only in the landward slopes, and among them, three, one, and one showed SL, SP, and RD traits, respectively. Only one Archaeospora OTU that has SL trait occurred in the landward slopes. In the landward slopes OTU richness of the SP and SP/RD types was not significantly different and higher than that of the RD type, while richness of the SL type was not different from that of the other types ( P < 0.05) (Fig. 3 a). In the seaward slopes several SP/RD-type OTUs and only a few SP types occurred in all and two plots, respectively, whereas the RD and SL types were absent. In the landward slopes b-diversity was highest in the SL-type OTUs, followed by the SP and SP/RD types, and that of the SP/RD type in the seaward slopes was lowest (Fig. 3 b). The lower diversity as well as the absence of SL-type OTUs in the seaward communities suggested that there is strong selection pressure on the communities, led to the idea that the fungi in the seaward slopes might be selected from those inhabit the landward slopes. To explore an OTU-distribution pattern across the diversity gradients between the two habitats, nestedness analysis was applied; significant nestedness was observed among the columns (NODF column = 68.00, Z = 13.66, P < 0.001) (Fig. 4 and Supplemental material Table S4), implying that the seaward communities are subsets of the landward communities. Discussion The mesh-separated compartment culture system successfully differentiated AM fungal traits responsible for regeneration after disturbance. To characterize soil-borne propagules of the fungi, the destructive method (i.e., wet sieving) has generally been employed in previous studies (e.g., Klironomos and Hart 2002 ; Varela-Cervero et al. 2016 ). It is likely, however, that the isolation of spore and hyphae by destructive manners would lead to underestimation of inoculum potential via damaging/losing the propagules. The compartment culture system had originally been established three decades ago to assess the impact of destruction of extraradical hyphae on their inoculum potential (Jasper et al. 1989 ) and has recently been applied to physiological studies on fungal nutrient uptake (e.g., Hodge & Fitter, 2010 ; Kikuchi et al., 2016). The present study demonstrated that the application of this technique enabled us to assess the potential of extraradical mycelia in a non-destructive manner, in which extraradical mycelia were separated from the mycorrhizal roots simply by removing the mesh bag, minimizing the damages of the propagules. This culture system, however, cannot separate spores and extraradical hyphae that may differ in inoculum potential; the latter inocula would be more important in the genera Rhizophagus , Claroideoglomus , and Acaulospora spp. because not only spores but also extraradical hyphae acted as major inoculum source in extraradical matrices, even after fragmentation (Klironomos and Hart 2002 ). Accordingly, it should be noted that the propagules of the fungi with the SP trait, particularly those of the fungi in these genera, might consist of not only spores but also extraradical hyphae. Given that spores are likely to survive a longer period than detached hyphae, further experiments that take into account a time factor are necessary to assess the significance of extraradical hyphae in regeneration. The present study demonstrated that a majority of AM fungi inhabit the dune ecosystem is capable of regenerating rapidly via soil-borne propagules within two months, that is, disturbance-tolerant fungi, and further that all fungi in the seaward slopes showed this trait, supporting the first hypothesis. But, unexpectedly, many of the SP-type fungi could also regenerate directly from the colonized roots, that is, SP/RD-type fungi, indicating that not only spores/extraradical hyphae but also intraradical mycelia are important for rapid regeneration after disturbance. It has been well documented that the production of abundant spore is a prevalent trait in the members of Glomeraceae (Chagnon et al. 2013 ; Klironomos and Hart 2002 ), consistent with our finding that 20 out of the 38 OTUs with the SP trait belong to this family (i.e., those assigned to Rhizophagus , Glomus , and uncultured Glomeraceae ). On the other hand, the higher biomass ratios of intraradical to extraradical mycelia are also a feature of Glomeraceae fungi (Hart and Reader 2002 ). This could contribute to rapid regeneration from colonized roots, which is the "disturbance-avoidance strategy" (Chagnon et al. 2013 ). In fact, 14 of the 17 OTUs with the RD trait belong to the Glomeraceae . Given the significance of carbon supply from intraradical mycelia in the bridging/healing of fragmented extraradical hyphae (de la Providencia et al. 2007 ), photosynthetic activity of the host would be a major constraint of the reconstruction of extraradical hyphal networks and sporulation. In this study, the intact plants that retained photosynthetic activity were used as inoculum source for raising RD-type fungi, which might accelerate/enhance the colonization of the assessment plants. Potential of the colonized roots with or without the above-ground parts is of interest and to be assessed in the future. We first expected that the members in the family Gigasporaceae that consists of the three genera Gigaspora , Scutellospora , and Racocetra would be slow regenerators because they colonize roots mainly via spores, rarely via the colonized roots, and produce spores at later stages of their life cycles (Biermann and Linderman 1983 ; Jasper et al. 1993 ; Klironomos and Hart 2002 ; Oehl et al. 2009 ), which are typical of the competitor traits (Chagnon et al. 2013 ). In line with these studies, no Scutellospora OTUs showed the RD trait, confirming that their intraradical mycelia may not be major inoculum source. However, 12 of the 13 Scutellospora OTUs showed the SP trait, suggesting that they are capable of regenerating rapidly via extraradical hyphae detached from the roots (Jasper et al. 1993 ) and/or via mature spores produced within two months, although the latter assumption is inconsistent with the observations that S . calospora produced very few spores at least 11 weeks after inoculation (Jasper et al. 1993 ). All the slow regenerator fungi occurred exclusively in the landward slopes, supporting the second hypothesis that slow regenerators are enriched in a less severely disturbed habitat. The 22 SL-type OTUs were distributed across diverse genera in the Glomeromycotina, and remarkably, b-diversity of the slow regenerator communities was significantly higher than those of the rapid regenerator (SP/RD and SP) communities, reflecting that most of the SL-type OTUs were unique to each plot. This patchy distribution of the slow regenerators across the slopes suggests that stochastic processes, rather than environmental factors such as disturbance severity, may play an important role in structuring the communities (Dumbrell et al. 2010 ; Lekberg et al. 2012 ). It is speculated, therefore, that more diverse SL-type fungi will be detected in this ecosystem by more intensive sampling, given the high b-diversity of the communities. The absence of SL-type OTUs in the seaward slopes, however, should be interpreted by taking into account that the seaward samples were collected destructively, which could potentially reduce the inoculum potential of SL-type fungi. In fact, two of the 22 SL-type OTUs, 008_Rhz and 206_Div, were detected in the roots of L. mollis grown in the seaward slopes in the previous study (Kawahara and Ezawa 2013 ), implying that these fungi are capable of colonizing the seaward slopes. The impact of the destructive collection on their potential, however, might be minimum because these two OTUs could also be detected in trap culture using the destructively collected soil samples (Kawahara and Ezawa 2013 ). In this context, it seems likely that not all of the SL-type fungi have the competitor traits. There is a possibility that some of them have the ruderal traits but could not colonize the roots within two months due to rapid occupation of the niches by the dominant rapid regenerators. Detailed characterization of the slow regenerator fungi, e.g., their spore productivity and resilience of hyphal networks, is necessary to examine this possibility, although isolation of this type of fungi may not be easy due to the coexistence of the rapid regenerators. Our finding that the AM fungi with the SP and/or RD traits are prevalent in the dunes provides experimental support for the idea that disturbance-tolerant fungi are selected across the ecosystem (Kawahara and Ezawa 2013 ). In addition, the nestedness pattern along the richness gradient indicates that the rapid regenerators in the seaward slopes were selected from the landward community, suggesting that selection pressure is even stronger in the seaward slopes than in the landward slopes. The lower OTU richness and b-diversity in the seaward communities further suggest that the communities converged towards the common members that preferentially inhabit coastal dune ecosystems. This raises a new idea that AM fungal communities associated with coastal dune vegetation converge across the globe, given the low endemism of AM fungi (Davison et al. 2015 ). Further surveys are necessary to examine this idea. Conclusion We employed Ishikari sand dune ecosystem as a model and experimentally characterized the disturbance-tolerance traits of AM fungi by taking into account the time necessary for regeneration after disturbance. In the landward community, although many of the fungi showed the traits involved in disturbance tolerance, disturbance-sensitive fungi (i.e., slow regenerators) were also present. Whereas the seaward community consisted only of disturbance-tolerant fungi that were a subset of the landward community. These observations suggest that rapid regeneration from soil-borne propagules and colonized roots are key traits not only for the survival of the fungi but also for the resilience and maintenance of vegetation in dune ecosystems. Taken together, our findings provide a new insight into the life-history strategies of AM fungi in frequently/severely disturbed ecosystems and will also contribute to the practical application of AM fungi to restoration of post-disturbed ecosystems. Declarations Acknowledgement This work was conducted at the open research facility of Hokkaido Agricultural Research Center, NARO. We also acknowledge Ministry of Education, Culture, Sports, Science and Technology, Japan for the scholarship to AC. Competing interests The authors have no relevant financial or non-financial interests to disclose. Author contributions All authors contributed to the study conception, design, and writing the manuscript. Data availability The sequence reads have been deposited in the Sequence Read Archive of National Center for Biotechnology Information under the accession number PRJNA949820. 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Geoderma 269:126–137. https://doi.org/10.1016/j.geoderma.2016.02.004 Hungr O, Leroueil S, Picarelli L (2014) The Varnes classification of landslide types, an update. Landslides 11:167–194. https://doi.org/10.1007/s10346-013-0436-y Jansa J, Mozafar A, Anken T, Ruh R, Sanders IR, Frossard E (2002) Diversity and structure of AMF communities as affected by tillage in a temperate soil. Mycorrhiza, 12:225–234. https://doi.org/10.1007/s00572-002-0163-z Jasper DA, Abbott, LK, Robson AD (1989) Soil disturbance reduces the infectivity of external hyphae of vesicular-arbuscular mycorrhizal fungi. New Phytol 112:93–99. https://doi.org/10.1111/j.1469-8137.1989.tb00313.x Jasper DA, Abbott LK, Robson AD (1993) The survival of infective hyphae of vesicular–arbuscular mycorrhizal fungi in dry soil: An interaction with sporulation. New Phytol 124:473–479. https://doi.org/10.1111/j.1469-8137.1993.tb03838.x Kawahara A, Ezawa T (2013) Characterization of arbuscular mycorrhizal fungal communities with respect to zonal vegetation in a coastal dune ecosystem. Oecologia 173:533–543. https://doi.org/10.1007/s00442-013-2622-y Kikuci Y, Hijikata N, Ohtomo R, Handa Y, Kawaguchi M, Saito K, Masuta C, Ezawa T (2016) Aquaporin-mediated long-distance polyphosphate translocation directed towards the host in arbuscular mycorrhizal symbiosis: Application of virus-induced gene silencing. New Phytol 211:1202–1208. https://doi.org/10.1111/nph.14016 Klironomos JN, Hart MM (2002) Colonization of roots by arbuscular mycorrhizal fungi using different sources of inoculum. Mycorrhiza 12:181–184. https://doi.org/10.1007/s00572-002-0169-6 Kurothe RS, Kumar G, Singh R, Singh HB, Tiwari SP, Vishwakarma AK, Sena DR, Pande VC (2014) Effect of tillage and cropping systems on runoff, soil loss and crop yields under semiarid rainfed agriculture in India. Soil Tillage Res 140:126–134. https://doi.org/10.1016/j.still.2014.03.005 Lanfranco L, Fiorilli V, Gutjahr C (2018) Partner communication and role of nutrients in the arbuscular mycorrhizal symbiosis. New Phytol 220:1031–1046. https://doi.org/10.1111/nph.15230 Lekberg Y, Schnoor T, Kjøller R, Gibbons SM, Hansen LH, Al-Soud WA, Sørensen SJ, Rosendahl S (2012) 454-sequencing reveals stochastic local reassembly and high disturbance tolerance within arbuscular mycorrhizal fungal communities. J Ecol 100:151–160. https://doi.org/10.1111/j.1365-2745.2011.01894.x Neary DG (2019) Forest soil disturbance: Implications of factors contributing to the wildland fire nexus. Soil Sci Soc Am J 83:S228–S243. https://doi.org/10.2136/sssaj2018.12.0471 Niwa R, Koyama T, Sato T, Adachi K, Tawaraya K, Sato S, Hirakawa H, Yoshida S, Ezawa T (2018) Dissection of niche competition between introduced and indigenous arbuscular mycorrhizal fungi with respect to soybean yield responses. Sci Rep 8:2–5. https://doi.org/10.1038/s41598-018-25701-4 Oehl F, Sieverding E, Ineichen K, Mader P, Wiemken A, Boller T (2009) Distinct sporulation dynamics of arbuscular mycorrhizal fungal communities from different agroecosystems in long-term microcosms. Agric Ecosyst Environ, 134:257–268. https://doi.org/10.1016/j.agee.2009.07.008 Oksanen AJ, Blanchet FG, Friendly M, Kindt R, Legendre P, Mcglinn D, Minchin PR, Hara RBO, Simpson GL, Solymos P, Stevens MHH, Szoecs E (2020) Package ‘vegan’ Rey F (2003) Influence of vegetation distribution on sediment yield in forested marly gullies. Catena 50:549–562. https://doi.org/10.1016/S0341-8162(02)00121-2 Schnoor TK, Lekberg Y, Rosendahl S, Olsson PA (2011) Mechanical soil disturbance as a determinant of arbuscular mycorrhizal fungal communities in semi-natural grassland. Mycorrhiza 21:211–220. https://doi.org/10.1007/s00572-010-0325-3 Smith SE, Read DJ (2008) Mycorrhizal Symbiosis, 3rd edn. Academic Press, London Smith VH (2003) Eutrophication of freshwater and coastal marine ecosystems: A global problem. Environ Sci Pollut Res 10:126–139. https://doi.org/10.1065/espr2002.12.142 Strona G, Galli P, Seveso D, Montano S, Fattorini S (2014) Nestedness for Dummies (NeD): A user-friendly web interface for exploratory nestedness analysis. J Stat Softw 59. https://doi.org/10.18637/jss.v059.c03 Suescún D, Villegas JC, León JD, Flórez CP, García-Leoz V, Correa-Londoño GA (2017) Vegetation cover and rainfall seasonality impact nutrient loss via runoff and erosion in the Colombian Andes. Reg Environ Chang 17:827–839. https://doi.org/10.1007/s10113-016-1071-7 Ulrich W, Gotelli NJ (2007) Null model analysis of species nestedness patterns. Ecology 88:1824–1831. https://doi.org/10.1890/06-1208.1 Van Eynde E, Dondeyne S, Isabirye M, Deckers J, Poesen J (2017) Impact of landslides on soil characteristics: Implications for estimating their age. Catena 157 : 173–179. https://doi.org/10.1016/j.catena.2017.05.003 Varela-Cervero S, López-García Á, Barea JM, Azcón-Aguilar C (2016) Differences in the composition of arbuscular mycorrhizal fungal communities promoted by different propagule forms from a Mediterranean shrubland. Mycorrhiza 26:489–496. https://doi.org/10.1007/s00572-016-0687-2 Supplementary Files Supplementalmaterial1.docx Supplementalmaterial2.xlsx Cite Share Download PDF Status: Published Journal Publication published 24 Oct, 2023 Read the published version in Plant and Soil → Version 1 posted Reviewers agreed at journal 06 Apr, 2023 Editor invited by journal 02 Apr, 2023 Editor assigned by journal 02 Apr, 2023 First submitted to journal 30 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-2758973","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":189746609,"identity":"453f128b-ff55-47e0-830b-9ef3ee363146","order_by":0,"name":"Anjar Cahyaningtyas","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anjar","middleName":"","lastName":"Cahyaningtyas","suffix":""},{"id":189746610,"identity":"66596350-8559-4464-ac05-71241f72d5b9","order_by":1,"name":"Tatsuhiro Ezawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYNCCChDBw8DYwMDMwMYA4eAEPEBFDAxnSNbC2IakhSCwZz9/+DPvPDu77Q28Bx/ObLOW52NgfviBQeYOblt4ktmkebclJ885wJdsuLEt3bCNgc1YgoHnGR6HJbMx825jTgaqMpN82HYY5EgzoPhh3Fr4HzN/5p1TD9di38bA/g2/FolkBmnehsN2YC0b2w4ntgEZ+LXceGwmOefY8QQJZh5jwxnn0pPbmHmKJRLw+IW9P/Hxhzc11fYS7D2GD3vKrG3nt7dv/PCxB3eIwUBiAzxGQIzEngMEtdij8X8Q1jIKRsEoGAUjBgAAjvVH0cmeLyAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6188-7635","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tatsuhiro","middleName":"","lastName":"Ezawa","suffix":""}],"badges":[],"createdAt":"2023-03-31 03:39:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2758973/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2758973/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-023-06337-4","type":"published","date":"2023-10-24T15:01:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":35543139,"identity":"fb7adb93-e7b8-4501-bda9-809e367d5b75","added_by":"auto","created_at":"2023-04-10 17:36:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1071690,"visible":true,"origin":"","legend":"\u003cp\u003ea) Location of Ishikari coastal dune in Hokkaido, Japan. Scale, 250 km. b) Sampling plots and ID in the seaward (1S – 5S) and landward (1L – 5L) slopes in the dunes. The image was generated by Google Earth (https://www.google.co.jp/intl/ja/earth/) using the GPS data of the plots (Supplemental material Table S1). Scale, 100 m. c) \u003cem\u003eMiscanthus sinensis\u003c/em\u003e grown in a landward slope. d) \u003cem\u003eLeymus mollis\u003c/em\u003e grown in a seaward slope.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2758973/v1/42a6d65657bb330c0a508b1d.png"},{"id":35543136,"identity":"5f72946a-bb16-4ec9-9a98-7197fade2fd4","added_by":"auto","created_at":"2023-04-10 17:36:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55506,"visible":true,"origin":"","legend":"\u003cp\u003eOTU-habitat (sample) biplots of nonmetric multidimensional scaling (NMDS) of a) the arbuscular mycorrhizal fungal OTUs that assigned to the genera \u003cem\u003eRhizophagus\u003c/em\u003e (grey symbols) and \u003cem\u003eGlomus\u003c/em\u003e (open symbols) in the family \u003cem\u003eGlomeraceae\u003c/em\u003e, b) those that assigned to uncultured \u003cem\u003eGlomeraceae\u003c/em\u003e, c) those that assigned to the genera \u003cem\u003eScutellospora\u003c/em\u003e (grey symbols), Aculospora (open symbols), and \u003cem\u003eDiversispora\u003c/em\u003e (closed symbol) in the order \u003cem\u003eDiversisporales\u003c/em\u003e, and d) those assigned to the genera \u003cem\u003eParaglomus\u003c/em\u003e (grey symbols), \u003cem\u003eArchaeospora\u003c/em\u003e(closed symbol), and \u003cem\u003eClaroideoglomus\u003c/em\u003e (open symbols). Only average values of the landward (L) and seaward (S) sample scores are plotted with standard deviations (vertical and horizontal bars). Disturbance-tolerance traits of the OTUs are indicated as follows: squares, soil propagule-mediated/root-direct regenerator; circles, soil propagule-mediated regenerator; diamonds, root-direct regenerators; triangles, slow regenerator. The read count data were transformed to logarithmic values and subjected to the analysis using Bray-Curtis dissimilarity index as a distance metric (two dimensions, stress = 0.0612).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2758973/v1/5f5f0df0deb392f6b198dc85.png"},{"id":35543137,"identity":"469982bb-7ca8-4d0c-82ac-92a48f73445e","added_by":"auto","created_at":"2023-04-10 17:36:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49424,"visible":true,"origin":"","legend":"\u003cp\u003eRichness (a) and b-diversity (b) of the arbuscular mycorrhizal fungal OTUs that showed the soil propagule-mediated/root-direct regenerator (SP/RD), soil propagule-mediated regenerator (SP), root-direct regenerator (RD), and slow regenerators (SL) traits occurred in the landward and seaward slopes. Richness represents the numbers of OTU. b-diversity represents Bray-Curtis dissimilarity indices calculated only for the trait categories in which the fungi were detected in three or more samples by pairwise comparison between all combinations of the samples (n.d., not determined). Welch's \u003cem\u003et-\u003c/em\u003etest, followed by Bonferroni corrections, were applied for the assessment of statistical significance: different letters indicate significant difference at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2758973/v1/70201e0e32e4053a0cf0c90e.png"},{"id":35543138,"identity":"17887361-44b7-46bf-9325-487c1308a524","added_by":"auto","created_at":"2023-04-10 17:36:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":100074,"visible":true,"origin":"","legend":"\u003cp\u003eMaximally stacked matrix of the arbuscular mycorrhizal (AM) fungal communities in the landward and seaward slopes. The rows and columns represent the OTUs and samples, respectively. All community composition data, including those of the three trait categories, were pooled within the individual sampling plots and transformed to presence-absence data (Supplemental material Table S4). The columns and rows were sorted by OTU richness and occurrence (number of sampling plot in which the OTU occurred), respectively. OTU richness is indicated above the matrix. Traits of the OTUs are indicated at the right columns: SP, soil propagule-mediated regenerator; RD, root-direct regenerator; SL, slow regenerator. Significant nestedness was observed among the columns: NODF\u003csub\u003ecolumn\u003c/sub\u003e, 68.00; \u003cem\u003eZ\u003c/em\u003e-score, 13.66 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2758973/v1/1c117d220af680d0467ca65c.png"},{"id":45453727,"identity":"d6c8e1f2-5d9f-43f7-b245-bb61c991bbf3","added_by":"auto","created_at":"2023-10-30 15:05:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1378768,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2758973/v1/6013fd89-a299-42e0-96bb-25e1e54abb96.pdf"},{"id":35543141,"identity":"7daefd1d-f823-4e41-a396-29124feaf82f","added_by":"auto","created_at":"2023-04-10 17:36:47","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":165707,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2758973/v1/ef23d5c05b854b8755ba1eb6.docx"},{"id":35543140,"identity":"f30e4184-1e05-47d9-a3a7-6f73eb8b67cf","added_by":"auto","created_at":"2023-04-10 17:36:47","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":42403,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2758973/v1/59284be3aa7eb49522e78f0f.xlsx"}],"financialInterests":"","formattedTitle":"Disturbance tolerance of arbuscular mycorrhizal fungi: trait-based characterization along a disturbance gradient in a coastal dune ecosystem","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil disturbance occurs through natural processes, e.g., volcanic ash fall (Crisafulli et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; del Moral \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), landslide in steep slopes (Hungr et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Van Eynde et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), wildfire (Hosseini et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Neary \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and sand movement by wind and tidal waves in coastal dunes (Delgado-Fernandez and Davidson-Arnott \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which cause environmental issues, such as erosion (Kurothe et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rey \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Suesc\u0026uacute;n et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and eutrophication of ground and marine water ecosystems (Smith \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Arbuscular mycorrhizal (AM) fungi form symbiotic association with most land plants and deliver mineral nutrients to the host (Smith and Read \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In the association the fungi construct hyphal networks in the soil, that is, extraradical mycelia, from which water and nutrients are taken up and translocated to arbuscules in the root cortex where the fungal symbionts release nutrients to the host (Ezawa and Saito \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In exchange for the nutrients, the fungi receive lipid and sugar as carbon source (Lanfranco et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil disturbance has a serious impact on mycorrhizal association via destruction of extraradical hyphal networks, which inhibits nutrient exchange between the symbionts and reduces new colonization mediated by the networks. It has been demonstrated that soil disturbance acts as selection pressure for the fungi that inhabit agroecosystems (Jansa et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), semi-natural grassland (Schnoor et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), coastal dunes (Corkidi and Rinc\u0026oacute;n \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Kawahara and Ezawa \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and volcanic slopes (Atunnisa and Ezawa \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fujiyoshi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), indicating that sensitivity to soil disturbance is different among AM fungal species. Rapid regeneration of hyphal networks after disturbance would be an important trait for disturbance-tolerant fungi; they would have short life cycles (Declerck et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), high-hyphal growth rates (Avio et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hart and Reader \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and efficient hyphal-healing mechanisms (de la Providencia et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Chagnon et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) proposed a concept of AM fungal life-history strategy according to the C-S-R (competitor, stress-tolerator, and ruderal) framework, which was originally raised by Grime (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) to classify plant life-history strategies. Competitor fungi (C) construct a high-density of hyphal networks in the soil and produce spores at later growth stages. Stress-tolerator fungi (S) can tolerate biotic stresses, such as high-soil acidity and low temperature, have long-lived mycelia, and grow slowly. Ruderal fungi (R) grow rapidly, show high rates of hyphal turnover, produce abundant spores at early growth stages or constitutively, and allocate more biomass in the roots than in the soil. In this context, the ruderal traits are likely involved in disturbance tolerance, whereas competitor fungi would be susceptible to soil disturbance. Disturbance-tolerant/sensitive traits of the fungi, however, have not yet been experimentally characterized.\u003c/p\u003e \u003cp\u003eIn this study, disturbance-tolerance traits of AM fungi were characterized by employing Ishikari coastal dunes as a model ecosystem in which vegetation is zonally distributed along soil-disturbance gradients (Kawahara and Ezawa \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). We addressed two hypotheses that i) disturbance-tolerant AM fungi that are capable of establishing new colony rapidly from soil-borne propagules (rapid regenerators) are enriched in a more severely disturbed habitat and ii) disturbance-sensitive fungi that require longer time to establish colony after disturbance (slow regenerators) are enriched in a less severely disturbed habitat. To test these hypotheses, a nylon mesh-separated compartment culture system was applied for assessing inoculum potential of extraradical and intraradical mycelia of the fungi separately.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003eSampling sites\u003c/p\u003e \u003cp\u003eA sampling area of 100 \u0026times; 1,000 m was defined along Ishikari coastal dune, Hokkaido, Japan (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). This ecosystem belongs to the subarctic zone with the annual mean temperature and rainfall are 8.3˚C and 651.0 mm, respectively. The primary dunes are 2\u0026ndash;6 m in height and located about 50\u0026ndash;100 m inland from the coastal line. Vegetation in the dune has been described in Kawahara and Ezawa (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Briefly, the landward slopes of the primary dune are 80\u0026ndash;150 m in width and largely dominated by the C4 perennial grass \u003cem\u003eMiscanthus sinensis\u003c/em\u003e Andersson (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In addition, \u003cem\u003eRosa rugosa\u003c/em\u003e Thunb. and \u003cem\u003eLathyrus japonicus\u003c/em\u003e Willd. are also patchily distributed. The seaward slopes are 40\u0026ndash;70 m in width and dominated by \u003cem\u003eLeymus mollis\u003c/em\u003e (Trin. ex Spreng.) Pilg. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). \u003cem\u003eCalystegia soldanella\u003c/em\u003e (L.) Riom. et Schult, \u003cem\u003eArabis stelleri\u003c/em\u003e var. japonica (A. Gray) Fr. Schm., \u003cem\u003eGlehnia littoralis\u003c/em\u003e F. Schmidt ex Miq, \u003cem\u003eCarex kobomugi\u003c/em\u003e Ohwi., \u003cem\u003eLinaria japonica\u003c/em\u003e Miq., and \u003cem\u003eIxeris repens\u003c/em\u003e (L.) A. Gray are also patchily distributed in the slopes. The habitats of \u003cem\u003eM. sinensis\u003c/em\u003e and \u003cem\u003eL. mollis\u003c/em\u003e are clearly segregated between the slopes, and this typical zonal distribution is observed more than 5 km along the coastal line. The topsoil layer of the seaward slopes is constantly disturbed by wind thus unstable. Whereas the thick root system of \u003cem\u003eM. sinensis\u003c/em\u003e stabilized the landward slopes, and thus an organic layer (3\u0026ndash;5 cm) originated from \u003cem\u003eM. sinensis\u003c/em\u003e litter has been developed. The soil chemical properties in the two slopes are described in Kawahara and Ezawa (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e); it is noteworthy that sodium (Na) concentration in the root-zone soils were not significantly different between the seaward and landward slopes (140 and 128 mg-Na kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, on average).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSoil sampling\u003c/p\u003e \u003cp\u003eWithin the area, five sampling plots were defined at 200 m intervals along the coastal line in each of the seaward and landward slopes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Four individuals of \u003cem\u003eM. sinensis\u003c/em\u003e and \u003cem\u003eL. mollis\u003c/em\u003e were selected in each sampling plot (5 \u0026times; 5 m), and root-zone soil samples, including root fragments, were collected from the four plants on June 17, 2020 by using a stainless-steel core sampler [5 \u0026times; 5 cm (diam/height), 100 mL in vol] for \u003cem\u003eM. sinensis\u003c/em\u003e and with a small shovel for \u003cem\u003eL. mollis\u003c/em\u003e (ca. 100\u0026ndash;200 g soil plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The core sampler was not used for the latter because the root-zone soil (sand) was too fragile to collect by the sampler due to the low density of \u003cem\u003eL. mollis\u003c/em\u003e roots. Twenty kilograms of sand was also collected from the seaward slope where no vegetation was present and autoclaved for 2 h as a base medium for soil trap culture.\u003c/p\u003e \u003cp\u003eTrap culture\u003c/p\u003e \u003cp\u003eThe soil samples collected by the core samplers (landward slopes) and those collected by the shovel (seaward slopes) were transferred to cylinder-shaped 37-\u0026micro;m nylon mesh bags [5 \u0026times; 5 cm (diam/height)] (non-destructively for the landward samples) in 350-mL plastic pots in which the base medium was filled around the mesh bag (four-replicated samples per plot and five plots in each of the two slopes in a total of 40 pots). \u003cem\u003eM. sinensis\u003c/em\u003e seeds (Snow Brand Seed Co., Ltd., Sapporo) were sown onto all soil samples in the nylon mesh bag, covered with a thin layer of sterilized sand, and grown in a temperature/light/humidity-controlled greenhouse (day/night temperature 26/20˚C; 14-h day length; 60% relative humidity). The seedlings were grown with tap water for the first month and with liquid fertilizer made of Peters Professional 25-5-20 (ICL, St. Louis, MO) at 50 \u0026micro;M phosphate once a week thereafter and thinned to three plants per pot one month after sowing. After two months, the seedlings were removed together with the mesh bag from two out of the four-replicated pots. These seedlings were used as donor plants of \"root-direct regenerator\" fungi, whereas spore/mycelia formed in the medium outside the mesh bag (remained in the pots) were used as inocula of \"soil propagule-mediated regenerator\" fungi as follows. The hole formed after the removal of the mesh bag was filled with the base medium to which five three-week-old new seedlings of \u003cem\u003eM. sinensis\u003c/em\u003e grown in the base medium were transplanted and grown for two months as assessment plants for soil propagule-mediated regenerators (two pots per plot) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Roots of the donor plants (i.e., the seedlings from the mesh bags) were washed on a stainless mesh with gently pressured tap water to remove adhering spores and extraradical mycelia, and the plants were transplanted outside the mesh bag of a new pot filled with the base medium. Then five three-week-old \u003cem\u003eM. sinensis\u003c/em\u003e seedlings were transplanted inside the mesh bag and grown for two months as assessment plants for root-direct regenerators (two pots per sampling plot). To raise \"slow regenerator\" fungi, five three-week-old \u003cem\u003eM. sinensis\u003c/em\u003e seedlings were transplanted outside the mesh bag in the remaining two pots and grown for further four months without removing the donor plants in the mesh bag. In all treatments the two pots from each sampling plot were treated as technical replication, and thus the data from the two were combined prior to analysis as described in the later section.\u003c/p\u003e \u003cp\u003eAfter harvesting the assessment plants, the roots were washed with pressured tap water, cut into 1-cm segments, randomized in water, blotted on a paper towel, immersed in RNA\u003cem\u003elater\u003c/em\u003e (Thermo Fisher Scientific, Tokyo) for more than 48 h at room temperature to fix nucleic acid, blotted on a paper towel to remove excess RNA\u003cem\u003elater\u003c/em\u003e, transferred to a 3-mL tube with an O-ring sealed cap (Yasui Kikai, Osaka), and stored at -80\u0026deg;C for DNA extraction.\u003c/p\u003e \u003cp\u003eMolecular identification\u003c/p\u003e \u003cp\u003eThe frozen roots in the 3-mL tube were ground with a metal cone in the presence of liquid nitrogen at 2,500 rpm for 2 \u0026times; 5 s using Multi-Beads Shocker (Yasui Kikai, Osaka), and DNA was extracted and purified from approx. 100 mg of ground sample by Maxwell RSC Instrument (Promega, Madison, WI) using Maxwell RSC PureFood GMO and Authentication Kit (Promega) according to the manufacturer's instructions, stored at -30˚C, and used as template for PCR amplification. The divergent domain 2 of large-subunit ribosomal RNA gene (LSU rDNA) was amplified in a 25-\u0026micro;L reaction mixture of Expand High-Fidelity PCR System (Roche Diagnostics, Tokyo), 0.5 nmol \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e each of FLd3 (forward) and FLR2 (reverse) primers that were linked to TruSeq-type forward- and reverse-adapter sequences (Illumina, Tokyo), respectively, at the 5'-end (Niwa et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and 0.2\u0026ndash;2 \u0026micro;L template DNA using C1000 TouchTM Thermal Cycler (BIO-RAD, Tokyo) with the following program: initial denaturation at 94˚C for 2 min, followed by 30 cycles of denaturation at 94˚C for 15 s, annealing at 48˚C for 40 s, polymerization at 72˚C for 1 min, and final elongation at 72˚C for 10 min. The PCR products were sequenced on the Illumina MiSeq platform (2 \u0026times; 300 bp), and high-quality paired end reads (read 1 and read 2) were merged with a minimum overlap length of 10 nt using FLASH (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ccb.jhu.edu/software/FLASH/\u003c/span\u003e\u003cspan address=\"http://ccb.jhu.edu/software/FLASH/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) at Bioengineering Lab (Sagamihara, Kanagawa, Japan). The merged reads were subjected to BLASTn searches against the fungal LSU rDNA database consisted of 82,494 operational taxonomic units (OTUs) of fungi, including 524 OTUs of AM fungi, constructed by Niwa et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and assigned to the OTUs at \u0026ge;\u0026thinsp;95% similarities over 330-bp alignment with an E-value cut-off of 1e-30.\u003c/p\u003e \u003cp\u003eData processing and trait definition\u003c/p\u003e \u003cp\u003eThe read numbers of the OTUs were subjected to pairwise correlation analysis within the same genera/families of AM fungi, and pairs that showed a correlation coefficient more than 0.9 were merged into single OTUs. The OTUs occurred uniquely in the soil propagule-mediated regenerator (SP) and root-direct regenerator (RD) treatments across all samples, irrespective of the occurrence in the slow regenerator (SL) treatment, were categorized as SP and RD, respectively, and those that occurred in both treatments were defined as SP/RD. Only the OTUs occurred uniquely in the SL treatment were categorized as SL. Total read numbers in each sample were standardized to 5,000 reads, whereas zero-data were assigned to all OTUs in the samples from which no PCR product was obtained (i.e., total read number was zero per sample). In this step, OTUs with less than one read were excluded from the datasets. Then total read number from the same sampling plots [two technical replications \u0026times; three (SP, RD, and SL) treatments] were standardized to 30,000 reads per plot, irrespective of whether zero-data samples were present or not. The read number data were transformed to logarithmic values (log\u003csub\u003e2\u003c/sub\u003e) or binary data (presence/absence) prior to analysis.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eF\u003c/em\u003e-test for equality of variance and Welch's \u003cem\u003et\u003c/em\u003e-test were conducted in the R 3.6.3 platform (R Core Team 2020), in which Bonferroni correction was applied for multiple comparisons. Non-metric multidimensional scaling (NMDS) were performed with the vegan package (Oksanen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) in the R platform using Bray-Curtis dissimilarity index as a distance metric. Bray-Curtis index was also employed for b-diversity analysis and applied only to the trait categories in which fungal OTUs were detected from three or more plots. Community nestedness was assessed with a maximally stacked matrix constructed with the presence-absence dataset, in which the order of the columns (plots) and rows (OTUs) were sorted by OTU richness and the frequency of OTU, respectively, on the web interface Nestedness for Dummies (Strona et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://purl.oclc.org/ned\u003c/span\u003e\u003cspan address=\"http://purl.oclc.org/ned\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), employing nested overlap and decreasing fill (NODF) as a metric (Almeida-Neto et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) with the proportional row and column totals null model (Ulrich and Gotelli \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The metric NODF ranges from 0 (non-nested) to 100 (fully nested), and a positive \u003cem\u003eZ\u003c/em\u003e-score indicate nestedness, and a negative \u003cem\u003eZ\u003c/em\u003e-score indicates anti-nestedness.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003eOTU definition and trait assignment\u003c/p\u003e \u003cp\u003ePCR products were obtained from all samples from the landward slopes, except for one of the RD samples, and from five SP, five RD, and 10 SL samples from the seaward slopes. The amplicon sequencing generated 50,617 paired reads per sample on average, which were quality-filtered and generated 41,460 merged reads per sample on average. About 86% of the merged reads were assigned to the AM fungal OTUs, and among them, two \u003cem\u003eRhizophagus\u003c/em\u003e OTUs, 003_Rhz and 011_Rhz, and two Uncultured Glomeraceae OTUs, 107_UnG and 157_UnG, were merged, respectively, into single OTUs (Supplemental material Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In the landward slopes 35, 15, and 53 OTUs occurred in the SP, RD, and SL treatments, respectively (60 OTUs in total), whereas 8, 5, and 5 OTUs occurred in the SP, RD, and SL treatments, respectively (10 OTUs in total), in the seaward slopes. Based on their occurrence patterns, 15, 23, 2, and 22 OTUs were categorized as SP/RD, SP, RD, and SL types, respectively (Supplemental material Table S3).\u003c/p\u003e \u003cp\u003eTrait-based characterization of the landward and seaward communities\u003c/p\u003e \u003cp\u003eNMDS species (OTU)-habitat biplots at the genus level provided an overview of taxon-trait distributions in the two slope habitats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea \u0026ndash; d). In these biplots, OTUs that are located closer to the center of habitat (sample) plots occur more specifically/abundantly in the habitat, while those that are located between the sample plots are broadly distributed in both habitats. Overall, diverse OTUs with various traits occurred in the landward slopes, whereas the seaward slopes were dominated by several SP/RD- and SP-type OTUs of limited taxa. Many of \u003cem\u003eRhizophagus\u003c/em\u003e OTUs were the SP/RD type, and two of them were broadly distributed across the habitats (Supplemental material Table S3). The \u003cem\u003eGlomus\u003c/em\u003e OTUs consisted of the SP/RD, SP, and SL types and distributed only in the landward slopes. The OTUs assigned to uncultured \u003cem\u003eGlomeraceae\u003c/em\u003e contained all types, and those of the SP/RD type were distributed in both slopes. A number of \u003cem\u003eScutellospora\u003c/em\u003e OTUs occurred in the landward slopes, in which all, except for one with the SL trait, showed the SP trait. The three \u003cem\u003eAcaulospora\u003c/em\u003e OTUs showed the SP traits, and one of the OTUs occurred specifically in the seaward slopes. All three \u003cem\u003eDiversispora\u003c/em\u003e OTUs showed the SL trait and occurred only in the landward slopes. The two \u003cem\u003eParaglomus\u003c/em\u003e OTUs with the SP/RD traits occurred both in the seaward slopes, but the other two with SP and SL traits were distributed only in the landward slopes. All five \u003cem\u003eClaroideoglomus\u003c/em\u003e OTUs occurred only in the landward slopes, and among them, three, one, and one showed SL, SP, and RD traits, respectively. Only one \u003cem\u003eArchaeospora\u003c/em\u003e OTU that has SL trait occurred in the landward slopes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the landward slopes OTU richness of the SP and SP/RD types was not significantly different and higher than that of the RD type, while richness of the SL type was not different from that of the other types (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the seaward slopes several SP/RD-type OTUs and only a few SP types occurred in all and two plots, respectively, whereas the RD and SL types were absent. In the landward slopes b-diversity was highest in the SL-type OTUs, followed by the SP and SP/RD types, and that of the SP/RD type in the seaward slopes was lowest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe lower diversity as well as the absence of SL-type OTUs in the seaward communities suggested that there is strong selection pressure on the communities, led to the idea that the fungi in the seaward slopes might be selected from those inhabit the landward slopes. To explore an OTU-distribution pattern across the diversity gradients between the two habitats, nestedness analysis was applied; significant nestedness was observed among the columns (NODF\u003csub\u003ecolumn\u003c/sub\u003e = 68.00, \u003cem\u003eZ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.66, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplemental material Table S4), implying that the seaward communities are subsets of the landward communities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe mesh-separated compartment culture system successfully differentiated AM fungal traits responsible for regeneration after disturbance. To characterize soil-borne propagules of the fungi, the destructive method (i.e., wet sieving) has generally been employed in previous studies (e.g., Klironomos and Hart \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Varela-Cervero et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is likely, however, that the isolation of spore and hyphae by destructive manners would lead to underestimation of inoculum potential via damaging/losing the propagules. The compartment culture system had originally been established three decades ago to assess the impact of destruction of extraradical hyphae on their inoculum potential (Jasper et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) and has recently been applied to physiological studies on fungal nutrient uptake (e.g., Hodge \u0026amp; Fitter, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kikuchi et al., 2016). The present study demonstrated that the application of this technique enabled us to assess the potential of extraradical mycelia in a non-destructive manner, in which extraradical mycelia were separated from the mycorrhizal roots simply by removing the mesh bag, minimizing the damages of the propagules. This culture system, however, cannot separate spores and extraradical hyphae that may differ in inoculum potential; the latter inocula would be more important in the genera \u003cem\u003eRhizophagus\u003c/em\u003e, \u003cem\u003eClaroideoglomus\u003c/em\u003e, and \u003cem\u003eAcaulospora\u003c/em\u003e spp. because not only spores but also extraradical hyphae acted as major inoculum source in extraradical matrices, even after fragmentation (Klironomos and Hart \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Accordingly, it should be noted that the propagules of the fungi with the SP trait, particularly those of the fungi in these genera, might consist of not only spores but also extraradical hyphae. Given that spores are likely to survive a longer period than detached hyphae, further experiments that take into account a time factor are necessary to assess the significance of extraradical hyphae in regeneration.\u003c/p\u003e \u003cp\u003eThe present study demonstrated that a majority of AM fungi inhabit the dune ecosystem is capable of regenerating rapidly via soil-borne propagules within two months, that is, disturbance-tolerant fungi, and further that all fungi in the seaward slopes showed this trait, supporting the first hypothesis. But, unexpectedly, many of the SP-type fungi could also regenerate directly from the colonized roots, that is, SP/RD-type fungi, indicating that not only spores/extraradical hyphae but also intraradical mycelia are important for rapid regeneration after disturbance. It has been well documented that the production of abundant spore is a prevalent trait in the members of \u003cem\u003eGlomeraceae\u003c/em\u003e (Chagnon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Klironomos and Hart \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), consistent with our finding that 20 out of the 38 OTUs with the SP trait belong to this family (i.e., those assigned to \u003cem\u003eRhizophagus\u003c/em\u003e, \u003cem\u003eGlomus\u003c/em\u003e, and uncultured \u003cem\u003eGlomeraceae\u003c/em\u003e). On the other hand, the higher biomass ratios of intraradical to extraradical mycelia are also a feature of \u003cem\u003eGlomeraceae\u003c/em\u003e fungi (Hart and Reader \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). This could contribute to rapid regeneration from colonized roots, which is the \"disturbance-avoidance strategy\" (Chagnon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In fact, 14 of the 17 OTUs with the RD trait belong to the \u003cem\u003eGlomeraceae\u003c/em\u003e. Given the significance of carbon supply from intraradical mycelia in the bridging/healing of fragmented extraradical hyphae (de la Providencia et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), photosynthetic activity of the host would be a major constraint of the reconstruction of extraradical hyphal networks and sporulation. In this study, the intact plants that retained photosynthetic activity were used as inoculum source for raising RD-type fungi, which might accelerate/enhance the colonization of the assessment plants. Potential of the colonized roots with or without the above-ground parts is of interest and to be assessed in the future.\u003c/p\u003e \u003cp\u003eWe first expected that the members in the family \u003cem\u003eGigasporaceae\u003c/em\u003e that consists of the three genera \u003cem\u003eGigaspora\u003c/em\u003e, \u003cem\u003eScutellospora\u003c/em\u003e, and \u003cem\u003eRacocetra\u003c/em\u003e would be slow regenerators because they colonize roots mainly via spores, rarely via the colonized roots, and produce spores at later stages of their life cycles (Biermann and Linderman \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Jasper et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Klironomos and Hart \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Oehl et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), which are typical of the competitor traits (Chagnon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In line with these studies, no \u003cem\u003eScutellospora\u003c/em\u003e OTUs showed the RD trait, confirming that their intraradical mycelia may not be major inoculum source. However, 12 of the 13 \u003cem\u003eScutellospora\u003c/em\u003e OTUs showed the SP trait, suggesting that they are capable of regenerating rapidly via extraradical hyphae detached from the roots (Jasper et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and/or via mature spores produced within two months, although the latter assumption is inconsistent with the observations that \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecalospora\u003c/em\u003e produced very few spores at least 11 weeks after inoculation (Jasper et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll the slow regenerator fungi occurred exclusively in the landward slopes, supporting the second hypothesis that slow regenerators are enriched in a less severely disturbed habitat. The 22 SL-type OTUs were distributed across diverse genera in the Glomeromycotina, and remarkably, b-diversity of the slow regenerator communities was significantly higher than those of the rapid regenerator (SP/RD and SP) communities, reflecting that most of the SL-type OTUs were unique to each plot. This patchy distribution of the slow regenerators across the slopes suggests that stochastic processes, rather than environmental factors such as disturbance severity, may play an important role in structuring the communities (Dumbrell et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lekberg et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). It is speculated, therefore, that more diverse SL-type fungi will be detected in this ecosystem by more intensive sampling, given the high b-diversity of the communities.\u003c/p\u003e \u003cp\u003eThe absence of SL-type OTUs in the seaward slopes, however, should be interpreted by taking into account that the seaward samples were collected destructively, which could potentially reduce the inoculum potential of SL-type fungi. In fact, two of the 22 SL-type OTUs, 008_Rhz and 206_Div, were detected in the roots of \u003cem\u003eL. mollis\u003c/em\u003e grown in the seaward slopes in the previous study (Kawahara and Ezawa \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), implying that these fungi are capable of colonizing the seaward slopes. The impact of the destructive collection on their potential, however, might be minimum because these two OTUs could also be detected in trap culture using the destructively collected soil samples (Kawahara and Ezawa \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this context, it seems likely that not all of the SL-type fungi have the competitor traits. There is a possibility that some of them have the ruderal traits but could not colonize the roots within two months due to rapid occupation of the niches by the dominant rapid regenerators. Detailed characterization of the slow regenerator fungi, e.g., their spore productivity and resilience of hyphal networks, is necessary to examine this possibility, although isolation of this type of fungi may not be easy due to the coexistence of the rapid regenerators.\u003c/p\u003e \u003cp\u003eOur finding that the AM fungi with the SP and/or RD traits are prevalent in the dunes provides experimental support for the idea that disturbance-tolerant fungi are selected across the ecosystem (Kawahara and Ezawa \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In addition, the nestedness pattern along the richness gradient indicates that the rapid regenerators in the seaward slopes were selected from the landward community, suggesting that selection pressure is even stronger in the seaward slopes than in the landward slopes. The lower OTU richness and b-diversity in the seaward communities further suggest that the communities converged towards the common members that preferentially inhabit coastal dune ecosystems. This raises a new idea that AM fungal communities associated with coastal dune vegetation converge across the globe, given the low endemism of AM fungi (Davison et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Further surveys are necessary to examine this idea.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe employed Ishikari sand dune ecosystem as a model and experimentally characterized the disturbance-tolerance traits of AM fungi by taking into account the time necessary for regeneration after disturbance. In the landward community, although many of the fungi showed the traits involved in disturbance tolerance, disturbance-sensitive fungi (i.e., slow regenerators) were also present. Whereas the seaward community consisted only of disturbance-tolerant fungi that were a subset of the landward community. These observations suggest that rapid regeneration from soil-borne propagules and colonized roots are key traits not only for the survival of the fungi but also for the resilience and maintenance of vegetation in dune ecosystems. Taken together, our findings provide a new insight into the life-history strategies of AM fungi in frequently/severely disturbed ecosystems and will also contribute to the practical application of AM fungi to restoration of post-disturbed ecosystems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis work was conducted at the open research facility of Hokkaido Agricultural Research Center, NARO. We also acknowledge Ministry of Education, Culture, Sports, Science and Technology, Japan for the scholarship to AC.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study conception, design, and writing the manuscript.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe sequence reads have been deposited in the Sequence Read Archive of National Center for Biotechnology Information under the accession number PRJNA949820.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlmeida-Neto M, Guimar\u0026atilde;es P, Guimar\u0026atilde;es PR Jr, Loyola RD, Ulrich W (2008) A consistent metric for nestedness analysis in ecological systems: Reconciling concept and measurement. 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Mycorrhiza 26:489\u0026ndash;496. https://doi.org/10.1007/s00572-016-0687-2\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"arbuscular mycorrhizal fungi, coastal dune, community nestedness, disturbance-tolerance trait, soil disturbance","lastPublishedDoi":"10.21203/rs.3.rs-2758973/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2758973/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eArbuscular mycorrhizal fungi that regenerate rapidly after disturbance play a significant role in resilience of vegetation after disturbance. We experimentally characterized disturbance-tolerance traits of the fungi that inhabit a coastal dune ecosystem.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRoot-zone soils, including root fragments, were collected from the seaward and landward slopes that differ in disturbance severity. The native grass \u003cem\u003eMiscanthus sinensis\u003c/em\u003e (donor plants) were grown in the soils for two months, and from a half of the plants, disturbance-tolerant fungi that regenerate from spores and extraradical hyphae (soil propagule-mediated regenerators, SP) and those that regenerate from the colonized roots (root-direct regenerators, RD) were trapped separately with new seedlings (assessment plants). The other half of the donor plants were further grown for four months together with assessment plants, during which the fungi trapped by the assessment plants were categorized as disturbance-sensitive slow regenerators (SL). DNA was extracted from the assessment plants, and fungal taxa were identified based on LSU rDNA sequences.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll fungi occurred in the seaward soil samples showed the SP and/or RD traits, whereas those occurred in the landward samples showed not only SP and/or RD traits but also SL traits. The seaward fungal communities were nested within the landward communities, implying that they were selected from the landward communities.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese observations suggest that rapid regeneration from soil-borne propagules and colonized roots are key traits of the fungi for survival in dune ecosystems, providing a new insight into the life-history strategies of AM fungi in frequently and severely disturbed ecosystems.\u003c/p\u003e","manuscriptTitle":"Disturbance tolerance of arbuscular mycorrhizal fungi: trait-based characterization along a disturbance gradient in a coastal dune ecosystem","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-10 17:36:42","doi":"10.21203/rs.3.rs-2758973/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-04-06T13:44:26+00:00","index":0,"fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2023-04-02T22:26:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-02T12:04:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2023-03-31T03:14:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7fe824b0-7ddb-4359-a9b3-66c840ba447e","owner":[],"postedDate":"April 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-30T15:03:15+00:00","versionOfRecord":{"articleIdentity":"rs-2758973","link":"https://doi.org/10.1007/s11104-023-06337-4","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2023-10-24 15:01:36","publishedOnDateReadable":"October 24th, 2023"},"versionCreatedAt":"2023-04-10 17:36:42","video":"","vorDoi":"10.1007/s11104-023-06337-4","vorDoiUrl":"https://doi.org/10.1007/s11104-023-06337-4","workflowStages":[]},"version":"v1","identity":"rs-2758973","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2758973","identity":"rs-2758973","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}